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The impact of Squoxin on aquatic invertebrates and an assessment of its fate in the aquatic environment Staley, George Stephen 1977

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THE IMPACT OF SQUOXIN ON AQUATIC INVERTEBRATES AND AN ASSESSMENT OF ITS FATE IN THE AQUATIC ENVIRONMENT by George Stephen Staley B.Sc, University of Arizona, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA in the Department of Zoology February, 1977 S T E P H E N S T A L E Y / 1977 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced d e g r e e at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ABSTRACT Squoxin, 1,1'-methylenedi-2-naphthol, i s a pesticide developed to eradicate the northern squawfish, Ptychocheilus  oregonensis. Investigations concerning the acute t o x i c i t y of the p e s t i c i d e to aquatic invertebrates and an assessment of i t s fate i n aquatic ecosystems are reported. Squoxin was found to be much less toxic to aquatic invertebrates than i t was to squawfish. I t was proposed that the t o x i c i t y of squoxin to aquatic invertebrates i s inversely proportional to t h e i r adaptations to habitats having low lev e l s of oxygen a v a i l a b i l i t y . The most sensitive invertebrate species tested was the b l a c k f l y larva, Simulium canadense, which i s di s t r i b u t e d i n streams having high current v e l o c i t y and abundant dissolved oxygen. These larvae exhibit an LC50 value of 60 ug/l i n 4 8 hours. Chaoborus t r i v i t t a t u s larvae, known to tolerate anoxic conditions, were re s i s t a n t to treatments up to 10 mg/1, exhibiting a maximum response of 30 percent i n 96 hours. The degradation of Squoxin was monitored i n surface waters of diverse o r i g i n . Degradation was most severe in water having a high pH and high a l k a l i n i t y . Degradation was also found to occur due to the presence of dissolved organic compounds of high molecular weight. Freshwater bacteria did not exhibit any a b i l i t y to u t i l i z e Squoxin as a carbon source i n short term tests. A 100 ug/l Squoxin treatment depressed the natural hetero-trophic a c t i v i t y of bacteria by nearly 25 percent based on i i i 14 studies of uptake of C-glucose over a 2 hour period. 14 C-Squoxm residues i n i t i a l l y adsorbed to phyto-plankton and organic sediment i n simple laboratory micro-cosms. However, the residues steadily desorbed from these components and became increasingly soluble throughout the test period. Daphnia pulex and Hyalella azetca i n the microcosms took up Squoxin rapidly i n the f i r s t 24 hours afte r treatment. Uptake then l e v e l l e d o f f and tissue concentration of Squoxin increased only at a very slow rate during the remainder of the test. Only small amounts of Squoxin were transferred to organisms feeding on contaminated food items w*hen compared to the dose obtained from a contact exposure. C. t r i v i t t a t u s fed contaminated D. pulex retained only 6 percent of the ingested Squoxin residues. These residues, however, appeared to be retained for a longer period than tissue residues gained through a contact exposure. Invertebrates exhibited an a b i l i t y to excrete Squoxin residues given time and an uncontaminated environ-ment. D. pulex excreted over 90 percent of the toxicant i n 96 hours; H. azteca required 275 hours. It was postulated that because of Squoxin's water s o l u b i l i t y , low p a r t i t i o n c o e f f i c i e n t , rapid degradation, and the a b i l i t y of a wide variety of organisms to excrete i t , the toxicant would not be b i o l o g i c a l l y magnified to a s i g n i f i c a n t degree i n aquatic ecosystems. i v TABLE OF CONTENTS Page LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x Chapter 1. INTRODUCTION 1 2. MATERIALS AND METHODS 7 2.1 General 7 2.2 Degradation 10 2.20 Colorimetry 10 2.21 Sephadex Column Chromatography . 