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Effects of chlorinated hydrocarbons on the heterotrophic activity of aquatic microorganisms Boyd, Walter Sean 1978

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EFFECTS OF CHLORINATED HYDROCARBONS ON THE HETEROTROPHIC ACTIVITY OF AQUATIC MICROORGANISMS by WALTER SEAN BOYD B . S c , U n i v e r s i t y o f Dalhousie, 1971 B.A., U n i v e r s i t y o f Dalhousie, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA ~ \ Walter Sean Boyd, 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or pub l i ca t ion of this thesis for financial gain shall not be allowed without my written permission. Department of The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 1 . ABSTRACT This study i n v e s t i g a t e d the short-term i n h i b i t o r y e f f e c t s o f 8 c h l o r i n a t e d compounds on a q u a t i c microorganism h e t e r o t r o p h i c a c t i v i t y . A technique i n v o l v i n g the measurement o f s u b s t r a t e uptake r a t e s u s i n g r a d i o -a c t i v e l y l a b e l e d glucose was employed. A l l compounds s t u d i e d have been i d e n t i f i e d as f o r e i g n p o l l u t a n t s i n aquatic environments and i n c l u d e the h i g h l y t o x i c ones DDT, d i e l d r i n and a PCB ( A r o c l o r 1254). The a q u a t i c microorganisms were predominatly b a c t e r i a . On a short-term b a s i s , the PCB and t e t r a c h l o r o p h e n o l were the most t o x i c p o l l u t a n t s , decreasing the maximum glucose uptake r a t e (Vmax) by 50 percent a t 250 ppb c o n c e n t r a t i o n s . The remaining p o l l u t a n t s had l i t t l e e f f e c t under 2500 ppb. In a d d i t i o n , t e t r a c h l o r o p h e n o l was the o n l y com-pound t o s i g n i f i c a n t l y i n c r e a s e glucose turnover time ( T t ) . Results from other experiments were: the t o x i c i t y o f t e t r a c h l o r o -phenol v a r i e d according to the (environmental) water temperature; t e t r a -chlorophenol a f f e c t e d the uptake o f glucose more than t h a t o f two amino a c i d s , a l a n i n e and glutamic a c i d ; w i t h respect to d i - , t r i - , and t e t r a -c hlorophenol, n e i t h e r the c h l o r i n e percentage per p o l l u t a n t molecule, nor the number o f p o l l u t a n t molecules per mole o f water, v a r i e d l i n e a r l y w i t h the p o l l u t a n t ' s a b i l i t y to i n h i b i t glucose uptake; the combined t o x i c i t i e s o f v a r i o u s p o l l u t a n t s reduced uptake to only 9 percent below the average o f t h e i r separate e f f e c t s ; and none o f the 4 p o l l u t a n t s t e s t e d f o r long-term e f f e c t s (DDT, PCB, d i e l d r i n and tetrachorophenol) i n h i b i t e d uptake a f t e r 4 days. I t was concluded t h a t the two t e s t s (Type I and Type I I as described i n S e c t i o n 2) were quick and r e l i a b l e techniques f o r a s s e s s i n g the s h o r t and long-term e f f e c t s o f a c o n c e n t r a t i o n o f a p o l l u t a n t on h e t e r o t r o p h i c i i . a c t i v i t y . I t was a l s o determined t h a t other p o l l u t a n t s should be t e s t e d f o r t h e i r long-term e f f e c t s under a v a r i e t y o f c o n d i t i o n s ( f o r example, t h e i r e f f e c t s on d i f f e r e n t substrates and on the sediment m i c r o b i a l community). i i i . TABLE OF CONTENTS Page LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x Chapter 1. INTRODUCTION 1 1.1 GENERAL 1 1.2 MICROORGANISM HETEROTROPHY AND ITS SIGNIFICANCE TO AQUATIC SYSTEMS 2 1.3 STRESS ON AQUATIC SYSTEMS 3 1.4 CHLORINATED ORGANIC POLLUTANTS IN AQUATIC SYSTEMS 5 1.5 CHLORINATED POLLUTANTS AND AQUATIC MICROORGANISMS 7 1.6 PAST RESEARCH USING THE HETEROTROPHIC ACTIVITY TECHNIQUE 9 1.7 SIGNIFICANCE OF THE HETEROTROPHIC ACTIVITY TECHNIQUE 1 1 1.8 PRINCIPLES OF THE HETEROTROPHIC ACTIVITY TECHNIQUE 1 2 2. MATERIALS AND METHODS 1 7 2.1 THE SUBSTRATE 1 7 2.2 THE POLLUTANTS ' 1 7 2.3 THE SAMPLING STATIONS 1 8 2.4 BASIC EXPERIMENTAL PROCEDURE 19 2.5 TYPE I AND TYPE I I EXPERIMENTS 2 2 I v . Page 2.6 PRELIMINARY EXPERIMENTS 2 4 2.6.1 Substrate uptake w i t h time 24 2.6.2 Comparison o f water samples 24 2.6.3 V a r i a b i l i t y i n and between experiments 26 2.6.4 Checking a Type I experiment 27 2.'6.5 D i f f e r e n t i a l e f f e c t s o f a p o l l u t a n t on f r e s h and aged water samples 27 2.6.6 E f f e c t s o f a p o l l u t a n t w i t h a changing s u b s t r a t e range 28 2.7 A SET OF TYPE I EXPERIMENTS 28 2.8 A SET OF TYPE I I EXPERIMENTS 28 2.9 VARIABLE TEMPERATURE EXPERIMENTS 3 0 2.10 VARIABLE CHLORINE PERCENTAGE.. EXPERIMENTS 3 0 2.11 VARIABLE SUBSTRATE EXPERIMENT 3 1 2.12 COMBINATION EXPERIMENTS 3 1 2.13 LONG-TERM EXPERIMENT 3 1 3. RESULTS AND DISCUSSION 3 3 3.1 PRELIMINARY EXPERIMENTS 3 3 3.1.1 Substrate uptake w i t h time 3 3 3.1.2 Comparison o f water samples 3 3 3.1.3 V a r i a b i l i t y i n and between experiments 3 ^ 3.1.4 Checking a Type I experiment 4 2 3.1.5 D i f f e r e n t i a l e f f e c t s o f a p o l l u t a n t on f r e s h and aged water 4 2 3.1.6 E f f e c t s o f a p o l l u t a n t w i t h a changing s u b s t r a t e range 42 Page 3.1.7 D i f f e r e n c e s i n Trout Lake and Nitobe Pond microbe communities 46 3.1.8 The c a r r i e r e f f e c t s 48 3.1.9 Summary' o f p r e l i m i n a r y experiments 48 3.2 A SET OF TYPE I EXPERIMENTS 49 3.3 A SET OF TYPE I I EXPERIMENTS 56 3.4 VARIABLE TEMPERATURE EXPERIMENTS 61 3.5 VARIABLE CHLORINE PERCENTAGE EXPERIMENTS 6 4 3.6 VARIABLE SUBSTRATE EXPERIMENT 6 7 3.7 COMBINATION EXPERIMENTS 6 9 3.8 LONG-TERM EXPERIMENT 69 4. SUMMARY AND CONCLUSIONS 75 5. FUTURE STUDIES 7 7 LITERATURE CITED 7 9 APPENDICES 8 5 v i . LIST OF TABLES TABLE Page 1 A summary o f the important parameters f o r each experiment 29 2 A comparison o f water sample uptake parameters 35 3 A comparison o f microbe heterotrophy from Trout Lake and Nitobe Pond 3 5 4 The e f f e c t s o f PCB (@ 385 ppb) on f r e s h and aged water samples 44 5 Vary i n g e f f e c t s o f PCB on Vmax and Tt w i t h a changing s u b s t r a t e range 44 6 The p o l l u t a n t concentrations causing a 25 percent decrease i n the c o n t r o l V 55 :1 R e s u l t s o f the Type I I experiments on PCB, te t r a c h l o r o p h e n o l and d i c h l o r o a c e t i c a c i d 59 8 Results o f the v a r i a b l e c h l o r i n e percentage experiment 65 9 The e f f e c t s o f t e t r a c h l o r o p h e n o l (@ 1000 ppb) on the uptake o f three substrates 65 10 The e f f e c t s o f t e t r a c h l o r o p h e n o l (@ 1000 ppb) on Vmax and Tt f o r a l a n i n e and glutamic a c i d 68 11 Results o f the combination .experiment 70 v i i . LIST OF FIGURES FIGURE Page 1 N u t r i e n t r e c y c l i n g and decomposition i n an aquatic ecosystem... 4 2 S a t u r a t i o n curves -*-3 3 A s a t u r a t i o n curve and i t s l i n e a r t r a n s f o r m a t i o n 16 4 A t y p i c a l i n c u b a t i o n v e s s e l and f i l t e r i n g apparatus used 20 5 General curve f o r a Type I experiment 23 6 Experimental s a t u r a t i o n curves and t h e i r l i n e a r transforma-t i o n s ^ 7 Graphs o f glucose uptake and uptake r a t e s versus i n c u b a t i o n time 3 4 8 S a t u r a t i o n curves f o r the average o f two samples from Trout Lake and Nitobe Pond 3 7 9 Type I experiments w i t h PCB 3 9 10 Type I experiments w i t h t e t r a c h l o r o p h e n o l 4 0 11 Type I experiments w i t h d i c h l o r o a c e t i c a c i d 4 1 12 Checking a Type I experiment 4 3 13 Va r y i n g s a t u r a t i o n curves w i t h a changing glucose range 4 5 14 D i f f e r e n c e s between Trout Lake and Nitobe Pond 4 ? 15 Type I experiments w i t h PCB and t e t r a c h l o r o p h e n o l 51 16 Type I experiments w i t h l-chloro-3-methyl-2-butene and 1, 3, 5-trichlorobenzene 52 17 Type I experiments w i t h DDT and d i e l d r i n 53 18 Type I experiments w i t h hexachloroacetone and d i c h l o r o a c e t i c a c i d 54 19 The e f f e c t s o f PCB and t e t r a c h l o r o p h e n o l on Vmax (Type I I experiments) 57 .viii. FIGURE D Page 20 The effects of PCB and tetrachlorophenol on Tt (Type II experiments) 58 21 The effect of tetrachlorophenol on glucose uptake at different incubation temperatures 62 22 The decrease in the controls due to tetrachlorophenol at different incubation temperatures 63 23 Decrease in controls due to the percent df chlorine per pollutant molecule, and the number of pollutant molecules per mole of water 66 24 Long-term experiment with PCB and tetrachlorphenol 72 25 Long-term experiment with DDT and dieldrin 73 26 Non-competitive and competitive inhibition 86 ACKNOWLEDGEMENTS I would like to thank Drs. W. E. Neill, B. C. McBride and J . E. Phillips for their constructive criticism of this manuscript. Mrs. L. MacDonald, of the Environmental Engineering Laboratory, Dept. of Civil Engineering, provided valuable assistance in the laboratory. Special thanks goes to my research supervisor, Dr. K. J. Hall, who gave much of his time and energy (not to mention advice) in bringing this study to a successful completion. Financial support provided by NRC grant A8935 is gratefully acknowledged. Chapter 1 INTRODUCTION 1.1 GENERAL There a r e , a t pr e s e n t , many d i f f e r e n t kinds and amounts o f f o r e i g n p o l l u t a n t s e n t e r i n g n a t u r a l water environments. Even though some have been found to be harmful to l a r g e r a q u a t i c l i f e , t h e i r e f f e c t ( s ) upon the microorganism compartment i s , to a great e x t e n t , unknown. The purpose o f t h i s t h e s i s i s twofold: 1. To study a technique i n v o l v e d i n measuring microorganism h e t e r o t r o p h i c a c t i v i t y . With t h i s technique i t i s ' p o s s i b l e to determine the s u b l e t h a l e f f e c t s o f a p o l l u t a n t upon the dynamics o f s u b s t r a t e uptake by an aquatic microbe community. 2. To determine, under a v a r i e t y o f c o n d i t i o n s , the short-term t o x i c i t i e s ' ' " o f 8 c h l o r i n a t e d organic compounds u s i n g t h i s technique. Before d e s c r i b i n g the a c t u a l experiments and t h e i r r e s u l t s , s e v e r a l t o p i c s r e l a t e d to t h i s area o f research w i l l be discussed. I n gen e r a l , these t o p i c s concern microorganism heterotrophy, c h l o r i n a t e d organic p o l l u t a n t s , and the h e t e r o t r o p h i c a c t i v i t y technique. In t h i s study, " t o x i c i t y " r e f e r s to the a b i l i t y o f a p o l l u t a n t to i n h i b i t s u b s t r a t e uptake. 2. 1.2 MICRORGANISM HETEROTROPHY2 AND ITS SIGNIFICANCE TO AQUATIC SYSTEMS The p o o l o f d i s s o l v e d organic matter (DOM) i n n a t u r a l freshwater bodies ranges from 0.2 to 50 mg l " 1 (Wright and Hobbie, 1966; A l l e n , 1969). This p o o l c o n s i s t s o f a wide v a r i e t y o f sugars, amino a c i d s and o r g a n i c a c i d s r e s u l t i n g from p l a n t and animal e x c r e t i o n s and p a r t i c u l a t e o r g a n i c matter (POM) decomposition ( G u i l l a r d and Wangersky, 1958; Wetzel, 1975). Algae are p o t e n t i a l DOM heterotrophs, but d i f f e r from a q u a t i c b a c t e r i a i n t h e i r manner o f s u b s t r a t e a s s i m i l a t i o n . A ccording t o Wright and Hobbie (1966), algae r e l y upon d i f f u s i o n as t h e i r uptake mechanism, a l l o w i n g s i g n i f i c a n t uptake only a t h i g h s u b s t r a t e l e v e l s (>500 pgr.1" ^ ) . B a c t e r i a , on the other hand, possess a system i n v o l v i n g d i f f e r e n t kinds and numbers o f uptake s i t e s l o c a t e d i n or on the c e l l membrane (Wright and Hobbie, 1966; Weimer, 1972). These s i t e s are a c t i v e i n t h e i r t r a n s -p o r t o f s u b s t r a t e from the o u t s i d e to the i n s i d e o f the c e l l ^ and each i s r e s p o n s i b l e f o r the uptake o f one o r , a t the most, a few s u b s t r a t e s present i n the surrounding medium. Due to t h i s a c t i v e uptake system, b a c t e r i a are extremely e f f i c i e n t a t removing low co n c e n t r a t i o n s o f DOM from the water. Since most su b s t r a t e s are below 100 yg 1 i n a l l n a t u r a l waters, the above uptake phenomena favour an almost complete b a c t e r i a l dominance o f a q u a t i c microbe heterotrophy (Wright and Hobbie, 1966). 2 Heterotrophic a c t i v i t y i s simply the r a t e a t which heterotrophy proceeds. 3 In t h e i r i n v e s t i g a t i o n o f E . ; c o l i , f o r example, Cohen and Monod (1957) found i t t o have between 30 and 50 t r a n s p o r t systems. ^This process r e q u i r e s energy. A p o r t i o n o f the t r a n s p o r t e d s u b s t r a t e i s r e s p i r e d as CO- and the r e s t i s i n c o r p o r a t e d i n t o c e l l m a t e r i a l . 3. The h e t e r o t r o p h i c process i s s i g n i f i c a n t i n t h a t i t i s the primary-method by which a q u a t i c , a e r o b i c b a c t e r i a o b t a i n t h e i r f u e l f o r energy requirements and b u i l d i n g b l o c k s f o r growth (Sistrom, 1962). When a c e r t a i n b i o m a s s / c e l l i s reached, (under the r i g h t c o n d i t i o n s ) the c e l l s d i v i d e and the p o p u l a t i o n i s able to m a i n t a i n i t s e l f . A l s o , because i t i s i n t i m a t e l y a s s o c i a t e d w i t h the processes o f decomposition (Sistrom, 1962; Ruschke, 1968) and n u t r i e n t r e c y c l i n g ( S e k i , 1964; Kuznetsov, 1968; S e k i and Kennedy, 1969; Romanenko, 1970; Weimer, 1972), the microbe community, and t h e r e f o r e i t s heterotrophy, i s an i n t e g r a l p a r t o f most aquatic ecosystems. Figure 1 shows the decomposi-t i o n o f POM by the b a c t e r i a , r e s u l t i n g i n reduced d e t r i t a l biomass and DOM. I t a l s o i n d i c a t e s the r e s p i r a t i o n o f CX^ by the microbe compartment and i t s u t i l i z a t i o n as a food source by other a q u a t i c organisms. Both processes r e s u l t i n the r e c y c l i n g o f n u t r i e n t s . I n c e r t a i n slow-moving systems, the microbes help impede the bottom sediments from b u i l d i n g up and i n others ( f o r example, i n a marsh-estuarine environment) they supply a l a r g e p r o p o r t i o n o f the energy/nutrient requirements f o r the higher t r o p h i c l e v e l s (Odum, 1971; Smith, 1974). 1.3 STRESS ON AQUATIC SYSTEMS The d i f f e r e n t species o f m i c r o f l o r a i n an a q u a t i c community are c o n t i n u a l l y competing f o r the a v a i l a b l e resources and adapting to the e x i s t i n g environmental c o n d i t i o n s o f temperature, pH, C^AX^ t e n s i o n s , and n u t r i e n t l e v e l s . A t any one time, t h i s r e s u l t s i n a p a r t i c u l a r community s t r u c t u r e and h e t e r o t r o p h i c a c t i v i t y . I f s t r e s s e d , f o r example due to some m o d i f i c a t i o n i n an a b i o t i c parameter or the presence o f a 4. t e r t i a r y consumers FIGURE 1. N u t r i e n t r e c y c l i n g and decomposition i n an a q u a t i c ecosystem. p o l l u t a n t , t h i s s t r u c t u r e and/or a c t i v i t y might change, l e a d i n g to an a l t e r a t i o n i n the r a t e s o f decomposition and n u t r i e n t r e c y c l i n g w i t h i n the system. Horvath and Brent (1972) d e s c r i b e the p o t e n t i a l l y harmful e f f e c t s t h a t thermal p o l l u t i o n c o u l d have on a fresh-water system. They suggest t h a t a c h r o n i c , h i g h water temperature would l e a d t o : 1. the d i s r u p t i o n o f the microorganism p o p u l a t i o n balance and an a l t e r a t i o n o f the metabolic c a p a b i l i t i e s o f the remaining microbes; . 2. the o b s t r u c t i o n o f the n a t u r a l decomposition and p u r i f i c a t i o n processes, l e a d i n g to an accumulation o f organic m a t e r i a l s and unde s i r a b l e substances; 3. the increase o f the e u t r o p h i c a t i o n process, l e a d i n g to anaerobic areas, f i s h K i l l s , u n d e s i r a b l e t a s t e s and odours, a l g a l blooms and lowered a e s t h e t i c values. From the above, i t can be seen t h a t a s h i f t i n one a b i o t i c parameter, l i k e temperature, c o u l d cause a s i g n i f i c a n t change i n the f u n c t i o n i n g o f a microbe community ( i n p a r t i c u l a r ) and i t s a q u a t i c system (as a whole). The presence o f a f o r e i g n p o l l u t a n t may have an e q u a l l y damaging e f f e c t . 1 . 4 CHLORINATED ORGANIC POLLUTANTS IN AQUATIC SYSTEMS Since they are n a t u r a l s i n k s , water bodies r e a d i l y c o l l e c t f o r e i g n chemicals. C h l o r i n a t e d compounds enter water systems i n many d i f f e r e n t ways and because o f t h e i r r e c a l c i t r a n t nature some b u i l d up t o l e v e l s which become t o x i c to a q u a t i c l i f e . A recent example o f t h i s con-cerns the p o l y c h l o r i n a t e d b i p h e n y l s (PCB's). They were f i r s t s y n t h e s i z e d about, 50 years ago, proved to have e x c e p t i o n a l chemical c h a r a c t e r i s t i c s , 6. and t h e r e f o r e u s e f u l i n l u b r i c a n t s , p l a s t i c s , c a r t i r e s and e l e c t r i c a l appliances (Boyle, 1975; Bourquin and Cassidy, 1975; Maki and Johnson, 1975). A f t e r some time, PCB's were di s c o v e r e d i n environmental samples from a l l over the world. They were found to be t o x i c to most marine l i f e , even at very low concentrations i n the water, and t h e i r c h r o n i c e f f e c t on fishv eating b i r d s was to cause them t o l a y t h i n - s h e l l e d eggs. The inherent danger o f PCB's i s due t o t h e i r l i p i d - s o l u b l e , non-degradable c h a r a c t e r i s e t i c s which a l l o w them to magnify tremendously throughout the food c h a i n . PCB's reached the environment i s s e v e r a l ways: through d i r e c t discharge i n t o streams by manufacturing p l a n t s , through i n d i r e c t l e a c h i n g from garbage dumps, and v i a atmospheric f a l l o u t a f t e r i n c i n e r a t i o n . PCB's have a l s o been found to be major urban, storm-water p o l l u t a n t s r e s u l t i n g from surface contamination ( H a l l e t a l . , 1976). Other c h l o r i n a t e d compounds, l i k e the p e s t i c i d e s DDT and d i e l d r i n , e n t e r water systems as a r e s u l t o f s p r a y i n g and dumping p r a c t i c e s (Middleton, 1973; T a r d i f f , 1973). S t i l l others get there by way o f i n d u s t r i a l / u r b a n e f f l u e n t (Middleton, 1973; T a r d i f f , 1973) and many, according to Glass (1975), J o l l e y e t a l . (1975 a and b ) , Stevens and Robeck (1975), Glaze e t al., (1976) and J o l l e y (1976), are formed as a r e s u l t o f the c h l o r i n a t i o n process used t o k i l l pathogens i n water s u p p l i e s , to d i s i n f e c t e f f l u e n t s from sewage treatment p l a n t s and to remove b i o l o g i c a l s urface f i l m s i n e l e c t r i c power-p l a n t c o o l i n g systems. Due to the c h l o r i n a t i o n process at waste treatment p l a n t s , a wide v a r i e t y o f c h l o r i n a t e d organic compounds are formed and discharged i n t o the r e c e i v i n g waters. Rozen e t al,. (1972). detected 77 and 38 organic compounds (some c h l o r i n a t e d ) i n the e f f l u e n t from primary and secondary 7. treatment p l a n t s , r e s p e c t i v e l y . P i t t et_ a l . (1974) determined t h a t over 100 r e f r a c t o r y compounds can be present i n e f f l u e n t from sewage treatment p l a n t s . They suggested t h a t the c h l o r i n a t e d compounds were a d i r e c t r e s u l t o f the c h l o r i n a t i o n process. Glaze et_ al_. Q-976) discov e r e d a t o t a l o f 40 c h l o r i n a t e d compounds to be a r e s u l t o f waste c h l o r i n a t i o n . Mori (1976) found t h a t the amount o f c h l o r i n e i n v o l a t i l e organic compounds doubled when mu n i c i p a l wastes are c h l o r i n a t e d . In h i s study he noted 20 new c h l o r i n a t e d compounds r e s u l t i n g from the c h l o r i n a t i o n process. Jolley (1973) c a l c u l a t e d t h a t about 1 percent o f the c h l o r i n e a p p l i e d t o primary e f f l u e n t ends up as s t a b l e , n o n - v o l a t i l e organochlorine compounds and Mori (1976) f e e l s t h a t these compounds c o u l d have p o s s i b l e damaging e f f e c t s i n n a t u r a l waters. Since the use o f t h i s p u r i f i c a t i o n technique i s e s c a l a t i n g , i t i s probable t h a t these new c h l o r i n a t e d p o l l u t a n t s are being r e l e a s e d i n t o r e c e i v i n g waters at an e v e r - i n c r e a s i n g r a t e . 1.5 CHLORINATED POLLUTANTS AND AQUATIC MICROORGANISMS Research t o date i n d i c a t e s t h a t some f o r e i g n p o l l u t a n t s have damaging e f f e c t s on a q u a t i c microorganisms. With respect t o algae, 14 E r i k s o n (1972) found t h a t p o p u l a t i o n growth and ;-;C uptake by the phytoplankton, T h a l a s s i o s i r a pseudonana, were i n h i b i t e d over an e n t i r e range o f added copper. In separate s t u d i e s , F i s h e r and Wurster (1973) and Larson and T i l l b e r g (1975) found t h a t growth and v i a b i l i t y o f v a r i o u s species o f algae were i n h i b i t e d by f a i r l y low concentrations o f p o l y -c h l o r i n a t e d b i p h e n y l . With respect t o b a c t e r i a , T r u d g i l l e t a l . (1971)found t h a t most species they s t u d i e d were a f f e c t e d by r e l a t i v e l y low concentrations o f i n s e c t i c i d e s . Hicks and Corner (1973) s t u d i e d the e f f e c t s o f DDT on 8. B a c i l l u s megaterium and discovered t h a t some h i g h doses caused death t o the c e l l s . Bourquin and Cassidy (1975) examined the e f f e c t s o f p o l y c h l o r -i n a t e d b i p h e n y l formulations on the growth o f 85 i s o l a t e s o f e s t u a r i n e b a c t e r i a . They found t h a t growth was i n h i b i t e d by the p o l l u t a n t and some species were a f f e c t e d to a g r e a t e r extent than others.. Although the exact manner i n which the b a c t e r i a are a f f e c t e d i s unknown, i t i s p o s s i b l e t h a t , i n some cases, t h e i r substrate-uptake system i s i n h i b i t e d . Vaccaro and Jannasch (1966) discovered t h a t i t was p o s s i b l e t o modify the enzyme-related t r a n s p o r t constant " K t " o f c e r t a i n i s o l a t e s simply by changing the temperature. P r e s c o t t (1970 a and b) determined t h a t s p e c i f i c enzyme systems o f the marine bacterium, Aeromonas p r o t e o l y t i c a , are g r e a t l y a f f e c t e d by temperature, n u t r i e n t s , i n o r g a n i c ions and c e r t a i n p o l l u t a n t s . J a n i c k i and K i n t e r (1971) and Beck (1972) found t h a t some h e r b i c i d e s and DDT i n h i b i t the a c t i v i t y o f s p e c i f i c enzymes used f o r m i c r o b i a l b i o s y n t h e s i s . According t o Ware and Roan (1970), p e s t i c i d e s tend t o adsorb onto m i c r o b i a l c e l l w a l l s . T h i s , they f e e l , i s due t o the low water s o l u b i l i t y o f most p e s t i c i d e s and the f a c t t h a t microbes have very h i g h s u r f a c e - t o -biomass r a t i o s * Smith (1974) suggests t h a t the l i p i d - s o l u b l e c h a r a c t e r -i s t i c o f the c h l o r i n a t e d hydrocarbons, l i k e PCB and DDT, enhances t h e i r a b i l i t y to penetrate and accumulate i n microbe membranes which are composed, to a l a r g e e x t e n t , o f l i p i d (Sistrom, 1962; Swanson, 1969; N o v i k o f f and Holtzman, 1970). Hicks and Corner (1973) n o t i c e d t h a t the b a c t e r i a i n t h e i r study bound about 1.7 pg o f DDT/mg o f c e l l dry weight. Most o f t h i s (75 percent) was concentrated i n the c e l l membrane and they b e l i e v e d t h a t the l e t h a l a c t i o n o f DDT was r e l a t e d to the r e s u l t i n g a l t e r e d membrane chemistry. F i n a l l y , c h l o r i n e i s a s t r o n g o x i d i z i n g agent and many c h l o r i n a t e d compounds are known to have complex molecular 9. s t r u c t u r e s which have damaging e f f e c t s on c e r t a i n enzymes and p h y s i o l o g i c a l f u n c t i o n s , even at low concentrations (Mount, 1973). The research o f the above authors suggests t h a t some c h l o r i n a t e d p o l l u t a n t s might a f f e c t the growth and v i a b i l i t y o f aquatic m i c r o f l o r a by b i n d i n g t o t h e i r membranes and i n t e r f e r i n g w i t h the f u n c t i o n i n g o f t h e i r substrate-uptake systems. This i n t e r f e r e n c e , due t o some physico-chemical i n t e r a c t i o n between the p o l l u t a n t molecules and the a c t u a l t r a n s p o r t enzymes, would have the e f f e c t o f i n h i b i t i n g the t o t a l microbe community heterotrophy. I t i s w e l l known t h a t c e r t a i n organochlorine compounds p e r s i s t i n the environment f o r a lo n g time (Chambers et^ a l ^ . , 1963; Alexander, 1964; Gustafson, 1970; Ware and Roan, 1970). T h i s , coupled w i t h the f a c t t h a t microorganisms have evolved without exposure to these new p o l l u t a n t s (So they are u n l i k e l y t o have defence mechanisms, acclima-t i o n c a p a b i l i t i e s and e x c r e t i o n pathways t o deal w i t h them), suggests t h a t a b u i l d - u p . o f these new c h l o r i n a t e d p o l l u t a n t s might very w e l l have d e l i t e r i o u s e f f e c t s upon the microorganism compartment i n the r e c e i v i n g water. As a r e s u l t , the community s t r u c t u r e and n u t r i e n t r e c y c l i n g process w i t h i n the system would l i k e l y change, e v e n t u a l l y a f f e c t i n g other compartments and u p s e t t i n g any p r e v i o u s l y e x i s t i n g balances between them. 1.6 PAST RESEARCH USING THE HETEROTROPIC ACTIVITY TECHNIQUE Since 1943, when Z o b e l l and Grant edetermined t h a t b a c t e r i a were capable o f u t i l i z i n g low concentrations o f organic s u b s t r a t e s i n the water, there has been a steady increa s e i n the 'refinement and a p p l i c a t i o n o f the experimental technique used to measure microorganism heterotrophy. In 1957, Cohon and Monod i n v e s t i g a t e d a number o f b a c t e r i a l transport systems responsible for the uptake of certain substrates. Parsons and Strickland Q-962) applied the Michaelis-Menten enzyme kinetics equation to calculate the "relative heterotrophic potential" in maxine waters. They measured the short-term uptake of labelled 14c-glucose and acetate and estimated the natural concentrations of these substrates in the water and their relative rates of incorporation into microbial cells. Wright and Hobbie (1965) elaborated upon the technique used to measure the net uptake of a substrate at low concentrations and in 1966 they studied the distinction between algal and bacterial heterotrophy in fresh-water systems. This was followed by Munro and Brock (1968) with a similar study on sea-water. Hobbie and Crawford (1969 a) perfected a technique which allowed the efficient capture of the respired CO2 from the microorganism community. This made i t possible to accurately measure the gross heterotrophic activity of a water sample. Fischer (1966), Harrison et al. (1971) and Hall et al. (1972) studied the heterotrophic activity of microorganisms in lake sediments. Hobbie and Crawford (1969 b) and Albright et al. (1973) -used the heterotrophic technique to measure the degree of eutrophication of different water bodies. Within the last decade there have been a number o.fi: otr other studies incorporating this technique: Menzel and Vaccaro (1964), Vaccaro and (Jannasch (1966), Allen (1968), Hobbie et/?.al. (1968 a and b), Stumm-Zolinger (1968), Allen (1969), Vaccaro (1969), Sorokin (1970), Takahaschi and Ichimura (1971), Varma and Nepal (1972), Hobbie (1973), Paerl (1974), Hall (1975) and Hobbie and Rublee (1975). This technique, then, has developed very r a p i d l y i n t o a unique method f o r s t u d y i n g the dynamics o f h e t e r o t r o p h i c behavior o f the micro-organism communities i n va r i o u s water environments. 