11 2.22 U l t r a f i l t r a t i o n 12 2.2 3 Microbial Degradation 12 2.3 Toxic i t y 13 2.30 Microbial A c t i v i t y 13 2.31 Invertebrates 14 2.4 Bioconcentration Potential 15 2.4 0 Microcosms 15 2.41 Feeding 16 2.42 Toxicant Excretion 18 3. RESULTS AND DISCUSSION 20 3.1 Degradation 2 0 3.10 E f f e c t of Water Chemistry. . . . 20 3.11 E f f e c t of Dissolved Organic Compounds 25 3.12 E f f e c t of Bacteria 32 . V Page 3.2 T o x i c i t y . . . . . 34 3.20 B a c t e r i a 34 3.21 I n v e r t e b r a t e s 3 8 3.3 B i o c o n c e n t r a t i o n P o t e n t i a l 49 3.30 General 49 3.31 Microcosms 51 3.32 Uptake Through Feeding 6 3 3.3 3 T o x i c a n t E x c r e t i o n 6 7 3.34 The P o t e n t i a l f o r B i o -c o n c e n t r a t i o n 7 6 4. SUMMARY AND CONCLUSIONS 81 4.1 Summary 81 4.2 P a r t i t i o n i n g of Squoxin 82 LITERATURE CITED 86 APPENDICES 91 v i LIST OF TABLES TABLE Page I Chemistry of water used i n degradation studies. 22 14 II Uptake and mineralization of C-Squoxm by freshwater bacteria 33 III E f f e c t of Squoxin on heterotrophic a c t i v i t y of freshwater bacteria 35 IV T o x i c i t y of Squoxin to invertebrates; summary of LC50 values 41 14 V Uptake of C-Squoxin by Hyalella azteca from contaminated organic sediment 6 4 VI Retention of Squoxin by Chaoborus t r i v i t t a t u s larvae fed contaminated Daphnia pulex 6 6 VII Residue determination i n Chaoborus t r i v i t t a t u s larvae, pupae, and imagos 75 v i i : LIST OF FIGURES FIGURE Page 1 Chemical structure of Squoxin and chemical constants 8 2 Degradation of Squoxin i n d i s t i l l e d water, Loon Lake water and P l a c i d Lake water 21 3 Degradation of Squoxin i n Capilano River water, Musqueam Creek water and Camosun Bog water 2 3 4 Spectral scan of waters i n FIG. 3 28 5 Sephadex column chromatography of a concen-trated sample of Burns Bog water with the 14 addition of C-Squoxin 2 9 6 Degradation of Squoxin i n f i l t r a t e and reten-tate of Camosun Bog water f i l t e r e d on an Amicon u l t r a f i l t r a t i o n unit 31 7 E f f e c t of Squoxin on uptake and u t i l i z a t i o n of 14 C-glucose by freshwater bacteria 36 8 To x i c i t y of Squoxin to Chaoborus t r i v i t t a t u s larvae and Simulium canadense larvae 39 9 To x i c i t y of Squoxin to Hyalella azteca and Anisogammarus ramellus 40 10 Toxi c i t y of Squoxin i n Daphnia pulex 42 11 Toxi c i t y of Squoxin to Daphnia pulex juveniles) 12 P a r t i t i o n i n g of Squoxin i n laboratory micro-4 cosms containing Daphnia pulex, 2.5 X 10 cells/ml C h l o r e l l a sp 53 13 P a r t i t i o n i n g of Squoxin i n laboratory micro-5 cosms containing Daphnia pulex, 1.0 X 10 c e l l s / ml C h l o r e l l a sp 54 v i i i FIGURE Page 14 P a r t i t i o n i n g of Squoxin i n laboratory micro-5 cosms containing Daphnia pulex, 2.0 X 10 cells/ml C h l o r e l l a sp 55 15 P a r t i t i o n i n g of Squoxin i n laboratory micro-cosms containing Hyalella azteca, organic sediment 57 16 Plot of uptake of Squoxin by D. pulex i n micro-cosms 5 9 17 P l o t of uptake of Squoxin by H. azteca i n microcosms 6 0 18 Excretion of Squoxin residues by D. pulex . . . 68 19 Excretion of Squoxin residues by D. pulex; percentage plot 70 20 Excretion of Squoxin residues by H. azteca. . . 72 21 Excretion of Squoxin residues by C. t r i v i t -tatus 74 22 P a r t i t i o n i n g of Squoxin i n a hypothetical l e n t i c ecosystem 84 i x ACKNOWLEDGEMENTS Many people contributed material or moral support to the completion of t h i s thesis. I would l i k e to thank Drs. T. G. Northcote and W. E. N e i l l for constructive c r i t i c i s m of the ongoing research and the ensuing manuscript. J. W. Cartwright and T. G. Halsey of the B.C. Fish and W i l d l i f e Branch, Department of Recreation and Conservation, provided administrative support as well as many useful discussions. My research supervisor, Dr. K. J. H a l l , was a constant source of advice, d i r e c t i o n , and technical expertise without whom i t would have been d i f f i c u l t to bring t h i s study to a successful conclusion. F i n a n c i a l support provided by the Fish and Wild-l i f e Branch through i t s Regional Management Office i n Kamloops and the Fisheries Research and Technical Services Section at the University of B r i t i s h Columbia i s g r a t e f u l l y acknowledged. 