1.7 SIGNIFICANCE OF THE HETEROTROPHIC ACTIVITY TECHNIQUE Se c t i o n 1.5 presented s e v e r a l examples on the e f f e c t s o f some p o l l u t a n t s upon aq u a t i c b a c t e r i a . There are, however, two o b j e c t i o n s t o the experimental technique used. F i r s t , the growth media f o r the assay c u l t u r e s and the chemical, p h y s i c a l and b i o l o g i c a l c o n d i t i o n s under which the e f f e c t s o f the p o l l u t a n t i s measured, are completely d i f f e r e n t from the n a t u r a l ones. Second, u s u a l l y the l e t h a l e f f e c t o n l y , t h a t i s , the conce n t r a t i o n o f the p o l l u t a n t which k i l l s o r i n h i b i t s microbe reproduc-t i o n , i s measured. These o b j e c t i o n s are s a t i s f i e d when the s u b l e t h a l e f f e c t (upon some dynamic aspect a s s o c i a t e d w i t h m i c r o b i a l existence) i s measured. This aspect should be r e p r e s e n t a t i v e o f the a c t u a l i n t e n s i t y a t which the microorganism community i s f u n c t i o n i n g under " i n s i t u " c o n d i t i o n s . Heterotrophicc'actJivJi.'ty appears to be a good measure o f t h i s i n t e n s i t y . There have been s e v e r a l s t u d i e s i n c o r p o r a t i n g t h i s i d e a . A l b r i g h t et a l . (1972), A l b r i g h t and Wilson (1974) and D i e t z e t a l . (1976) measured the s u b l e t h a l e f f e c t s o f va r i o u s m e t a l l i c s a l t s and organic compounds upon the v i a b i l i t y and net h e t e r o t r o p h i c a c t i v i t y o f fresh-water m i c r o f l o r a . They found t h a t some concentrations o f the p o l l u t a n t s decreased the h e t e r o t r o p h i c a c t i v i t y without k i l l i n g the c e l l s . In other words, something was happening to the dynamics o f m i c r o b i a l e x i s t e n c e which, at these low p o l l u t a n t c o n c e n t r a t i o n s , was impossible to detect u s i n g the method o f p l a t e counts. With this new technique, the effect of sublethal concentrations of a pollutant on the intensity at which a microorganism community is functioning can be determined under conditions very similar to those in the natural environment. 1.8 PRINCIPLES OF THE HETEROTROPHIC ACTIVITY TECHNIQUE Substrate uptake for a single enzyme follows the Michaelis-Menten kinetics equation: V = (Vmax • S)/(Kt + S) Here, V (in yg 1 - 1 hr~i) is the rate of uptake, or the hetero-trophic activity, at the substrate concentration S(in yg l - i ) . Vmax (in yg 1"! hr-1) is the maximum rate of uptake when the enzyme is saturated with substrate. Kt(in yg l" 1) is a transport constant, defined as the substrate concentration when V is one-half Vmax. As the substrate concen-tration S increases, V increases to Vmax according to the hyperbolic-saturation curve shown in Figure 2A. With respect to the uptake of a particular substrate, a species of microorganism in the community can be treated as a homogeneous pool of transport enzymes and s t i l l adhere to the Michaelis-Menten equation. Figure 2B shows the saturation curves for two different species of microbes with respect to the uptake of one substrate. Under any set of abiotic conditions, each microbe species has a characteristic Kt and Vmax value, specific to the uptake of that particular substrate. These values determine the shape of the saturation curve for each species. The smaller Kt i s , the more effective the species is at removing the substrate from the water when the concentration is low (species 1). The higher Vmax is, the more effective i t is at high substrate concentrations (species 2 ) . 13. A. Vmax V Wmax ~> B. V - I " V-l FIGURE 2 . Enzyme Depending upon the n a t u r a l l e v e l s i n the environment, then, i n t e r s p e c i f i c c o m p e tition would tend t o s e l e c t f o r those species which are able t o remove the d i f f e r e n t s u b s t r a t e s most e f f e c t i v e l y * . A c cording t o Vaccaro and Jannasch (1966), Hobbie e_t a l . (1968 a and b) and H a l l e t a l . (1972), the Michaelis-Menten k i n e t i c s equation describes the dynamics o f s u b s t r a t e uptake, by a microorganism community q u i t e w e l l . Here, the Kt and Vmax values would be, r e s p e c t i v e l y , the average o f and the sum o f the separate parameters f o r each s p e c i e s . This i s shown as the community s a t u r a t i o n curve i n Figure 2B^. E x p e r i m e n t a l l y , t h i s community s a t u r a t i o n curve (or gross uptake) can be d e r i v e d by s e p a r a t e l y measuring i t s two components—the amount o f s u b s t r a t e i n c o r -porated i n t o c e l l m a t e r i a l (net uptake) p l u s the amount r e s p i r e d as CO2 ( m i n e r a l i z a t i o n ) . This i s shown i n Fi g u r e 2£. Community h e t e r o t r o p h i c a c t i v i t y can be measured a t any su b s t r a t e c o n c e n t r a t i o n d e s i r e d . I t i s u s u a l , though, to measure i t a t e i t h e r the n a t u r a l l e v e l o r the s a t u r a t i o n l e v e l . At the n a t u r a l l e v e l , one i s measuring the normal h e t e r o t r o p h i c a c t i v i t y (or a c t u a l i n t e n s i t y ) o f the microbe community. Since i t i s d i f f i c u l t t o measure the low l e v e l s o f most s u b s t r a t e s i n the water, the turnover time T t , which i s the time i t takes the community t o completely remove the n a t u r a l s u b s t r a t e , i s determined. At the s a t u r a t i o n l e v e l , one i s measuring the maximum h e t e r o t r o p h i c a c t i v i t y Vmax (or the maximum p o t e n t i a l i n t e n s i t y ) o f the community. T h e o r e t i c a l l y , a l l the t r a n s p o r t s i t e s should be s a t u r a t e d w i t h s u b s t r a t e 5 T h i s _ s i t u a t i o n i s v e r y s i m i l a r t o t h a t between b a c t e r i a and algae i n s e c t i o n 1.3. ^This h y p o t h e t i c a l microbe community i s made up o f o n l y two sp e c i e s . and assimilating as fast as possible. Both Tt and Vmax are calculated by transforming the Michaelis-Menten, community saturation curve to a straight line. One way to do 7 this is by plotting S/V against S (see Figure 3). Here, S is the concen-tration of added substrate. The intercept of the line corresponds to Tt (in hours) and the reciprocal of the slope of the line corresponds to -1 -1 Vmax (in yg 1 hr ). The point where the line cuts the S axis is -(Kt + Sn) and this value is a measure of the maximum value of Sn, the natural concentration of the substrate. There are two modifications to this type of transformation. Both are, in effect, the same as the one above. One plots t/f against S,where t is the incubation time and f is the fraction of the added substrate taken up. The other transformation plots Cyt/c against S. Here, C is the corrected number of counts for 1 yCi of "^ C labeled substrate in the counting apparatus used, y is the quantity of "^ C added i n yCi, t is the incubation time, and c is the radioactivity in counts/min that was taken up or respired by the microbe community. The procedure for determining the effects of a pollutant on the heterotrophic activity is given in the next section. 7 This type of transformation has been found to give favourable plots, especially when the error in V is not too large (Dowd and Riggs. 1965). s FIGURE 3. A. A t y p i c a l gross community s a t u r a t i o n curve. B. A t r a n s f o r m a t i o n o f the s a t u r a t i o n curve to a s t r a i g h t l i n e to o b t a i n Vmax and Tt. The equation f o r t h i s l i n e i s : S = Kt + Sn + S_ . V Vmax Vmax Chapter 2 MATERIALS AND METHODS 2.1 THE SUBSTRATE Glucose was the substrate used in' the majority of experiments (alanine and glutamic acid were used for one experiment). Even though glucose represents only a fraction of the total DOM, Vaccaro and Jannasch (1966) consider i t to be a suitable substrate for measuring heterotrophic activity because i t is readily utilized by aquatic bacteria and is present in small amount in a l l waters. Most species of bacteria possess transport enzymes specific for glucose and many will utilize i t before other sub-strates (Ssistrom, 1962). Also, most researchers in the past have used glucose in their studies of aquatic microbe heterotrophy. Two batches of aliquots were prepared from the glucose isotope C^C - glucose (U), 150-250 mCi/mM New England Nuclear). The isotopes in the first batch were diluted to I uCi/ml with autoclaved distilled water, membrane filtered and 1 ml aliquots sealed in ampoules and frozen. The second batch was prepared in the same manner, but made up to 2 yCi/ml. Stock glucose was added to the labeled glucose, in the proper amount, whenever a particular concentration was desired in a reaction vessel. The alanine and glutamic acid came in prepackaged 1 ml ampoules (@ 10 mCi/mM). 2.2 THE POLLUTANTS Five different organochlorine chemicals and 3 known pollutants were chosen for this study. They represented different groups of organic compounds and a l l have been identified in waste-water effluent. They are: (1) l-chloro-3-methyl-2-butenej an unsaturated a l i p h a t i c hydrocarbon. (2) D i c h l o r o a c e t i c a c i d ; an a c i d . (3) 1, 3, 5-trichlorobenzene; an aromatic hydrocarbon. (4) 2, 3, 4, 6 - t e t r a c h l o r o p h e n o l ; a phenol. (5) Hexachloracetone; a ketone. (6) A r o c l o r 1254; a p o l y c h l o r i n a t e d b i p h e n y l . (7) 1 , 1 , l - t r i c h l o r o - 2 , 2-bis (p-chlorophenyl) ethane; a p e s t i c i d e c a l l e d DDT. (8) 1, 2, 3, 4, 10, 10-hexachloro-6, 7-expoxy-l, 4, 4a, 5, 6, 7, 8, 8a-octahydro-endo-exo-1, 4: 5, 8-dimethanonaphthalene; a p e s t i c i d e c a l l e d d i e l d r i n . The range o f concentrations under study was between 1 and 5000 + p a r t s per b i l l i o n (ppb). Most o f these p o l l u t a n t s have been found i n water environments i n f a i r l y , low concentrations (0.1 to 10 ppb) but some numbers 4, 6 and 7) have been found up to 300 ppb (Riseb.rough et a l . , 1968; Duke et ai.-, 1970; F i s h e r , 1975; Glaze e t a l . , 1976). U s u a l l y a s e r i e s o f p o l l u t a n t concentrations was needed f o r each experiment. This was accomplished by weighing out a known amount o f the pure compound and s u c c e s s i v e l y d i s s o l v i n g and d i l u t i n g i t i n 10 ml f l a s k s w i t h the proper c a r r i e r ( u s u a l l y reagent a l c o h o l or acetone) u n t i l the d e s i r e d c o n c e n t r a t i o n ( s ) was achieved. 2.3 THE SAMPLING STATIONS Most o f the water samples f o r t h i s study were taken from two d i f f e r e n t p l a c e s . The f i r s t , Trout Lake, i s l o c a t e d i n John Hendry Park i n the east end o f Vancouver, B.C. I t i s approximately 5 ha i n area, w i t h a maximum depth o f 4 m and has s e v e r a l storm d r a i n s e n t e r i n g i t from the surrounding p l a y i n g f i e l d s and s t r e e t s . A c a t t a i l marsh grows around most o f the lake's perimeter and there i s , no doubt, a f a i r l y h i g h POM c o n t r i b u t i o n from the l o c a l w i n t e r i n g duck p o p u l a t i o n . Samples were taken from the northwestern side o f the l a k e . The second sampling s t a t i o n was the southern s i d e o f Nitobe Pond, which i s l o c a t e d i n the A s i a n Gardens at U.B.C. i n Vancouver. I t i s l e s s than 0.2 ha i n area w i t h a maximum depth o f about 1.5 m. The water column i s s e a l e d o f f from the sediments by a l a y e r o f cement and there i s a constant i n f l o w o f f r e s h water to the system. Two other sampling s t a t i o n s were the nor t h e a s t e r n p o r t i o n o f Deer Lake, a f a i r l y e u t r o p h i c water body l o c a t e d i n Burnaby, B.C., and sur f a c e r u n o f f water a t Wreck Beach, again l o c a t e d a t U.B.C. The sampling procedure went as f o l l o w s : the c l e a n 1 l i t e r p l a s t i c sampling b o t t l e was r i n s e d twice w i t h the l a k e (or pond) water and then h e l d about 0.5m under the water s u r f a c e (about 1 m from shore) u n t i l i t was completely f i l l e d . I t was then capped, tr a n s p o r t e d back to the l a b (by c a r from Trout Lake) and r e f r i g e r a t e d a t a temperature c l o s e to t h a t o f the sampling s t a t i o n . The time i n t e r v a l between sampling and exper-imental use was u s u a l l y l e s s than 1.5 h r . 2.4 BASIC EXPERIMENTAL PROCEDURE A stopper/cup arrangement, c o n t a i n i n g a folded, g lass f i b e r f i l t e r (Reeve Angel 934 AH-2.4 cm), f i t t e d to a 25 ml Erlenmeyer f l a s k , was used as the i n c u b a t i o n v e s s e l (Figure 4A) . To t h i s v e s s e l was added a volume 8 of the sample water (<10 ml) , a known amount o f p o l l u t a n t p l u s c a r r i e r (or c a r r i e r i f i t was a c o n t r o l ) and a known amount o f the l a b e l e d glucose s o l u t i o n , so t h a t the t o t a l volume i n the f l a s k was 10 ml. The f l a s k was 8 ' In order to ensure a uniform d i s t r i b u t i o n o f water samples (with respect to microbe biomass) i n each r e a c t i o n v e s s e l , the water i n the 1 l i t e r sampling f l a s k was thoroughly mixed, u s i n g a s t i r r i n g magnet and apparatus, before p i p e t t i n g . stopper/cup arrangement glass f i b e r f i l t e r — 25 ml Erlenmeyer f l a s k water sample FIGURE 4. A. A t y p i c a l i n c u b a t i o n v e s s e l . B. The f i l t e r i n g apparatus used. 21. then incubated and shaken i n the dark f o r a given time p e r i o d ( u s u a l l y between 1.5 to 2 hours) i n a constant temperature water bath (Blue M, Model MSB-3222A-1). D i r e c t l y a f t e r i n c u b a t i o n , the microorganism community was k i l l e d by i n j e c t i n g 0.2 ml o f 5N H2SQ4 through the stopper, u s i n g a 1 ml s y r i n g e . The f l a s k was shaken f o r another 20 to 30 minutes and the CC^ c o l l e c t e d by the glass f i b e r f i l t e r a lready s a t u r a t e d w i t h 0.2 ml o f hyamine hydroxide. The water was f i l t e r e d through a 0.22 u M i l l i p o r e f i l t e r (25 mm diameter) u s i n g the apparatus shown i n Figure 4B. The two f i l t e r s , one c o n t a i n i n g the evolved CO2 (community m i n e r a l i z a t i o n ) and the other c o n t a i n i n g the p a r t i c u l a t e matter (net uptake), were p l a c e d 9 i n separate s c i n t i l l a t i o n v i a l s and 6 ml o f Bray's s c i n t i l l a t i o n s o l u t i o n added to each. The r a d i o a c t i v i t y i n each v i a l was then counted i n a l i q u i d s c i n t i l l a t i o n counter (Nuclear Chicago, Isocap 300) and the counts c o r r e c t e d f o r quenching and converted to d i s i n t e g r a t i o n s p er minute (DPM) and f i n a l -l y to an uptake r a t e o f yg 1 ^ h r ^ :glucose The a d d i t i o n o f the p a r t -i c u l a t e uptake r a t e to the r e s p i r e d CC^ r a t e gave the t o t a l glucose up--take r a t e , or gross h e t e r o t r o p h i c a c t i v i t y f o r t h a t r e a c t i o n v e s s e l . U s u a l l y 20 to 24 r e a c t i o n v e s s e l s were used i n each experiment. In g e n e r a l , the fundamental purpose o f a l l experiments was to compare the average gross h e t e r o t r o p h i c a c t i v i t y o f d u p l i c a t e or t r i p l i -cate experimental r e a c t i o n v e s s e l s ( c o n t a i n i n g 0.1 ml o f the c a r r i e r p l u s the proper amount o f p o l l u t a n t ) t o t h a t o f the c o n t r o l s ( c o n t a i n i n g o n l y 0.1 ml o f the c a r r i e r ) , a l l measured at one glucose l e v e l . 9 Bray's s c i n t i l l a t i o n s o l u t i o n r e c i p e : (1) 60 gram naphthalene (2) 4 gram PPO (2, 5-diphenyloxazole) (3) 0.2 gram POPOP (1, 4-bis-[2-(5-phenyloxazolyi)]-benzene) (4) 100 ml methanol (spec, grade) (5) 20 ml ethylene g l y c o l (6) Dioxane to 1 l i t e r Blanks ( i . e . r e a c t i o n v e s s e l s i n which the microbe community was k i l l e d by adding a c i d before incubation) were omitted i n the m a j o r i t y o f experiments f o r two reasons. F i r s t , the a d s o r p t i o n o f the l a b e l e d s u b s t r a t e was r e l a t i v e l y low and l i k e l y to occur, and d i d so i n the i n i t i a l experiments, to the same extent i n the experimentals as i n the c o n t r o l s . Second, there were a l i m i t e d number o f spaces a v a i l a b l e f o r f l a s k s i n the incuba t o r , and they were used f o r maximizing the r e p r o d u c i b i l i t y w i t h i n each experiment; The form o f the a c t u a l experiments d i d not s t r a y much from the above b a s i c procedure. Sometimes, complete experiments were d u p l i c a t e d whenever r e p r o d u c i b i l i t y was sought. In some types o f experiments, o n l y the p a r t i c u l a t e uptake was measured. Here, i n s t e a d o f k i l l i n g ; the sample a f t e r i n c u b a t i o n , i t was f i l t e r e d d i r e c t l y . In a l l experiments except one, o n l y the "short-term" e f f e c t s ( i . e . the time o f i n c u b a t i o n between 1.5 to 2 hours) o f a p o l l u t a n t were measured. 2.5 TYPE I AND TYPE I I EXPERIMENTS Two b a s i c types o f experiments were conducted during t h i s study: 1. Type I experiment: This experiment measures the e f f e c t o f i n c r e a s i n g concentrations o f a p o l l u t a n t on the gross h e t e r o t r o p h i c a c t i v i t y a t one su b s t r a t e l e v e l . A t y p i c a l p l o t f o r t h i s experiment i s presented i n Figure 5. The " e x p e r i -mental percentage o f the c o n t r o l " a c t i v i t y i s p l o t t e d a g a i n s t "the l o g o f the p o l l u t a n t c o n c e n t r a t i o n " . This experiment i s s i g n i f i c a n t i n th a t i t approximates the p o l l u t a n t c o n c e n t r a t i o n where the h e t e r o t r o p h i c a c t i v i t y i s i n i t i a l l y i n h i b i t e d . I t i s a l s o i d e a l i n comparing the r e l a t i v e e f f e c t s o f d i f f e r e n t p o l l u t a n t s to one another. 2. Type I I experiment: This type of experiment c o n s i s t s o f d e r i v i n g two s a t u r a t i o n curves, one f o r the c o n t r o l and one f o r the experimental, w i t h a known p o l l u t a n t concentration present (see s e c t i o n 1.7 f o r s a t u r a t i o n c u r v e s ) . Figures 6A and B show the t y p i c a l s a t u r a t i o n curves and t h e i r l i n e a r transforma-tions'!"^. From these t r a n s f o r m a t i o n s , the e f f e c t o f one c o n c e n t r a t i o n o f a p o l l u t a n t on Vmax and Tt can be determined. 2.6 PRELIMINARY EXPERIMENTS These experiments were designed to f a m i l i a r i z e the researcher w i t h the b a s i c technique used to determine the h e t e r o t r o p h i c a c t i v i t y o f a water sample. They are presented here because of t h e i r b e a r i n g upon the a c t u a l p o l l u t a n t - a d d i t i o n experiments. They might a l s o be o f some b e n e f i t to those u n f a m i l i a r w i t h t h i s type o f work. 2.6.1 Substrate uptake w i t h time The purpose of t h i s experiment was to c a l c u l a t e i f glucose uptake by a microorganism community v a r i e s l i n e a r l y w i t h time. I t was important to be assured o f the same uptake r a t e over a range o f i n c u b a t i o n p e r i o d s . Here, a Trout Lake water sample was used and the i n c u b a t i o n temperatures and glucose concentrations kept constant at 15°C and 100 p g l ^ r e s p e c t i v e -l y . The gross uptakes f o r d u p l i c a t e samples were measured a t i n c u b a t i o n times o f 0.5, 1.0, 1.5, 2.0, and 2.5 hours. 2.6.2 Comparison o f water samples The f i r s t experiments i n t h i s p a r t measured the k i n e t i c values Vmax, Tt arid (Kt + Sn) f o r water samples from Trout Lake, Deer Lake, In determining the l i n e a r i t y o f each t r a n s f o r m a t i o n , r , or the c o e f f i c i e n t o f determination (that i s , the sqaure o f the "degree o f f i t " o f the given p o i n t s to the l e a s t squares s t r a i g h t l i n e ) was c a l c u l a t e d u s i n g a Texas Instrument pocket c a l c u l a t o r (Model SR51). 25. FIGURE 6. A. S a t u r a t i o n curves f o r the gross h e t e r o t r o p h i c a c t i v i t i e s o f the experimental and c o n t r o l v e s s e l s . B. L i n e a r transformations f o r the above curves (Type I I experiment). 0 Wreck Beach r u n o f f , and d i s t i l l e d water. The glucose concentrations f o r each were 20, 40, 60, 80 and 100 yg l " 1 , the i n c u b a t i o n temperatures, 15°C and the i n c u b a t i o n times, 2 hours. These experiments took p l a c e i n December, 1975, and January, 1976. The second s e t o f experiments measured and compared the s a t u r a t i o n curves f o r the dominant microorganism species i n water samples from Trout Lake and Nitobe Pond. D u p l i c a t e experiments on separate water samples from each p l a c e were c a r r i e d out i n order to get a b e t t e r r e p r e s e n t a t i o n o f t h e i r r e s p e c t i v e m i c r o b i a l a c t i v i t i e s . The glucose concentrations were v a r i e d between 2.5 and 1000 ug 1 i n order to determine the "proper" s a t u r a t i o n curves f o r each system. 1"^ The i n c u b a t i o n temperature and times were 15°C and between 1.5 and 2 hours r e s p e c t i v e l y . These experiments took p l a c e i n May and June, 1976. 2.6.3 V a r i a b i l i t y i n and between experiments F i r s t , the v a r i a t i o n i n the r e s u l t s o f an experiment, due to experimental e r r o r o n l y , was determined. This was done by h o l d i n g the glucose c o n c e n t r a t i o n , and the i n c u b a t i o n temperature and time constant (at 50 yg 1 \ 15°C and 1.5 hours r e s p e c t i v e l y ) and measuring the gross uptake r a t e o f 18 d i f f e r e n t r e a c t i o n f l a s k s c o n t a i n i n g Trout Lake water. Second, three d i f f e r e n t s e t s o f Type I experiments were determined f o r between-experimental v a r i a t i o n . In the f i r s t s e t , 3 separate e x p e r i -ments on the e f f e c t s o f v a r y i n g concentrations o f PCB on the gross hetero-t r o p h i c a c t i v i t y o f Nitobe Pond water samples were c a r r i e d out. The same was done with, two other p o l l u t a n t s , d i c h l o r o a c e t i c a c i d and t e t r a c h l o r o -phenol. The glucose c o n c e n t r a t i o n , i n c u b a t i o n temperature and i n c u b a t i o n llrYhe more eu t r o p h i c a water body i s , the g r e a t e r the b a c t e r i a l biomass and Vmax ( A l b r i g h t and Wentworth, 1973). A c c o r d i n g l y , the higher Vmax,is, the g r e a t e r the s u b s t r a t e c o n c e n t r a t i o n necessary to produce s a t u r a t i o n . I f the s u b s t r a t e c o n c e n t r a t i o n i s too h i g h o r too low, then, the c u r v i n g p a r t (see s e c t i o n 3.16) o f the hyperbola w i l l not be a t t a i n e d . time f o r a l l 9 experiments were 20 yg l" , 15°C and 1.75 hours r e s p e c t i v e l y . 2.6.4 Checking a Type I experiment Here, three separate (constant s u b s t r a t e l e v e l / c o n s t a n t p o l l u t a n t l e v e l ) experiments, t e s t i n g the short-term e f f e c t s o f PCB on the gross h e t e r o t r o p h i c a c t i v i t y o f Trout Lake water, were c a r r i e d out. This was 12 done a t p o l l u t a n t l e v e l s o f 100, 1000 and 3400 ppb r e s p e c t i v e l y . The glucose c o n c e n t r a t i o n was 100 yg 1 \ the i n c u b a t i o n temperature 15°C and the i n c u b a t i o n time 2 hours. A type I experiment was then c a r r i e d out to check how w e l l i t compared to the previous s e t o f experiments. The glucose c o n c e n t r a t i o n was 100 yg 1 \ the i n c u b a t i o n temperature 15°C and the i n c u b a t i o n time 1.5 hours. 2.6.5 D i f f e r e n t i a l e f f e c t s o f a p o l l u t a n t on f r e s h and aged water samples This experiment compared a p o l l u t a n t ' s e f f e c t on the h e t e r o t r o p h i c a c t i v i t y o f f r e s h l y sampldd water to t h a t o f " o l d " samples kept i n the r e f r i g e r a t o r f o r some time. I f the e f f e c t s were s i m i l a r , then one water sample c o u l d have been used f o r a number o f experiments, s a v i n g a l o t o f time and e f f o r t f o r the researcher. The short-term effe'ct o f 385 ppb PCB was t e s t e d on the gross a c t i v i t y o f 4 r e a c t i o n v e s s e l s f o r each o f a f r e s h , a 1 week o l d , and a 5 week o l d Trout Lake water, sample. The glucose c o n c e n t r a t i o n i n each, f l a s k , and the i n c u b a t i o n temperature and times were 100 yg 1 \ 15°C and 2.5 hours r e s p e c t i v e l y . Experimental outline:.* i n one experiment, the e f f e c t s o f 100 ppb PCB on the gross h e t e r o t r o p h i c a c t i v i t y a t one glucose l e v e l was determined. This was done by measuring the a c t i v i t i e s o f 6 c o n t r o l f l a s k s and 6 e x p e r i -mental f l a s k s and comparing the r e s u l t s . The same t h i n g was done the next day w i t h 1000 ppb and then 3400 ppb. 2.6.6 E f f e c t s o f a p o l l u t a n t with, a changing s u b s t r a t e range In three separate experiments, the short--term e f f e c t s o f 1000 ppb PCB on Vmax and Tt o f Nitobe Pond water were measured (i.e. Type 11 experiments). A l s o , the range o f glucose co n c e n t r a t i o n s used was changed Cat f i r s t ) i n order to produce a proper s a t u r a t i o n curve. The concentra-t r a t i o n s f o r the f i r s t experiment were 20, 40, 60, 80 and 100 yg l - 1 , f o r the-second, 5, 10, 20, 40 and 80 yg l" 1, and f o r the t h i r d , 2.5, 5, 10 20 and 40 yg l" 1. The i n c u b a t i o n temperature and times were 15°C and 1.5 hours r e s p e c t i v e l y . 2.7 A SET OF TYPE I EXPERIMENTS^ A d i s c u s s i o n o f t h i s experiment i s presented i n s e c t i o n 2.5. Here, the short-term e f f e c t s o f the e i g h t p o l l u t a n t s from s e c t i o n 2.2 were s t u d i e d . In each experiment f i v e r e a c t i o n f l a s k s ( c o n t a i n i n g the l a b e l e d glucose and c a r r i e r ) were used as c o n t r o l s . The average c o n t r o l gross h e t e r o t r o p h i c a c t i v i t y was then compared to the average o f 3 experimental f l a s k s ( c o n t a i n i n g glucose, c a r r i e r and p o l l u t a n t ) a t each p o l l u t a n t c o n c e n t r a t i o n . 2.-vi . .'