1 Chapter 1 INTRODUCTION Interest i n the control of populations of undesirable f i s h has p a r a l l e l e d the development of intensive management of sport f i s h e r i e s . Chemical control of problem fishes began in 1913 with the treatment of trout lakes i n Vermont with copper sulphate (Lennon, 1970). Most toxicants, l i k e copper sulphate, have been l e t h a l to nearly a l l aquatic l i f e . Reclam-ation of a fishery often meant the creation of a b i o l o g i c a l desert followed by a long period of d e t o x i f i c a t i o n and sub-sequent restocking. Recent advances have begun to replace general t o x i -cants with chemicals which are s e l e c t i v e l y l e t h a l not only to f i s h , but to p a r t i c u l a r species of f i s h . The f i r s t such toxicant was TFM (3-trifluormethyl-4-nitrophenol) used to control the l a r v a l sea lamprey, Petromyzon marinus, i n the watersheds of the Great Lakes (Applegate et a l . , 1961). In 1968 another selective toxicant was patented by i t s p r i n c i p a l investigators. Craig MacPhee and Richard Ruelle of the University of Idaho found the compound, 1,1'-methylenedi-2-naphthol, which they l a t e r named Squoxin, to be s e l e c t i v e l y l e t h a l to the northern squawfish, Ptycho- cheilus oregonensis. Squawfish have long been considered a rapacious predator of salmon smolts i n freshwater (Clemens and Munro, 2 1934; Ricker, 1941; Foerster and Ricker, 1942; Thompson, 1959; Steigenberger and Larkin, 1974). In addition, t h e i r s i z e , d i e t and habitat requirements suggest they are primary competitors for the resources u t i l i z e d by both anadromous and resident sport f i s h (Keating et a_l. , 1972). Squawfish were found to be extremely susceptible to an acetone solution of Squoxin applied to the water. Depend-ing on water termperatures, the compound proved l e t h a l to 100 percent of squawfish i n a concentration range of 6 to 15 ^ug/1. Squawfish are apparently unaware of the chemical's presence in the water. A l l the f i s h tested were quiescent u n t i l toxic action became ph y s i c a l l y apparent. Labored v e n t i l a t i o n and loss of equilibrium, either f l o a t i n g v e r t i c a l l y or upside down, were two most common signs displayed. Fish removed from the presence of Squoxin aft e r these symptoms became mani-fest i n v a r i a b l y died (MacPhee and Ruelle, 1969). In addition to squawfish, MacPhee and Ruelle (1969) tested hatchery strains of chinook salmon (Oncorhynchus  tshawytscha), coho salmon (O. kisutch), steelhead trout (Salmo gairdneri) and brook trout (Salvelinus f o n t i n a l i s ) . They found these f i s h to be 3 to 100 times more tolerant of Squoxin. Johnson (1972) working with sockeye salmon (Oncorhynchus nerka) reported that although sockeye alevins would be affected to a small degree by the recommended f i e l d treatment of 100 jug/1, tolerance increased sharply a f t e r absorption of the yolk sac. Fry were able to withstand concentrations of 700 to 900jug/l. 3 Squoxin i s an apparent i d e a l solution to population control of a long-standing pest. The economics of control are sound. A k i l l i n g dose of the toxicant i s small enough that even large volumes of water can be treated for very l i t t l e cost. Hamilton et a l . (1970) estimated that the s u r v i v a l of coho salmon i n predator free enclosures was nearly 4 0 percent greater than unprotected populations. Since the primary predator i n t h i s case was squawfish, the small investment i n Squoxin to protect salmon from predation would y i e l d substantial returns even i f only a f r a c t i o n grew to marketable s