- of Type.;II 2.8 A SET OF TYPE I I EXPERIMENTS A d i s c u s s i o n o f t h i s experiment i s presented i n s e c t i o n 2.5. Here, only those p o l l u t a n t s which proved c o n s i d e r a b l y t o x i c under 1000 ppb i n the Type I experiments were t e s t e d — n a m e l y , PCB and t e t r a c h l o r o p h e n o l . The short-term e f f e c t s o f these p o l l u t a n t s on Vmax and Tt were determined a t 13Table 1 summarizes the important parameters a s s o c i a t e d w i t h each o f the experiments, 2.7 through t o 2.14. TABLE 1. A summary o f the important parameters f o r each experiment. EXPERIMENT WATER SOURCE SUBSTRATE CONCENTRATION(S) (yg l " 1 ) POLLUTANT CONCENTRATION(S) (ppb) INCUBATION TEMPERATURE(S) (°C) INCUBATION TIME(S) (hours) 2.7. . .. NITOBE POND 20 l-5000 + 15 1.5-2 2.8 NITOBE POND 2.5,5,10,20,40 • 10,100,1000 + 15 1.5-2.25 2.9 TROUT LAKE 20,6.1 500 0-44 1.3 2.10. NITOBE POND 11,8.2 1000 15 1.6 2.11 TROUT LAKE 20,40,60,80,100 1000 15 1.5 2.12 TROUT LAKE 7.3 1000 + 15 2.0 2.13 TROUT LAKE 20 1000 19 1-1.5 10, 100 and 1000 ppb concentrations. For the PCB, an e x t r a experiment at 3850 ppb and f o r the t e t r a c h l o r o p h e n o l , an e x t r a one at 340 ppb, were c a r r i e d out. At each glucose l e v e l , the gross h e t e r o t r o p h i c a c t i v i t i e s o f two c o n t r o l s and two experimentals were determined and s e p a r a t e l y averaged. For comparison, the e f f e c t o f a high c o n c e n t r a t i o n (6500 ppb) o f a more i n s i g n i f i c a n t p o l l u t a n t ( d i c h l o r o a c e t i c acid) was a l s o t e s t e d . 2.9 VARIABLE TEMPERATURE EXPERIMENTS Perhaps the t o x i c i t y o f a p o l l u t a n t i s a f f e c t e d by a change i n an a b i o t i c c o n d i t i o n l i k e temperature. In order to determine t h i s , two separate experiments were performed. These experiments measured the short-term e f f e c t s o f 500 ppb of t e t r a c h l o r o p h e n o l on the a c t i v i t y o f the water over a range o f i n c u b a t i o n temperatures (0, 15, 21, 34, and 44°C). The f i r s t experiment took place on October 14, 1976, and measured the e f f e c t s o f the p o l l u t a n t on the gross h e t e r o t r o p h i c a c t i v i t i e s at 20 yg 1 ^ glucose c o n c e n t r a t i o n s . The second took p l a c e on January 1, 1977, and measured the e f f e c t s on the net h e t e r o t r o p h i c a c t i v i t i e s at glucose concen-t r a t i o n s o f 6.1 yg 1 ^. ^ D u p l i c a t e f l a s k s f o r the c o n t r o l s and e x p e r i -mentals were measured at each temperature. 2.10 VARIABLE CHLORINE PERCENTAGE EXPERIMENT These two experiments were designed to determine whether or not i n c r e a s i n g c h l o r i n e s u b s t i t u t i o n on a p o l l u t a n t molecule a f f e c t e d the gross h e t e r o t r o p h i c a c t i v i t y to a gre a t e r exitent.. The short-term e f f e c t s of 1000 ppb of di-, t r i - and t e t r a c h l o r o p h e n o l were t e s t e d by averaging three f l a s k s ( f o r the experimental) and four ( f o r the c o n t r o l ) i n each experiment. 2.11 VARIABLE SUBSTRATE EXPERIMENT The short-term e f f e c t s o f a known p o l l u t a n t on the gross hetero-t r o p h i c a c t i v i t i e s o f two amino acids were determined. F i r s t , the r e l a t i v e e f f e c t s o f 1000 ppb o f t e t r a c h l o r o p h e n o l on the uptake o f L- a l a n i n e , L-glutamic a c i d and U glucose ( a l l at concentrations l e s s than 10 yg 1~1) were t e s t e d and compared. Next, two Type I I experiments, u s i n g the amino acid s as s u b s t r a t e , were c a r r i e d out at concentrations o f 20, 40, 60, 80 and 100 yg l' 1 and the e f f e c t o f 1000 ppb t e t r a c h l o r o -phenol on Vmax and Tt c a l c u l a t e d . 2.12 COMBINATION EXPERIMENTS In t h i s experiment, the s y n e r g i s t i c and/or a n t a g o n i s t i c e f f e c t s o f s e v e r a l p o l l u t a n t s were considered. The short-term e f f e c t s o f a l l combinations o f 1000 ppb concentrations o f PCB, t e t r a c h l o r o p h e n o l and d i e l d r i n on the net h e t e r o t r o p h i c a c t i v i t y were t e s t e d i n d u p l i c a t e . 2.13 LONG-TERM EXPERIMENT Tetrachlorophenol, PCB, DDT and d i e l d r i n were t e s t e d f o r t h e i r long-term e f f e c t s on net h e t e r o t r o p h i c a c t i v i t i e s . This experiment was an attempt at mimicing what might happen t o the t o x i c i t i e s o f the various p o l l u t a n t s over time i n the n a t u r a l environment. Fi v e samples o f water ( i n c l o s e d , 4.1 1, p l a s t i c c ontainers) were r e f r i g e r a t e d f o r 1.5 months c l o s e t o the lake temperature 6°C). Before i n c u b a t i o n , each o f the fou r p o l l u t a n t s ( d i s s o l v e d i n 10 ml o f the common c a r r i e r , reagent a l c o h o l ) were; added t o the d i f f e r e n t c o n t a i n e r s , so t h a t each had an i n i t i a l c o n c e n t r a t i o n o f 1000 ppb. To the f i f t h , the c o n t r o l , o n l y 10 ml o f the c a r r i e r was added. Every 2 t o 7 days ( f o r 32. a total of 12 times), the net uptake rates, for four subsamples of each container, were measured at an incubation temperature of 19°C. Chapter 3 RESULTS AND DISCUSSION 3.1 INITIAL EXPERIMENTS 3.1.1 Substrate uptake with time The uptake of glucose appeared to be linear with time (Figure 7A). The coefficient of determination, r 2 , for the relationship was 0.996. Figure 7B plots the uptake rate against incubation time. The "level" part of this graph falls between 1.5 and 2 hours, and was therefore selected as the incubation range for the majority of experiments. There did riot appear to be any obvious reason(s) for the higher uptake rates at 0.5 and 1.0 hour incubation.times. 3.1.2 Comparison of water samples The runoff water from Wreck Beach was apparently as micro-organism-free as the distilled water (Table 2). The Trout Lake water sample had more heterotrophic activity than the Deer Lake sample— a Vmax 5 times higher, indicating a proportionally higher bacterial biomass (see Albright and Wentworth, 1973), and a Tt about 3 times (y 42 hours) lower, indicating a faster glucose turnover time. Also, the Trout Lake sample had a (Kt + Sn) value 1.5 times higher than that of Deer Lake indicating either more natural glucose in the water and/or a higher Kt value for its microbe community. It must be kept in mind that the above results only apply to the water samples taken from one area in the system. A single sample does not accurately represent the activity of the whole waterbody, as bacterial biomass could change readily with the amount of POM present. 10. Oh 0.5 . 1.0 1.5 2.0 2.5 6.0 Glucose uptake r a t e ^l - J - h r " 1 ) 3.01 • • > 1 j 0.5 1.0 1.5 2.0 2.5 Incubation time (hours) FIGURE 7. A. Graph o f glucose uptake a g a i n s t i n c u b a t i o n time. B. Graph o f glucose uptake r a t e a g a i n s t i n c u b a t i o n time. TABLE 2. A comparison o f water sample uptake parameters. SAMPLE DATE' Vmax Tt Kt + Sn r2 (yg 1-1 hr-1) (hr) (yg 1 ) TROUT L. 10.12.75 1.653 19.9 33 0.776 DEER L. 12.12.75 0.322 61.7 20 0.903 WRECK B. 14. 1.76 0.0 00.0 OiO DISTILLED H20 12. 1.76 0.0 0.0 0.0 TABLE 3. A comparison o f microbe heterotrophy from Trout Lake and Nitobe Pond. SAMPLE DATE V Vmax Tt Kt + Sn 2 r (yg 1-1 h r " 1 ) (hr) r ( y g I " 1) TROUT L.(1) 20.4.76 1.787 90 161 .980 TROUT L.(2) 26.4.76 2.352 103 243 .937 AVERAGE1: 2.069 97 202 NITOBE P. (1) 6.5.76 0.553 7.6 4.2 .971 NITOBE P.(2) 7.5.76 0.608 < 1 < 1 .959 AVEMGE 0.580 v-4 * 2 36. There was a d i s t i n c t d i f f e r e n c e i n the Trout Lake water, taken from the same sampling area, between mid-December, 1975, and l a t e A p r i l , 1976 (see Trout L. i n Tables 2 and 3). Vmax had i n c r e a s e d o n l y 1.25 times w h i l e Tt i n c r e a s e d 4.85 times (77 h o u r s ) . This Tt in c r e a s e was probably due t o a r i s e i n the amount o f glucose (and other s u b s t r a t e s ) i n the s p r i n g o f th a t year.-^ The proper glucose ranges, t o achieve s a t u r a t i o n k i n e t i c s , f o r Trout Lake and Nitobe Pond were 50-—J500 .and 2.5 — ;4;P -•yg 1"^ r e s p e c t i v e l y (Figure 8 ) . This was probably due t o the dominance o f t h e i r h e t e r o t r o p h i c communities by two d i f f e r e n t s pecies o f b a c t e r i a . The p l o t s i n d i c a t e d a more Vmax e f f i c i e n t species i n Trout Lake and a more Kt e f f i c i e n t species i n Nitobe Pond. This d i f f e r e n c e , i n t u r n , >would probably have been due t o the d i f f e r e n c e i n the n a t u r a l s u b s t r a t e l e v e l s i n each water body. In Trout Lake a h i g h e r n u t r i e n t l e v e l would have been caused by the s p r i n g warming and mi x i n g o f the water and sediments w i t h the consequent i n c r e a s e i n phytoplankton and r e l e a s e o f DOM. This would have l e d to the s e l e c t i o n f o r a Vmax e f f i c i e n t s p e c i e s . I n c o n t r a s t , because o f Nitobe Pond's apparently h i g h e r f l u s h i n g r a t e and s e a l e d bottom, l e s s s u b s t r a t e would have been a v a i l a b l e i n the water, so t h a t s e l e c t i o n f o r a species w i t h a low Kt would have occurred. 3.1.3 V a r i a b i l i t y i n and between experiments The f i r s t p a r t o f t h i s s e c t i o n was designed t o check the v a r i a b i l i t y i n the r e s u l t s o f an experiment due t o experimental e r r o r only. The range i n the r e s u l t s was x ± 17 percent and the 95 percent confidence i n t e r v a l , "^This i s an assumption o n l y . The value (Kt + Sn) i n c r e a s e d by 169 yg 1"! (or 6.12 times) s i n c e December, and i t i s d i f f i c u l t to determine whether Kt or Sn o r b o t h were r e s p o n s i b l e f o r t h i s i n c r e a s e s i n c e n e i t h e r parameter was measured s e p a r a t e l y . FIGURE 8. S a t u r a t i o n curves f o r the average of two samples from each o f Trout Lake and Nitobe Pond. 38. y ± 4.25 percent. That i s , the gross uptake r a t e f o r any r e a c t i o n v e s s e l was w i t h i n 5 percent o f the mean, 95 percent o f the time. In the m a j o r i t y o f experiments, each a c t i v i t y (under one s e t o f experimental c o n d i t i o n s ) was the average o f 2, and sometimes 3 r e a c t i o n v e s s e l s . This technique, then, assured f a i r l y c o n s i s t e n t and r e p r o d u c i b l e r e s u l t s w i t h i n each experiment. The second p a r t was designed t o check the v a r i a b i l i t y between the r e s u l t s o f s e v e r a l Type I experiments. The r e s u l t s o f , t h i s p a r t are presented i n Figures 9, 10, and 11. P l o t s f o r each o f the three p o l l u t a n t s , namely PCB, t e t r a c h l o r o p h e n o l and d i c h l o r o a c e t i c a c i d , showed a f a i r degree of r e p r o d u c i b i l i t y and the average p l o t f o r each p o l l u t a n t f o l l o w e d a s i m i l a r l y shaped p a t t e r n , d i f f e r i n g o n l y i n the degree and p l a c e o f curva-t u r e . At each p o l l u t a n t c o n c e n t r a t i o n , the gross h e t e r o t r o p h i c a c t i v i t y o f the c o n t r o l was decreased t o a range o f a c t i v i t i e s i n the experimentals. This range v a r i e d anywhere from 5 percent to 40 percent i n the three f i g u r e s , and was h i g h e s t around the mid-point o f the descending p a r t o f the p l o t . This range would have taken i n t o account any v a r i a t i o n i n the b i o t i c and a b i o t i c c o n d i t i o n s i n the water samples, and any v a r i a t i o n i n v o l v e d between the a c M a l experiments. The v a r i a t i o n between Type I I experiments was not determined because i t was f e l t t h a t an experiment o f t h a t k i n d would have been j u s t as r e p r o d u c i b l e as ( i f not more than) the Type I experiments. The reason-i n g went as f o l l o w s : i n a Type I experiment, the drop i n the a c t i v i t y was measured at one glucose l e v e l and one p o l l u t a n t c o n c e n t r a t i o n . In a Type I I experiment, the c a l c u l a t e d changes i n Vmax and Tt were the r e s u l t s o f 5 separate measurements i n the change o f a c t i v i t y a t 5 d i f f e r e n t glucose l e v e l s and one p o l l u t a n t c o n c e n t r a t i o n ( r e f e r to: 'Figure 6) .. T h e o r e t i -c a l l y , then, these changes, which can be viewed as a s o r t o f averaging 100 SYMBOL DATE 25.5. 76 26.5. 76 •« 28.5. 76 1.0 2.0 3.0 4.0 5.0 Log PCB c o n c e n t r a t i o n (ppb) FIGURE 9. Type I experiments w i t h PCB % i n d i c a t e s the average experimental uptake r a t e as a percentage of the average c o n t r o l uptake rate. 40. SYMBOL DATE * 8.6.76 o g > 6 - 7 6 a 21.6.76 Log Tetrachlorophenol c o n c e n t r a t i o n (ppb) FIGURE 10. Type I experiments w i t h t e t r a c h l o r p h e n o l . 41. SYMBOL DATE '* 31.5.76 Log D i c h l o r o a c e t i c a c i d c o n c e n t r a t i o n (ppb) FIGURE 11. Type I experiments w i t h d i c h l o r o a c e t i c a c i d . 42. process, should have been reasonably accurate and r e p r o d u c i b l e from experiment to experiment. 3.1.4 Checking a TYPE I , experiment The r e s u l t s o f t h i s s e c t i o n are presented i n Figure 12. The p l o t f o r the Type I experiment c o i n c i d e d f a i r l y w e l l w i t h t h a t from the three separate experiments i n the PCB range 100 to 3400 ppb. This Type I e x p e r i -ment, then, proved to be a u s e f u l technique f o r determining the r e l a t i v e short-term e f f e c t s o f an i n c r e a s i n g c o n c e n t r a t i o n o f a p o l l u t a n t on the h e t e r o t r o p h i c a c t i v i t y o f a water sample. 3.1.5 D i f f e r e n t i a l e f f e c t s o f a p o l l u t a n t on f r e s h and aged water. From t h i s experiment, i t appears t h a t the PCB had more o f an i n h i b i t o r y e f f e c t on glucose uptake (about twice as much) i n the f r e s h l y sampled water than i n the aged water (Table 4 ) . Perhaps a more r e s i s t a n t s t r a i n o f microorganism dominated the. aging microbe community. Therefore, i t was determinedd more appropriate t o use only f r e s h l y sampled water f o r a l l experiments d e a l i n g w i t h the r e l a t i v e e f f e c t s o f a p o l l u t a n t on su b s t r a t e uptake. 