i z e . From a fishery management point of view there are overriding reasons for the eventual use of Squoxin. It i s easy to apply, does not disrupt the sport or commercial fishery and can be used i n waters containing wild or indigenous species. Such a compound deserves consideration within the broader e c o l o g i c a l context. This study was designed to consider the e f f e c t s of Squoxin on aquatic invertebrates. It was to consist of a series of acute t o x i c i t y bioassays on common aquatic .inverte-brate species. This information would then serve as a base-l i n e for designing a f i e l d study to monitor e f f e c t s of a Squoxin treatment on the invertebrate community of a small lake. Unfortunately, the f i e l d study had to be cancelled. The United States Environmental Protection Agency revoked the experimental permit granted to the American manufacturer 4 of the compound, pending review; there were no alternate i sources of supply. Under the e x i s t i n g s i t u a t i o n , the laboratory research was expanded i n scope. In addition to augmenting the b i o l o g i -c a l studies, a series of investigations on the rate of degradation of the compound i n water were added. The e f f e c t of Squoxin on the b a c t e r i a l community and t h e i r a b i l i t y to degrade the compound were also studied. The thesis i s organ-ized into three sections: 1) degradation of Squoxin due to water chemistry and bacteria; 2) t o x i c i t y to aquatic inverte-brates and e f f e c t upon b a c t e r i a l heterotrophic a c t i v i t y ; 3) assessment of bioconcentration pot e n t i a l based on i t s p a r t i t i o n i n g i n laboratory microcosms and i t s uptake and loss rates by aquatic invertebrates. This study, along with a l i t e r a t u r e review already completed (Staley and H a l l , 1974) , was designed to a s s i s t fishery managers i n evaluating the environmental costs and benefits of eradication of northern squawfish with the use of Squoxin. Squoxin i n aqueous solution i s r e a d i l y oxidized or otherwise converted into at least 11 degradation products (Burnard, Kiigemagi and Terriere, 1974a). None of these has been firmly i d e n t i f i e d except the dehydrogenation product 2-oxo-2H,1 1H-spiro (napthalene-1,2'-naphthol-2,1-b) furan (Terriere, pers. comm.). Sunlight and aeration are the p r i n c i p a l factors a f f e c t i n g degradation, but the rate i s also affected by the chemistry of the water to which i t i s applied. 5 In the f i e l d , where a l l these factors come into play, i t has been suggested that Squoxin 1s e f f e c t i v e h a l f l i f e would be 2 hours i n streams and less than 2 4 hours i n standing water (Keating et a l . , 1972). The e f f e c t of Squoxin on aquatic invertebrates has previously been assessed i n eithe r i n d i r e c t or anecdotal ways. F i e l d studies have considered the e f f e c t of Squoxin on insect d r i f t i n streams (Brusven and MacPhee, 1974), com-pared benthic samples i n streams before and afte r treatments (Keating et a_l. , 1972) or consisted of casual observations made during treatments (Keating et aJL. , 1972; Keating, 1972; Cartwright, 19 73). Laboratory studies included v i s u a l observation of aquatic insects i n a 2200 l i t e r p l a s t i c pool for 21 days afte r a 100 ug/l treatment with Squoxin (Keating et a l . , 1972) and observation of zooplankton for 7 days a f t e r treatment of up to 100 ug/l (Johnson, 1972). No research e f f o r t has ever defined a l e t h a l concentration for an aquatic invertebrate. Terriere and Burnard (1975) used t r i t i a t e d Squoxin to examine tissue l e v e l s of the pesticide i n rainbow trout (Salmo gairdneri) and squawfish. They found the relat i o n s h i p between dose and tissue l e v e l to be a li n e a r function i n trout given multiple exposures to 50 ug/l Squoxin. Squawfish died within 6 hours of one such treatment. Their tissues contained 1.5 times the f i n a l tissue