3.1.6 E f f e c t s o f a p o l l u t a n t w i t h a changing s u b s t r a t e range At the su b s t r a t e range 20 t o 100 yg l " 1 , both the c o n t r o l and the experimental gross h e t e r o t r o p h i c a c t i v i t i e s produced s t r a i g h t l i n e s (Figure 13A). Apparently the glucose s a t u r a t i o n l e v e l f o r the Nitobe Pond microkes>mhad halTeadyafteenereachedltedSligh^ produced when the s u b s t r a t e range was 5 t o 80 yg I " 1 (Figure 13B) and s t i l l b e t t e r curves were generated w i t h the range.2.5 t o 40 yg 1~* (Figure 13C). On c a l c u l a t i n g the e f f e c t s PCB had on Vmax and T t , three somewhat d i f f e r e n t r e s u l t s were obtained (Table 5 ) . These 'results suggested t h a t a substrate range too h i g h , underestimated the e f f e c t o f a p o l l u t a n t on Vmax 1 1 — 1 1 1 _ !-0 2.0 3.0 4.0 5.0 Log PCB c o n c e n t r a t i o n (ppb) FIGURE 12. Checking a Type I experiment w i t h 3 separate constant s u b s t r a t e l e v e l / c o n s t a n t p o l l u t a n t l e v e l experiments. TABLE 4. The e f f e c t s o f PCB (@ 385 ppb) on f r e s h and aged water samples. SAMPLE AVERAGE OF 4 AVERAGE OF 4 % DECREASE EXPERIMENTALS CONTROLS OF CONTROLS (dpm) (dpm) DUE TO PCB FRESH TROUT L. 1519 2685 43 4 1 WK. OLD TROUT L. 4418 5734 22*9 5 WK. OLD TROUT L. 4017 5439 26 1 TABLE 5. V a r y i n g e f f e c t s o f PCB on Vmax and Tt w i t h a changing s u b s t r a t e range. SAMPLE F-DATE GLUCOSE RANGE EXPERIMENTAL Tt INCREASE (yg 1-1) PERCENTAGE OF DUE TO PCB CONTROL Vmax(%) (hr) NITOBE P.(1) 6.5.76 ZC20.100 57.3 86 NITOBE P.(2) 7.5.76 5-80 54.6 64 NITOBE P.(3) 17.5.76 2.5-40 43.6 9 45. A. V 0.5 0.4 0.3 0.2 0.1 Control O-i _ _ — O— O"" -— - "•0«~—— Experimental 20 40 60 80 100 V 0.5 0.4 0.3 0.2 0.1 c r —-O-5 10 20 40 Control Experimental 80 C. V 0.5 0.4 0.3 0.2 0.1 2.5 5 10 20 Control O Experimental 40 SCygl"1) FIGURE 13. Varying saturation curves with a changing glucose range (PCB at 1000 ppb): A. 20-100 y g l " 1 B. 5- 80 ugl-1 C. 2.5-40 y g l - 1 and overestimated the e f f e c t on Tt (at l e a s t w i t h respect t o a more "proper" s u b s t r a t e range). I t i s q u i t e p o s s i b l e , though, t h a t some other reason was re s p o n s i b l e f o r the change. In any case, i t was concluded t h a t the b e s t glucose range was between 2.5 and 40 yg 1 ^ f o r the Type I I experiments u s i n g Nitobe Pond water, s i n c e t h i s range produced the be s t c o n t r o l s a t u r a -t i o n curves. 3.1.7 D i f f e r e n c e s i n Trout Lake and Nitobe Pond microbe communities I t was s t a t e d , i n s e c t i o n 3.13, t h a t there may have been a d i f f e r -ence i n the dominant microorganism species occupying Trout Lake and Nitobe Pond waters ( i n the s p r i n g o f t h a t year) because o f t h e i r d i f f e r e n t community s a t u r a t i o n curves. I f t h a t was the case, then a p o l l u t a n t l i k e PCB may have had more o f an e f f e c t on one species than on the other. This seemed to be t r u e i n a comparison o f the two p l o t s i n Figure 14A.' Each p l o t i s the average o f those i n Figures 8 and 12. A t a l l c o n c e n t r a t i o n s , the PCB decreased Nitobe Pond a c t i v i t y more than Trout Lake a c t i v i t y . The d i f f e r e n t i a l e f f e c t s mentioned above were, a t f i r s t , thought to have been caused by d i f f e r e n c e s i n p a r t i c u l a t e o r g a n i c matter (POM) co n c e n t r a t i o n i n the r e s p e c t i v e water bodies. I t was f e l t t h a t the h i g h e r l e v e l o f POM i n Trout Lake water would b i n d more o f the p o l l u t a n t and lower i t s i n h i b i t o r y " e f f e c t . This does not appear t o have been the case because another d i f f e r e n c e between the two water bodies was noted. This d i f f e r e n c e concerned the percentage o f r e s p i r e d CO^ from the experimentals as compared to t h a t o f the c o n t r o l s . F i gure 14B presents three p l o t s f o r Nitobe Pond water, r e s u l t i n g from the data o f Figure 8, and two p l o t s f o r Trout Lake water, r e s u l t i n g from the data o f Figure 12. There appeared to be a d i f f e r e n c e i n the e f f e c t PCB had on the r a t i o o f the experimental C0 9 A. 100 Trout Lake Nitobe Pond 1.0 2.0 3.0 4.0 5.0 B. 1.0 Ra t i o Experimental C o n t r o l C0 2% 0.5 h 1.0 2.0 3.0 4.0 Log PCB c o n c e n t r a t i o n (ppb) >Trout Lake >Nitobe Pond 5.0 FIGURE 14. D i f f e r e n c e s between Trout Lake and Nitobe Pond: A. With respect to PCB's e f f e c t on t h e i r gross hetero-t r o p h i c ' a c t i v i t i e s ; and w i t h respect to the e f f e c t o f PCB on the r a t i o of the experimental C0 2 percentage r e l a t i v e to the B c o n t r o l C0 2 percentages, p e r c e n t a g e s ^ t o the c o n t r o l CO^ percentages, i n the two waters. T h i s i n d i c a t e d something other than POM c o n c e n t r a t i o n caused the d i f f e r e n t i a l i n h i b i t o r y e f f e c t s between water bodies and t h a t t h i s very w e l l might have been a d i f f e r e n c e i n dominant microorganism species occupying the two water systems. 3.1.8 The c a r r i e r e f f e c t s In s e v e r a l i n i t i a l experiments, the short-term e f f e c t s o f the 0.1 ml acetone and a l c o h o l c a r r i e r s , i n n the-.-10 ml o f sample water, were measured. These one-part-per-hundred concentrations dropped the gross h e t e r o t r o p h i c a c t i v i t y by 39 - 7 percent and 17 - 7 percent r e s p e c t i v e l y . Compared to the l e v e l o f PCB or t e t r a c h l o r o p h e n o l which would produce s i m i l a r decreases, the t o x i c i t i e s o f these c a r r i e r s were r a t h e r low. I n 6 7 f a c t , these c a r r i e r s were approximately 10 to 10 times l e s s t o x i c than the PCB or the t e t r a c h l o r o p h e n o l . 3.1.9 Summary of p r e l i m i n a r y experiments These p r e l i m i n a r y experiments helped- f a m i l i a r i z e the researcher w i t h the h e t e r o t r o p h i c a c t i v i t y technique and the manner i n which hetero-trophy i s a f f e c t e d under d i f f e r e n t c o n d i t i o n s . There were a l s o s e v e r a l important r e s u l t s t h a t came out of these experiments: 1. D i f f e r e n t water bodies c o u l d have d i f f e r e n t dominant microorganism species and these species c o u l d have d i f f e r e n t uptake parameters a s s o c i a t e d w i t h t h e i r r e s p e c t i v e h e t e r o t r o p h i c a c t i v i t i e s . 2. A p o l l u t a n t might have a d i f f e r e n t e f f e c t on the h e t e r o t r o p h i c a c t i v i t y o f f r e s h l y sampled water than on t h a t o f an aged sample. I t i s , 16 The CO2 percentage i s simply the r a t e o f r e s p i r e d CO7 ( i . e . m i n e r a l i z a t i o n ) d i v i d e d by the gross h e t e r o t r o p h i c a c t i v i t y . This was the o n l y time, f o r any p o l l u t a n t , t h a t t h i s r a t i o changed s i g n i f i c a n t l y from 1.0. t h e r e f o r e , necessary to c a r r y out a l l experiments on f r e s h samples. 3. A p o l l u t a n t might a l s o have d i f f e r e n t i a l e f f e c t s on the a c t i v i t i e s o f d i f f e r e n t water bodies. I t i s , t h e r e f o r e , wise to c a r r y out a s e t o f experiments on water samples from the same p l a c e . Since t h i s phenomenon i s c o n t r o l l e d , i n p a r t at l e a s t , by the e x i s t i n g microorganism species i n the water, and s i n c e the microorganism community s t r u c t u r e i s l i k e l y t o change from day to day ( i . e . d i f f e r e n t dominant species a t d i f f e r e n t times) i t i s a l s o wise to c a r r y out a s e t o f experiments w i t h i n as s h o r t a time i n t e r v a l as p o s s i b l e . 4. A p o l l u t a n t ' s e f f e c t on Vmax and Tt might vary w i t h the s u b s t r a t e range used. Therefore, i t i s important t o determine the range t h a t w i l l produce the proper s a t u r a t i o n curve f o r the water body under study, and to use t h a t range throughout the s e t o f experiments. 3.2 A SET OF TYPE I EXPERIMENTS The r e s u l t s o f these Type I experiments w i t h the 8 p o l l u t a n t s are presented i n Figures 15, 16, 17 and 18. Each p o l l u t a n t had a short-term e f f e c t on the h e t e r o t r o p h i c a c t i v i t y as d i f f e r e n t minimum concentrations i n the water. PCB and t e t r a c h l o r o p h e n o l i n h i b i t e d glucose uptake a t concentrations under 10 ppb. D i c h l o r o a c e t i c a c i d , on the other hand, was . not t o x i c u n t i l i t s c o n c e n t r a t i o n rose above 3000 ppb. The remaining p o l l u t a n t s were intermediate between these two extremes. The r e l a t i v e t o x i c i t y o f a p o l l u t a n t i s u s u a l l y given as the c o n c e n t r a t i o n i n ppb or ppm which drops the gross uptake r a t e by a c e r t a i n percentage. However, p o l l u t a n t molecules d i f f e r i n weight and s i z e so t h a t 100 ppb o f one p o l l u t a n t may not have the same number o f molecules per mole o f water as another. Since p o l l u t a n t s a f f e c t the uptake systems., on a molecular b a s i s , i t seems l o g i c a l to compare these methods o f so. expressing c o n c e n t r a t i o n s . For comparison's sake, then, the p o l l u t a n t concentrations i n "ppb" and "molecules per mole o f water", which 17 dropped V by 25 percent , were e x t r a p o l a t e d from the curves (Table 6 ) . In the f i r s t case, the r e l a t i v e t o x i c i t i e s o f the 8 compounds went as f o l l o w s : t e t r a c h l o r o p h e n o l > PCB > d i e l d r i n > t r i c h l o r o b e n z e n e > l-chloro-3-methyl-2-butene > hexachloroacetone > DDT > d i c h l o r o -a c e t i c a c i d In the second-case: PCB > t e t r a c h l o r o p h e n o l > d i e l d r i n > t r i c h l o r o b e n z e n e > l-chloro-3-methyl-2-butene>hexachloroacetone > DDT > D i c h l o r o -a c e t i c a c i d . For both c o n c e n t r a t i o n systems, the order of descending t o x i c i t y was n e a r l y the same - the only d i f f e r e n c e occurred when PCB and t e t r a -chlorophenol exchanged pl a c e s f o r the most t o x i c p o s i t i o n . Each p o l l u t a n t had a p a r t i c u l a r k i n e t i c p r o f i l e a s s o c i a t e d w i t h the drop i n a c t i v i t y . Again, there was a c l e a r d i s t i n c t i o n between the PCB and t e t r a c h l o r o p h e n o l p r o f i l e s and t h a t o f d i c h l o r o a c e t i c a c i d . The p l o t s f o r the f i r s t two were o f a more gradual nature as compared to the l a t t e r . As soon as the d i c h l o r o a c e t i c a c i d reached a c o n c e n t r a t i o n above l o g 3.6, the h e t e r o t r o p h i c a c t i v i t y q u i c k l y dropped from 100 percent to zero. This h i g h c o n c e n t r a t i o n might have been the p o i n t a t which the acute e f f e c t s o f d i c h l o r o a c e t i c a c i d were f e l t , perhaps causing death to the microorganisms. This was not the case w i t h PCB. I t ' s p r o f i l e showed a remarkable a b i l i t y f o r the microorganism community to r e s i s t a h i g h c o n c e n t r a t i o n o f t h i s p o l l u t a n t . For example, there was o n l y a 2.5 percent drop i n a c t i v i t y (from 30.5 t o 28.0 percent) due to an increase o f 27,000 ppb (from 3,000 to 30,000 ppb o f PCB). 17 • 25 percent was chosen i n s t e a d o f 50 percent because DDT, d i e l d r i n and hexcachloroacetone had not dropped V to 50 percent w i t h i n the concentra-t i o n range s t u d i e d . FIGURE 16. Type I experiments w i t h l-chloro-3-methyl-2-butene and 1,3,5-trichlorobenzene. . 1 I I I t 1.0 2.0 3.0 4.0 5.0 Log p o l l u t a n t c o n c e n t r a t i o n (ppb). FIGURE 17. Type I experiments w i t h DDT and D i e l d r i n . 5 4 . FIGURE 18. Type I experiments w i t h hexachloroacetone and d i c h l o r o a c e t i c a c i d . 55. TABLE 6. The p o l l u t a n t concentrations causing a 25 percent decrease i n the c o n t r o l V. POLLUTANT APPROXIMATE CONCENTRATION FOR 25 PERCENT DROP IN V [ i n ppb] [ i n molecules/mole FLO] (x l O 1 5 ) 1. Tetrachlorophenol 2. PCB 3. D i e l d r i n 4. Trichlorobenzene 5. l-chloro-3-methyl-2-butene 6. Hexachloroacetone 7. DDT 8. D i c h l o r o a c e t i c a c i d 35 56 320 500 1260 4570 7000 7000 1.69 1.57 9.40 31.20 142.70 191.00 223.35 614.65 56. I t appears, then, t h a t only two p o l l u t a n t s (PCB and t e t r a c h l o r o -phenol) were s i g n i f i c a n t l y t o x i c to the gross h e t e r o t r o p h i c a c t i v i t y under 1000 ppb. Both dropped the a c t i v i t y by 50 percent a t concentrations around 250 - 50 ppb. For the remaining compounds, t h i s c o n c e n t r a t i o n was w e l l above 2500 ppb. The t o x i c i t y o f ceach p o l l u t a n t was no doubt a s s o c i a t e d w i t h the manner i n which i t was taken up by, o r adsorbed to the membranes o f , the microorganism c e l l s . In a d d i t i o n , t h i s t o x i c i t y was probably r e l a t e d to the degree o f physio-chemical i n t e r a c t i o n between the p o l l u t a n t molecule(s) and the uptake s i t e s , i t s a b i l i t y to p r e c i p i t a t e out a t h i g h c o n c e n t r a t i o n s , and i t s r a t e o f a d s o r p t i o n to the r e a c t i o n v e s s e l w a l l s and POM. 3.3 A SET OF TYPE I I EXPERIMENTS The r e s u l t s o f these Type I I experiments are presented i n Table 7 2 and Figures 19 and 20. The r 's f o r a l l experimental and c o n t r o l t r a n s -formations were above 0.950. The p l o t s f o r the PCB and t e t r a c h l o r o p h e n o l from Figure 19 com-pared w e l l w i t h those from Figure 15 o f the previous s e c t i o n , suggesting tha a s i n g l e Type I experiment i s a good approximation o f the r e s u l t s o f s e v e r a l Type I I experiments (that i s , i f one i s not i n t e r e s t e d i n the p o l l u t a n t ' s e f f e c t on T t ) . Together, Figures 19 and 20 gave a good i n d i c a t i o n o f how the a c t u a l h e t e r o t r o p h i c a c t i v i t y o f Nitobe Pond would have been a f f e c t e d i f sub-j e c t e d to a given dose o f one o f the p o l l u t a n t s . For example, i f i t was suddenly discharged i n t o the pond such t h a t i t s immediate and uniform c o n c e n t r a t i o n was 1000 ppb, the PCB's short-term e f f e c t on the micro-organisms would have been to drop t h e i r p o t e n t i a l , gross h e t e r o t r o p h i c a c t i v i t y (Vmax) by 55 percent. The t e t r a c h l o r o p h e n o l ' s e f f e c t , a t the 57. 