concentration obtained by the trout. This may have been due to a greater uptake pot e n t i a l of squawfish or the continuous rapid excretion of 6 the Squoxin by the trout. The l a t t e r seems to be a more at t r a c t i v e hypothesis as trout were able to purge 90 percent of the Squoxin from th e i r tissues within 48 hours aft e r the treatment ceased. Preliminary t o x i c o l o g i c a l tests with rats and rabbits indicate that Squoxin has a low order of t o x i c i t y to mammals. Its dermal LC50 i n rabbits i s at lea s t 5 g/kg and i t s o r al LC50 i n rats i s at least 10 g/kg. Rats have sur-vived a 30 day feeding period on a 1.25 percent Squoxin di e t (Terriere, pers. comm.). Squoxin appears to be a very promising compound for control of squawfish as an adjunct to a salmon enhancement program. The term " s e l e c t i v e l y l e t h a l " , however, implies that t h i s compound w i l l only be toxic to a s p e c i f i c organism. It must be r e a l i z e d that any toxic compound i s sel e c t i v e only within a defined concentration range. Squoxin must be considered a p o t e n t i a l l y dangerous compound subject to con-s t r a i n t s on i t s use within a framework of sound environmental management. 7 Chapter 2 MATERIALS AND METHODS 2.1 GENERAL Squoxin, 1,1'methylenedi-2-naphthol, was obtained from the A l d r i c h Chemical Company, Milwaukee, Wisconsin, i n c r y s t a l l i n e powder form. An ethanolic solution of the compound, 1 mg/ml 95 percent ethanol, freshly prepared, was u t i l i z e d i n a l l investigations. 14 Carbon-labelled Squoxin was prepared by refluxi n g 14 C-formaldehyde (Amersham-Searle), d i s t i l l e d water, 1 per-cent NaOH, and 2-naphthol at 100° C for 2 hours. The product was then cooled, a c i d i f i e d with 2N HC1 and the r e s u l t i n g c r y s t a l s f i l t e r e d from solution. The synthesis joins the 14 2-naphthol molecules with a C-methylene bridge (Figure 1). Thin layer chromatography revealed no unreacted 2-naphthol i n the product, which exhibited the same Rf value as the commercially prepared, unlabelled Squoxin (Appendix 1). The water used i n a l l tests involving invertebrates was Capilano River water (North Vancouver, B.C.). Prior to use the water was f i l t e r e d through Reeve-Angel GF/C glass f i b e r f i l t e r s , 934AH grade (no pore size s p e c i f i e d by manu-facturer) , to remove suspended s o l i d s and ref r i g e r a t e d at 15° C in a covered glass container. Chemical c h a r a c t e r i s t i c s of t h i s water are reported i n Table 1 and Squoxin 1s rate of degradation i s graphically displayed i n Figure 3. SQUOXIN O H O H •» AA -LABEL VJ 1, 1 '-METHYLENEDI-2-NAPHTHOL C H 2 ( C 1 0 H 6 ° H ) 2 MOLECULAR WEIGHT . . . . . 300.361 MELTING POINT 200 - 20 3 V pK a 9.722 SOLUBILITY 600 (ig/liter3 PARTITION COEFFICIENT . . 3.8 4 i Aldrich Chemical Co.; product information. 2Skytte Jensen, 1964. 3 Terriere, Kiigemagi and Chan, 1970. 4Nakaue, Caldwell and Buhler, 1972. FIGURE 1. Chemical structure of Squoxin, its molecular weight, formula, and physical/chemical properties as reported in various literature sources. 9 A l l assays, except those involving bacteria, were conducted i n a Percival constant temperature incubator (Percival, Boone, Iowa) at 15 ± 1° C. Light, supplied by a single "cool-white" fluorescent tube, was maintained on a regular 12 hour light/dark cycle. Wet weights of invertebrates were obtained by pour-ing the culture water containing the animals through a fine mesh nylon screen with a plexiglass frame. The screen was rinsed with clean water from a wash bottle and blotted dry from the underside with tissue. The organisms were trans-ferred to a tared sheet of weighing paper by tapping the inverted•screen and weighed immediately on a Mettler balance to ± 0.1 mg. Preparation of invertebrates for determination of r a d i o a c t i v i t y involved t r a n s f e r r i n g the weighed sample to a g l a s s ^ s c i n t i l l