58. FIGURE 20. The e f f e c t s o f a PCB and t e t r a c h l o r o p h e n o l on Tt (A, i n hours and B, i n percent o f c o n t r o l ) . TABLE 7. Results o f the Type I I experiments on PCB, t e t r a c h l o r o p h e n o l and d i c h l o r o a c e t i c a c i d . EXPERIMENTAL Tt INCREASE POLLUTANT CONCENTRATION DATEl'E PERCENTAGE OF % (ppb) CONTROL Vmax(&) o f hours c o n t r o l PCB 10 100 1000 3850 13.5.76 14.5.76 17.5.76 19.5.76 91.2 85.1 43.6 37.5 120 440 680 700 1 7 9 7 TETRACHLORO-PHENOL 10 100 340 1000 23.6.76 22.6.76 10.6.76 24.6.76 97.6 52,9 13.2 11.2 240 110 670 3980 4 1 68 159 DICHLORO-ACETIC ACID 6500 4.6.76 51.8 100 < 1 same c o n c e n t r a t i o n , would have been to decrease i t by 90 percent. More s i g n i f i c a n t , though, might have been the e f f e c t they would have had on the.normal a c t i v i t y (Tt) at the n a t u r a l glucose l e v e l . Whereas 1000 ppb PCB would have increased Tt by on l y 7 hours (or about 700 percent o f the c o n t r o l T t ) , an eq u i v a l e n t c o n c e n t r a t i o n o f t e t r a c h l o r o p h e n o l would have in c r e a s e d i t by 160 hours (or about 4000 percent o f the c o n t r o l ) . That i s , the t e t r a c h l o r o p h e n o l would have slowed the a c t u a l h e t e r o t r o p h i c a c t i v i t y down to approximately 2.5 percent o f i t s n a t u r a l l e v e l . This would have hindered m i c r o b i a l a c t i v i t y (with respect to glucose uptake a t l e a s t ) u n t i l such time as the p o l l u t a n t was rendered i n e f f e c t i v e . I f the long-term e f f e c t s were s i m i l a r , o r i f the p o l l u t a n t was being pumped i n t o the system a t a r a t e which kept i t s c o n c e n t r a t i o n h i g h , problems w i t h the n u t r i e n t r e c y c l i n g and decomposition processes-would have r e s u l t e d . F i n a l l y , the 6500 ppb c o n c e n t r a t i o n o f d i c h l o r o a c e t i c a c i d dropped Vmax by 48 percent and inc r e a s e d Tt by l e s s than 1 hour. L i k e the PCB, a hig h c o n c e n t r a t i o n o f t h i s p o l l u t a n t g r e a t l y a f f e c t e d the maximum a c t i v i t y but had l i t t l e o r no e f f e c t on the r a t e o f turnover o f the n a t u r a l glucose p o o l . D i e t z e t a l . (1976) s t u d i e d the s u b l e t h a l e f f e c t s o f va r i o u s p o l l u t a n t s upon the n a t i v e m i c r o f l o r a o f Georgia S t r a i t . One o f the p o l l u t a n t - s t u d i e s was a PCB ( A r o c l o r 1260). A t a 100 ppb c o n c e n t r a t i o n , Vmax was lowered to 50 percent o f the c o n t r o l value and Tt inc r e a s e d 4 times. These values are w i t h i n the range o f those i n t h i s study. In another study, A l b r i g h t e t a l . (1972) found that low concentrations o f m e t a l l i c s a l t s decreased Vmax c o n s i d e r a b l y . They r e f e r r e d to t h i s as non-competive i n h i b i t i o n (see appendix 1 f o r d e t a i l s ) . For example, NaCl, a t 1000 ppb, decreased Vmax by on l y 9 percent. A t 100 ppb concen-t r a t i o n s , NaAs(3 3, B a C l 2 • 2H 20, C d C l 2 ' 2.5 ILO, C r C l 2 * 6FL0, H g C l 2 , PbCl and Z n C l ? dropped Vmax by 43, 33, 74, 22, 87, 71 and 55 percent r e s p e c t i v e l y . At 10 ppb, CuC^ dropped I t by 52 percent and N i C ^ ' 6 ^ 0 , by 20 percent. The l a s t and most t o x i c o f the s a l t s was Ag2S0^, which decreased Vmax by 47 percent a t the low l e v e l o f 0.1 ppb. With respect to Tt increases a t the above p o l l u t a n t c o n c e n t r a t i o n s , the h i g h e s t was 741 percent caused by HgC^. The remaining s a l t s i n c r e a s e d Tt to any-where from 109 to 385 percent o f the c o n t r o l v a l u e s . From t h i s , i t appears t h a t low l e v e l s o f some heavy metal, s a l t s can I n h i b i t Vmax t o a greater extent than d i d the PCB and t e t r a c h l o r o p h e n o l i n t h i s study, w h i l e the e f f e c t s on Tt were s i m i l a r . 3 .4 VARIABLE TEMPERATURE EXPERIMENT The p l o t s i n Figure 21 A and B d i f f e r e d somewhat t o t h e i r optimum uptake temperatures. For the October water sample, i t was between 34 and 44°C, and f o r the January sample, between 22 and 35°C. There i s good reason to b e l i e v e t h a t t h i s was due to the d i f f e r e n t n a t u r a l water temperatures i n the f a l l and w i n t e r months (around 10°C and 4°C r e s p e c t i v e l y ) causing the microorganism communities to a d j u s t and perhaps change t h e i r s t r u c t u r e s a c c o r d i n g l y (at l e a s t w i t h respect to the dominant species p r e s e n t ) . Hence, a microorganism community, adapted more to a h i g h water temperature, would tend to dominate i n the warmer months and another, adapted to a lower water temperature, would tend to dominate i n the c o l d e r months. Figure 22 p l o t s the e f f e c t o f 500 ppb o f t e t r a c h l o r o p h e n o l on the h e t e r o t r o p h i c a c t i v i t y a t the d i f f e r e n t i n c u b a t i o n temperatures. The c o e f f i c i e n t o f determination f o r the October data was 0.904 and f o r the Janaury.-data, 0.929. The slopes f o r each, o f the re g r e s s i o n s were -0.993 and 0.126 r e s p e c t i v e l y . This i n d i c a t e d t h a t the f a l l community glucose uptake was i n c r e a s i n g l y i n h i b i t e d by a decreasing i n c u b a t i o n temperature and the w i n t e r uptake was ( s l i g h t l y , but) i n c r e a s i n g l y i n h i b i t e d by an 62.-1 1 ' " > 1 • 1 • 0 4 8 12 16 20 24 28 32 36 40 44 Incubation t e m p e r a t u r e (°C) FIGURE 21. The e f f e c t o f t e t r a c h l o r o p h e n o l (@ 500 ppb) on glucose uptake a t d i f f e r e n t i n c u b a t i o n temperatures. A. F a l l , 1976; B. Winter, 1977 6Q » 1 -J 1 1 1 i i i i • i 0 4 8 12 16 20 24 28 32 36 40 44 Incubation temperature (°C) FIGURE 22. The decrease i n the c o n t r o l s due to t e t r a c h l o r o p h e n o l a t d i f f e r e n t i n c u b a t i o n temperatures. i n c r e a s i n g temperature. A change from the n a t u r a l water temperature, then, seemed to s t r e s s the microorganism community to the extent t h a t i t s h e t e r o t r o p h i c a c t i v i t y was somewhat e a s i e r to impair due to the presence o f t e t r a c h l o r o p h e n o l . This might suggest an i n c r e a s e d , i n i t i a l e f f e c t o f thermal p o l l u t i o n (see S e c t i o n 1.3) upon a microbe community i f a p o l l u t a n t happened to be present i n the water at the same time. Also,: at t h e i r n a t u r a l temperatures, the f a l l microorganism community hetero-trophy was i n h i b i t e d more than t h a t o f the w i n t e r (a 42 percent drop versus a 24 percent drop) suggesting t h a t a p o l l u t a n t ' s e f f e c t on a q u a t i c microorganisms might change depending upon the time o f y e a r and the c o r r e s -ponding water temperature. 3.5 VARIABLE CHLORINE PERCENTAGE EXPERIMENT The r e s u l t s o f t h i s experiment are presented i n Table 8. Although the two experiments d i f f e r e d i n the percentage drop i n a c t i v i t y due :to the three p o l l u t a n t s , t h e i r r e s u l t s were, i n g e n e r a l , s i m i l a r . The d i -and t r i c h l o r o p h e n o l had the same e f f e c t (averages o f 86 percent and 88 percent o f the c o n t r o l s , r e s p e c t i v e l y ) but the t e t r a c h l o r o p h e n o l was more t o x i c ( i t dropped the c o n t r o l a c t i v i t i e s down to 34 p e r c e n t ) . There appeared to be a b e t t e r r e l a t i o n s h i p between the t o x i c i t y o f - t h e p o l l u t a n t andJ.its r c h l o r i n e percentage (Figure 23 A) than i t s number o f molecules per mole o f water (Figure 23 B). In the former, t e t r a c h l o r o p h e n o l w i t h a higher c h l o r i n e percentage, was much more t o x i c than:.the other two p o l -l u t a n t s , but the r e l a t i o n s h i p was not l i n e a r . In the l a t t e r , even though t e t r a c h l o r o p h e n o l had a lower number o f molecules per mole o f water, i t was s t i l l much more t o x i c than the other two, suggesting t h a t some other physico-chemical f a c t o r was i n v o l v e d . This i s a l s o supported by the f a c t t h a t the c o n c e n t r a t i o n o f c h l o r i n e i n the water was the same (•> 30 x 10 TABLE 8. Results o f the v a r i a b l e c h l o r i n e percentage experiment. POLLUTANT (1000 ppb) EXPERIMENTAL . •PERCENTAGE v O0 OFP^'-CONTROL V I . 1 2. AVERAGE DICHLOROPHENOL 93.6 78.3 86.0 TRICHLOROPHENOL 93.7 82.1 87.9 TETRACHLOROPHENOL 47.4 21.1 34.3 ^Numbers 1. and 2. are two separate experiments. TABLE 9. The e f f e c t s o f t e t r a c h l o r o p h e n o l (@ 1000 ppb) on the uptake o f three substrates SUBSTRATE EXPERIMENTAL ©PERCENTAGE V (%) OFPcFCONTROL. V GI. iCOSE 1.1 2. AVERAGE A T A ' M T W GLUCOSE' 18.9 20.6 19.7 ALANINE 62.7 49.1 55.9 GLUTAMIC ACID 50.2 56.3 53.3 Numbers 1. and 2. are d u p l i c a t e s w i t h i n the same experiment. A. 100 % decrease o f c o n t r o l V 50 + Dichlorophenol O T r i c h l o r o p h e n o l • Tetrachlorophenol I 10 20 30 40 50 60 70 80 90 100 Percent C h l o r i n e per p o l l u t a n t molecule 100 % decrease o f c o n t r o l V 50 \ T -+ 1 1 2 3 4 5 6 7 8 9 10 Number o f p o l l u t a n t molecules ( x l 0 1 6 ) / m o l e H 20 FIGURE 23. The percent decrease o f the c o n t r o l V s due t o : A. The percent o f c h l o r i n e per p o l l u t a n t molecule; B. and the number o f p o l l u t a n t moleucles p er mole o f water. atoms/mole tLO f o r a l l three p o l l u t a n t s . 3.6' VARIABLE SUBSTRATE EXPERIMENT The e f f e c t o f t e t r a c h l o r o p h e n o l on,the net uptake o f the d i f f e r e n t s u b strates are presented i n Table 9. The averages o f the two t r i a l s show tha t the p o l l u t a n t lowered the uptake o f glucose almost 3 times more than the uptake o f the two amino a c i d s . Perhaps the amino a c i d t r a n s p o r t i s mediated by a s e t o f enzymes d i f f e r e n t from those r e s p o n s i b l e f o r glucose. I f so, they might be l e s s a f f e c t e d by the t e t r a c h l o r o p h e n o l molecules. Another e x p l a n a t i o n might concern the number o f biochemical steps i n v o l v e d before the s u b s t r a t e becomes i n c o r p o r a t e d i n t o c e l l m a t e r i a l . Glucose would face more o f these steps w i t h respect to p r o t e i n b i o s y n t h e s i s than the two amino a c i d s and, t h e r e f o r e , c o u l d be i n h i b i t e d to a g r e a t e r extent (that i s , i f the p o l l u t a n t molecule i n t e r f e r e s a t each s t e p ) . The r e s u l t s o f the second p a r t o f t h i s experiment are presented i n Table 10. The experimental and c o n t r o l regressions had r 's above 0.900. From the t a b l e , i t appeared t h a t the glutamic a c i d was a f f e c t e d more than the a l a n i n e w i t h respect to Vmax, but a f f e c t e d l e s s w i t h respect to Tt. The o v e r a l l r e s u l t s from t h i s experiment might be s i g n i f i c a n t . Even though 1000 ppb t e t r a c h l o r o p h e n o l lowers glucose uptake c o n s i d e r a b l y (see s e c t i o n 3.3), the microorganisms may be able to s u r v i v e and maint a i n t h e i r l e v e l o f i n t e n s i t y by s w i t c h i n g to an uptake o f ot h e r , l e s s a f f e c t e d substrates s i m i l a r to alamine and glutamic a c i d . The maximum uptake r a t e s f o r the a l a n i n e and glutamic a c i d c o n t r o l s 18 were compared to t h a t f o r glucose. On a u n i t weight b a s i s , glucose, 18 The experiments f o r glucose were a l s o c a r r i e d out w i t h Trout Lake water, one year previous to t h i s s e t o f experiments (see s e c t i o n 3.13, Tables 2 and 3 ) , and the average Vmax was c a l c u l a t e d by decreasing the n a t u r a l value by 17 percent (the percentage drop due t o the a l c o h o l c a r r i e r ) 68. TABLE 10. The e f f e c t s o f te t r a c h l o r o p h e n o l (@ 1000 ppb) on Vmax and Tt f o r a l a n i n e and glutamic a c i d . SUBSTRATE DATE EXPERIMENTAL Tt INCREASE PERCENTAGE OF CONTROL Vmax % hours ALANINE GLUTAMIC ACID 9.2.77 10.2.77 107.1 75.0 200 50 150 7 a l a n i n e and glutamic a c i d had values o f 1.861, 1.111 and 2.358 yg 1 h r r e s p e c t i v e l y . Converting uptake to a b a s i s o f carbon, they had values o f 0.618, 0.449 and 0.954 yg C l ' - H i r " 1 r e s p e c t i v e l y . F i n a l l y , uptake, w i t h respect t o the number o f s u b s t r a t e molecules, gave values o f 5.342, 7.858 15 -1 -1 and 9.987 ( a l l x 10 ) molecules 1 h r . Therefore, whether expressed on a u n i t weight, carbon or molecular b a s i s , the uptake r a t e o f glutamic a c i d was more r a p i d than e i t h e r gluc©se o r a l a n i n e . This means t h a t a drop i n the glucose uptake r a t e due to a p o l l u t a n t may not be harmful to the microbe compartment i f there are other organic s u b s t r a t e s i n the water which are j u s t as e a s i l y a s s i m i l a t e d . 