a t i o n v i a l containing 0.2 to 0.5 ml NCS tissue, s o l u b i l i z e r (Nuclear Chicago) , depending on the size and number of organisms i n the sample. The v i a l was t i g h t l y capped and placed i n an oven at 40° C overnight. Upon cooling, 10 ml of toluene s c i n t i l l a t i o n solution (4 g PPO"*", 2 0.2 g POPOP i n 1 l i t e r toluene) were added. PPO = 2,5-diphenyloxazole POPOP = 1,4-bis-[2-(5-pheyloxazolyl)]-benzene 10 Detection and measurement of r a d i o a c t i v i t y present i n a sample was done by l i q u i d s c i n t i l l a t i o n spectrometry using a Searle Isocap 300 l i q u i d s c i n t i l l a t i o n counter.:, Counts per minute were converted to disintegrations per minute (DPM) using an external standard to determine the e f f i c i e n c y of counting. DPM were converted to nanograms Squoxin by the use of prepared standards. Sediment samples were f i r s t dried at 4 0° C, weighed and placed i n a s c i n t i l l a t i o n v i a l . Ten ml of toluene s c i n t i l l a t i o n solution was added along with enough Cabo-Sil (fin e l y divided s i l i c a ) to keep the p a r t i c l e s in suspension. Aqueous samples and any m i l l i p o r e f i l t e r s were placed i n a s c i n t i l l a t i o n v i a l containing 10 ml Bray's solution (Bray, 1960) . 2.2 DEGRADATION 2.2 0 Colorimetry Water used i n degradation studies was treated at a 3 l e v e l of 100 p.g/1 Squoxin. Loon Lake (near Cache Creek, B.C.) and Plac i d Lake (U.B.C. Research Forest) waters received no special treatment before addition of Squoxin. Capilano River, Musqueam Creek (University Endowment Lands) and Camosun Bog (University Endowment Lands) waters were run through several f i l t r a t i o n steps which removed a l l suspended sol i d s larger than 8 micrometers. Concentration of Squoxin 3 p g / l = 10" 6 g/1 = ppb _ 3 mg/1 = 10 g/1 = ppm 11 in a water sample was monitored at 6, 12, 24, 48, 72 and 96 hours aft e r treatment following the colorimetric method of Kiigemagi, Burnard, and Terriere (1975) u t i l i z i n g an H i t a c h i -Perkin-Elmer spectrophotometer with a 2 cm c e l l path (Appendix 2). Sp e c i f i c conductivity of the test water was deter-mined using a Radiometer CDM 3 Conductivity Meter temperature compensated to 25° C. A Fisher Acumet pH Meter was used to measure pH and to determine t o t a l a l k a l i n i t y by the potentio-metric method (APHA, 1971)-2.21 Sephadex Column Chromatography A 1 l i t e r sample of water from Burns Bog (Delta, B.C.) was f i l t e r e d through a GF/C glass f i b e r f i l t e r and evaporated under vacuum to 50 ml on a f l a s h evaporator at 50° C. A 5 ml 14 sample was taken and a 5 ml C-Squoxin solution (1 mg Squoxin/10 ml methanol) was added to i t . After reacting for 1 hour, the mixture was placed on top of a 30.5 cm chroma-tography column packed with Sephadex LH-20 g e l . The Sephadex had previously been l e f t to swell i n a 1 : 1 methanol: d i s t i l l e d water solution, which was also used to elute the 14 C-Squoxin-humic compound mixture. A t o t a l of 250 ml was taken o f f the bottom of the column i n 5 ml fracti o n s . Each f r a c t i o n was analyzed for t o t a l r a d i o a c t i v i t y and for absor-bance at 400 nM on a Bausch and Lomb Spectronic 600 spectrophotometer using a 1 cm c e l l path. 12 2.22 U l t r a f i l t r a t i o n Camosun Bog water was f i l t e r e d through M i l l i p o r e HAWP 0.45 micrometer (pore s i z e ) f i l t e r s and p l a c e d i n an Amicon u l t r a f i l t r a t i o n u n i t f i t t e d w ith a 20,000 nominal molecular weight f i l t e r . The 3 l i t e r sample was f i l t e r e d t o 1 l i t e r under n i t r o g e n a t 200 Kpa, thereby c o n c e n t r a t i n g the high-molecular weight compounds i n the r e t e n t a t e by a f a c t o r o f three and removing most of the i n o r g a n i c compounds to the f i l t r a t e . The f i l t r a t e was r e s e r v e d f o r degradation a n a l y s i s , w h ile the r e t e n t a t e was r e s t o r e d to 3 l i t e r s w i t h g l a s s double d i s t i l l e d water and f i l t e r e d a gain. The r e t e n t a t e from the second f i l t r a t i o n was r e - d i l u t e d t o 3 l i t e r s and both i t and the r e s e r v e d f i l t r a t e were t r e a t e d with 100 u g / l Squoxin. Degradation was monitored a t 6, 12, 24, 48 and 72 hours by the c o l o r i m e t r i c method. A t o t a l d i s s o l v e d o r g a n i c carbon a n a l y s i s was performed on both samples by the U.B.C. Department of Oceanography. 2.2 3 M i c r o b i a l Degradation Ten ml of Trout Lake (Vancouver, B.C.) water was p i p e t -te d i n t o each of a s e r i e s of 50 ml f l a s k s . T h e ' f l a s k s were f i t t e d with a rubber stopper h o l d i n g a s m a l l p l a s t i c cup sus-pended j u s t i n s i d e the body of the f l a s k . The cup, c o n t a i n g 0.2 4 ml o f Hyamme hydroxide (Rohm and Haas) and a g l a s s f i b e r f i l t e r acted as a C0 2 t r a p . C o n c e n t r a t i o n s of 20 to 200 pg/1 Hyamine hydroxide = p - ( d i i s o b u t y l - c r e s o x y e t h o x y e t h y l ) -dimethylbenzyl-ammonium hydroxide. Squoxin were obtained by adding varying amounts of a C-Squoxin standard solution. The bacteria i n half the t r e a t -ment series were then k i l l e d with 0.2 ml 5N I^SO^. A l l the flasks were placed i n a temperature controlled shaker bath at 15° C. After a 2 hour incubation time, the water was f i l t e r e d through a M i l l i p o r e HAWP 0.4 5 u f i l t e r and the f i l t e r placed i n a s c i n t i l l a t i o n v i a l . The f i l t e r contain-ing the Hyamine hydroxide was placed i n a separate v i a l and the r a d i o a c t i v i t y i n a l l samples counted. 2.3 TOXICITY 2.30 Microbial A c t i v i t y A series of samples of 10 ml of Trout Lake water i n 50 ml flasks were prepared as i n Section 2.23. The samples 14 were enriched with C-glucose at a concentration of 60 or 100 ug/l. One t h i r d of the flasks were given a 100 jag/1 Squoxin treatment, one t h i r d (ethanol controls) were given the equivalent amount of 95 percent ethanol that the treatment flasks received, and the remaining t h i r d received no t r e a t -ment. Experimental conditions, sampling time and procedure were i d e n t i c a l to those i n Section 2.23. Through the use of known standards, the r a d i o a c t i v i t y expressed i n DPMs was converted to pg glucose/1. The amount of glucose taken up by the c e l l s was added to the amount of glucose respired and the uptake rate for a l l samples was calculated i n pg glucose/ 1 / hour. 14 A second experiment compared the uptake rate of 14 20, 60 and 100 pg/1 C-glucose by bacteria treated with 100 pg/1 Squoxin and that of bacteria receiving no t r e a t -ment . 2.31 Invertebrates A l l 96 hour acute bioassays using aquatic inverte-brates were s t a t i c t e s t s ; however, the water was changed and the treatment renewed at 48 hours. Ten organisms, selected more or less at random, were placed i n each glass test chamber containing 400 ml of water. The organisms were acclimated for 12 ± 2 hours before treatment with Squoxin. Treatments were assigned at random using a random number table. Controls were treated with 95 percent ethanol equiva-lent to the volume of Squoxin/ethanol solution given the culture receiving the highest concentration i n the treatment serie s . The cultures were monitored at 2, 4, 8, 12, 24, 33, 48, 72 and 96 hours a f t e r treatment and organisms revealing no movement upon examination under a di s s e c t i n g microscope (the c r i t e r i o n of death) were removed. Average loading of organisms ranged from 0.01 g/1 water (Simulium canadense) to 0.2 g/1 water (Chaoborus t r i v i t t a t u s ) . Bioassays with Simulium canadense were run d i f f e r e n t l y because the larvae require moving water for s u r v i v a l . Freshly c o l l e c t e d larvae were placed i n 1 l i t e r beakers containing 