3.7 COMBINATION EXPERIMENT The short-term e f f e c t s o f the v a r i o u s combinations o f p o l l u t a n t s d i d not appear to be a d d i t i v e (Table 11). In f a c t , each combination lowered the net uptake r a t e to a value ^ 9 percent below the average o f each poll-l u t a n t s ' separate e f f e c t s . This suggested a c e r t a i n degree o f antagonism and was caused perhaps, by some chemical i n t e r a c t i o n between the p o l l u t a n t s i n the water or by the molecules o f the l e s s t o x i c p o l l u t a n t competing and i n t e r f e r i n g w i t h those o f the more t o x i c one a t the p l a c e ( s ) o f i n h i b i t i o n . This might i n d i c a t e t h a t an e c o l o g i c a l system p o l l u t e d w i t h a d i v e r s i t y o f organochlorine chemicals may not have i t s microorganism a c t i v i t y drop much below the average e f f e c t o f each p o l l u t a n t taken s e p a r a t e l y . 3.8 LONG-TERM EXPERIMENT One must be c a r e f u l when i n t e r p r e t i n g the r e s u l t s o f t h i s e x p e r i -ment. The c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s were d i f f e r e n t from those i n the n a t u r a l environment, and,.because o f t h i s , the r e s u l t s might be d i f f i c u l t t o apply to a l a k e system. A l s o , the a c t u a l causes f o r c e r t a i n TABLE 11. Results o f the combination experiment. POLLUTANTS) ' EXPERIMENTAL PERCENTAGE OF CONTROL V 1000 ppb PCB (1) 47.0 1000 ppb TETRACHLOROPHENOL (2) 20.3 1000 ppb DIELDRIN (3) 51.8 (1) + (2) 26.0 CI) + (3) 43.3 (2) + (3) 27.8 (I) + (2) + (3) 31.4 phenomena may be completely d i f f e r e n t from what one i n t e r p r e t s from the r e s u l t s . In Figures 24 and 25, the net uptake r a t e s o f a l l 5 c o n t a i n e r s were p l o t t e d as percentages r e l a t i v e to the i n i t i a l (day 0) r a t e i n the 19 c o n t r o l c o n t a i n e r . The p l o t f o r the c o n t r o l f l u c t u a t e d up and down, f i n a l l y c o l l a p s i n g t o near zero,at 47 days. The p l o t s f o r the e x p e r i -mental, however, seemed to f o l l o w a d i f f e r e n t , more damped p a t t e r n ( e s p e c i a l l y DDT, PCB and t e t r a c h l o r o p h e n o l ) . Each p o l l u t a n t had a sho r t -term e f f e c t ( l a s t i n g from 1 to 2 days) on the net h e t e r o t r o p h i c a c t i v i t y but t h i s soon decreased to zero and remained there f o r the r e s t o f the i n c u b a t i o n p e r i o d . Thus, an apparent e q u i l i b r i u m was e s t a b l i s h e d beginning a t day 4. There are a number o f p o s s i b l e explanations f o r the above r e s u l t s : -Perhaps the p o l l u t a n t s f i n a l l y became t i e d up i n o r adsorbed to the organic matter i n the water and to the w a l l s o f the c o n t a i n e r ; Grossbard (1972) f e e l s t h a t t h i s happensLto most w a t e r - i n s o l u b l e p o l l u t a n t s , and t h e r e f o r e would reduce t h e i r e f f e c t on the p l a n k t o n i c microorganisms. -Perhaps, according to A l b r i g h t (personal communication), a hardy s t r a i n o f microbe, more r e s i s t a n t to the p o l l u t a n t s , became dominant i n the d i f f e r e n t communities. There was a l r e a d y an i n d i c a t i o n o f a d i f f e r e n c e i n the dominant microbes between Trout Lake and Nitobe Pond, the former seemingly more r e s i s t a n t to a t l e a s t one p o l l u t a n t , and t h i s c o u l d have occurred i n an enclosed c o n t a i n e r . -Perhaps the e x i s t i n g species were a b l e , a f t e r s u r v i v i n g the i n i t i a l 19 The a c t i v i t y r e l a t i v e to the c o n t r o l value f o r each day was not recorded f o r the f o l l o w i n g reason: i n the l a s t two experiments the c o n t r o l V s decreased j to approximately 51 o f the d a i l y average up to t h a t p o i n t and t h i s would have given the f a l s e impression t h a t the experimental V s increased tremendously, when I n f a c t they d i d not. Tetrachlorophenol PCB 250 200 Experimental percentage o f i n i t i a l c o n t r o l V 150 lOOf 8 12 16 20 24 Time (days) 28 32 36 40 44 48 FIGURE 24. Long-term experiment w i t h 1000 ppb of PCB and t e t r a c h l o r o p h e n o l . 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (days) FIGURE 25, Long-term experiment w i t h 1000 ppb o f DDT and d i e l d r i n . s t r e s s , to u t i l i z e the p o l l u t a n t s as energy sources. S e v e r a l s t u d i e s (Focht, 1972; K e i l et a l . , 1972; O l o f f s and A l b r i g h t , 1972; P a t i l e t a l . , 1972; Thomann and Sguros, 1974; Subba-Rao and Alexander, 1976) have concluded t h a t many species o f b a c t e r i a are capable o f u t i l i z i n g c h l o r i n -ated organic compounds f o r growth. Any one, o r a combination, o f the above would have caused an increase i n h e t e r o t r o p h i c a c t i v i t y , tending to r e t u r n i t to the i n t i a l l e v e l . There may have been a balance o f forces a f t e r day 4 - those tending to decrease s u b s t r a t e uptake ( i . e . the p o l l u t a n t s and t h e i r degradation products) versus one or more o f the above for c e s tending to i n c r e a s e i t . The c o n t r o l ' s d e c l i n e might have been due t o the exhaustion o f some e s s e n t i a l n u t r i e n t i n the water. In summary, i t seems th a t these four c h l o r i n a t e d p o l l u t a n t s , administered i n a s i n g l e dose, had n e g l i g i b l e long-term e f f e c t s on the h e t e r o t r o p h i c a c t i v i t y o f the water column microorganisms. However, there i s much l e f t unexplained i n t h i s experiment, and i t i s , t h e r e f o r e , probably not a good t e s t o f the long-term e f f e c t s o f a p o l l u t a n t . Accord-i n g l y , i t i s d i f f i c u l t to t r a n s f e r the r e s u l t s o f t h i s experiment and to speculate as t o what might happen i n the r e a l environment. Chapter 4 SUMMARY AND CONCLUSIONS This study was concerned w i t h the short-term e f f e c t s o f v a r i o u s organochlorine compounds on the microorganism h e t e r o t r o p h i c a c t i v i t y i n the a q u a t i c environment. One o f the p o l l u t a n t s s t u d i e d , PCB, i s a chemical used i n e l e c t r i c a l a p p l i a n c e s , DDT and d i e l d r i n are t o x i c p e s t i c i d e s , and the r e s t , probably a l l r e s u l t i n g from the c h l o r i n a t i o n process, have been i d e n t i f i e d i n wastewater e f f l u e n t . In summary, s e v e r a l p o i n t s can be made from t h i s study: 1. PCB and t e t r a c h l o r o p h e n o l i n h i b i t e d the Vmax of Nitobe Pond water to a l a r g e degree a t low concentrations i n the water ( i . e . about 50 percent at 250 ppb). The e f f e c t s o f the remaining p o l l u t a n t s were f e l t a t only very h i g h l e v e l s . 2. Tetrachlorophenol was a s i g n i f i c a n t p o l l u t a n t i n t h a t i t in c r e a s e d Tt c o n s i d e r a b l y (4000 percent o f c o n t r o l ) a t a c o n c e n t r a t i o n o f 1000 ppb. 3. The n a t u r a l water temperature, at d i f f e r e n t times o f the yea r , seemed to a f f e c t the t o x i c i t y of a p o l l u t a n t . October microorganism heterotrophy was i n h i b i t e d more (by 500 ppb tetrac h l o r o p h e n o l ) than January hetero-trophy. In a d d i t i o n , the October microbe community's a c t i v i t y was a f f e c t -ed more at low temperature than at h i g h ones. The opposite was tr u e f o r a January study. 4. N e i t h e r the c h l o r i n e percentage per p o l l u t a n t molecule nor the number o f p o l l u t a n t molecules per mole o f water v a r i e d l i n e a r l y w i t h t h a t p o l l u -t a n t 's t o x i c i t y t o glucose uptake. However, t e t r a c h l o r o p h e n o l was more t o x i c than e i t h e r o f d i - o r t r i c h l o r o p h e n o l , suggesting some other physico-chemical mode o f i n h i b i t i o n . 5. The heterotrophic activity of two amino acids was affected by a pollutant to a lesser degree than that of glucose. 6. When a combination of two or three pollutants was tested, i t always had an effect about 9 percent below the average of the separate effects of each pollutant. 7. None of the four pollutants studied in the laboratory environment had long-term effects on the heterotrophic activity after 4 days. Since most pollutants are at sublethal levels in a l l waters, i t will be of increasing importance to be able to determine their effects at these levels. The methodology used in this study is one approach to investigate this. A Type I experiment is a quick and reliable technique for assessing the effects of a range of concentrations of an unknown pollutant. A Type II experiment is appropriate when one wants to study the effects of one pollutant concentration on the actual and potential microorganism community heterotrophic activity. Both experiments can be used to study the short-term and long-term effects. On a short-term basis, tetrachlorophenol proved to be quite toxic to glucose uptake at low concentrations in the water. There are count-less other foreign pollutants entering the environment and many, like tetrachlorophenol, are apt to have toxic effects on the microbial l i f e existing there. Both their short-term and long-term effects on micro-organism heterotrophic activity should be investigated? 77. Chapter 5 FUTURE STUDIES In s e c t i o n 3.6 i t was found t h a t the uptake o f two other s u b s t r a t e s , glutamic a c i d and a l a n i n e , was not a f f e c t e d t o the extent glucose was. I f t h i s i s the case w i t h many other s u b s t r a t e s i n the water, the h e t e r o t r o p h i c a c t i v i t y , and hence the decomposition and n u t r i e n t r e c y c l i n g processes, miightd not be a f f e c t e d to the extent o u t l i n e d i n s e c t i o n 1.3. Therefore, i t i s necessary t o study the e f f e c t s o f the p o l l u t a n t s on a v a r i e t y o f subs t r a t e s u t i l i z e d by h e t e r o t r o p h i c b a c t e r i a . Some p o l l u t a n t s are continuously being pumped i n t o water systems (see Boyle, 1975, f o r the s t o r y on PCB) so th a t t h e i r long-term c h r o n i c e f f e c t s might end up d i s r u p t i n g the system i n the same:? way as thermal p o l l u t i o n ( s e c t i o n 1.3). This i s an important area worth c o n s i d e r a t i o n . An experiment s i m i l a r t o the long-term one i n t h i s study c o u l d be s e t up. Instead o f i n c u b a t i n g the c l o s e d containers i n the r e f r i g e r a t o r , more open and l a r g e r ones co u l d be used outdoors, under more n a t u r a l c o n d i t i o n s . Each day a s p e c i f i c l o a d i n g o f p o l l u t a n t s c o u l d be a p p l i e d , the concentra-t i o n s i n the water analysed, the kinds and numbers o f b a c t e r i a determined and the h e t e r o t r o p h i c a c t i v i t y measured and compared t o a c o n t r o l . I t would a l s o be i n t e r e s t i n g to s e t up an experiment i n order t o determine whether r a d i o a c t i v e l y l a b e l l e d c h l o r i n a t e d p o l l u t a n t s are taken up and metabolized by aquatic m i c r o f l o r a . I f so, rat e s o f m e t a b o l i z a t i o n c o u l d be measured. One f i n a l study might d e a l with>. w a t e r - i n s o l u b l e p o l l u t a n t s and t h e i r e f f e c t on the sediment microbe community. Most-waternsystems are hetero-geneous w i t h : respect •;tQ;ifche3 m-oroMloirae andi.poilutant, d i s t r i b u t i o n . U s u a l l y , microbe biomass v a r i e s throughout the water column ( A l i v e r d i e v a -Gamidova, 1969; K r i s s and Chebatarev, 1970; Grossbard, 1972) arid i s great-e s t i n the top 1 cm o f the system's sediments ( H a r r i s o n e t a l . , 1971; H a l l e t a l . , 1972; Wetzel, 1975). A l s o , many p o l l u t a n t s a r e h i g h l y water-i n s o l u b l e and adsorb to s o i l f r a c t i o n s (Grossbard, 1972), tending to accumulate i n the sediments. The organochlorine p o l l u t a n t s used i n t h i s study have been found i n concentrations up to 300 ppb i n water ( s e c t i o n 2.2) and they probably c o l l e c t to higher l e v e l s i n the sediments ( f o r i n s t a n c e , H a l l e t a l . (1976) found PCB and DDT to be present i n stream sediments up to 780 and 135 yg/kg dry weight r e s p e c t i v e l y ) . The o v e r a l l r e s u l t o f a p o l l u t a n t i n a system, then, might be a h i g h i n h i b i t -t i o n o f the h e t e r o t r o p h i c a c t i v i t y i n some areas (the sediments) and a low i n h i b i t i o n i n others (the water column). 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This i n h i b i -t i o n r e s u l t s i n a lowering o f Vmax (Figure 2'6A). Competitive i n h i b i t i o n , on the other hand, occurs when a subs t r a t e analogue competes f o r the same a c t i v e uptake s i t e s on the enzymes. This doesn't a f f e c t Vmax, because as S increases the e f f e c t o f the analogue decreases and Vmax i s e v e n t u a l l y reached (Figure 26B). Since Vmax was a f f e c t e d by PCB and t e t r a c h l o r o p h e n o l , at a l l l e v e l s , non-competitive i n h i b i t i o n was probably t a k i n g p l a c e , , i n d i c a t i n g the p o s s i b l e mode o f i n t e r a c t i o n between the p o l l u t a n t molecule(s) and the t r a n s p o r t enzymes. FIGURE 26. A. Non-competitive i n h i b i t i o n o f s u b s t r a t e uptake. B. Competitive i n h i b i t i o n . 

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