800 ml water and a 2 cm magnetic spinbar enclosed i n glass. The beaker was then immersed i n a bath of flowing tap water at 13 C over a rotating magnet powered by another stream of tap water. The rotating magnets were a l l connected i n series to the same stream of water and t h e o r e t i c a l l y were spinning at the same speed. The current of water caused by the rotating spinbar i n the culture attracted the larvae which took up positions i n a c i r c u l a r pattern around i t , or, i n some cases, on i t . The larvae were then treated i n the same manner indicated above. Since the larvae could not be removed without i n j u r i n g them, the tes t was terminated at 48 hours and the results tabulated. The t o x i c i t y of Squoxin to juvenile Daphnia pulex was determined by sel e c t i n g large females carrying eggs and placing them i n a special container of water with food. These females were checked 4 8 hours l a t e r . The newly hatched larvae were removed and placed i n 50 ml water, ten to a container, for treatment. The experiment was terminated 48 hours aft e r treatment. Results of bioassays were calculated by probit analysis u t i l i z i n g Abbot's formula for adjustment of natural responsiveness of the control (Finney, 1971). 2.4 BIOCONCENTRATION POTENTIAL 2.4 0 Microcosms Microcosms simulating the interaction of Squoxin with the plankton community were prepared using 2 00 ml of water, 10 D. pulex, and three d i f f e r e n t concentrations of C h l o r e l l a sp.: 2.5 x 10 c e l l s / m l , 1.0 x 10 cells/ml and 2.0 x 10 14 c e l l s / m l . Each microcosm was treated with 500 ug/l C-Squoxin after an e q u i l i b r a t i o n period of 12 ± 2 hours. Replicate > cultures were sampled at each of the following times: 2, 4, 8, 12, 24, 33, 48 and subsequently every 24 hours up to a maximum of 168 hours. The following sampling procedure was followed: a l l Daphnia were removed and placed i n a s c i n t i l l a t i o n v i a l ; 10 ml of water were f i l t e r e d through a M i l l i p o r e HAWP 0.45 p f i l t e r at less than 15 Kpa vacuum; the f i l t e r was placed i n a v i a l and 1 ml of the f i l t r a t e was put i n a separate v i a l . Microcosms simulating interactions of Squoxin with sediment and benthic organisms were conducted with 400 ml of water i n which 10 Hyalella azteca and 10 ml of wet sedi-ment from Marion Lake (U.B.C. Research Forest) were added. Sample times of r e p l i c a t e cultures were as above. The cultures were sampled by removing the amphipods, f i l t e r i n g 10 ml of water and t r e a t i n g the components i n a manner analogous to the Daphnia cultures above. The wet sediment was placed in a tared aluminum weighing boat and placed i n an oven at 40° C. After i t was completely dry, the weight was determined and a 100 mg f r a c t i o n was placed i n a s c i n t i l l a t i o n v i a l to be prepared for counting. 2.1 Feeding Chaoborus t r i v i t t a t u s larvae were placed i n d i v i d u a l l y i n small glass jars containing 50 ml water. Daphnia pulex, previously treated for 24 ± 4 hours with 1000 ug/l C-Squoxin, were pipetted into each j a r containing the C. t r i v i t t a t u s . Ten Daphnia from the treatment were sampled to ascertain the amount of Squoxin that was taken up. The culture was examined 72 hours l a t e r and the ingestion of the Daphnia recorded. At t h i s time two Chaoborus were sampled to determine residue levels i n the tissues from the feeding. This procedure was followed u n t i l the Chaoborus had ingested 10 treated Daphnia. The C. t r i v i t t a t u s were then sampled at 72 hours, 144 hours and 288 hours a f t e r the l a s t feeding to determine the residue l e v e l i n the body. To determine uptake of Squoxin by Hyalella azteca grazing on organic sediment, 50 ml of organic sediment from Marion Lake were pipetted into a glass jar and water added to a t o t a l volume of 2 00 ml. The sediment/water mixture 14 was treated with 1000 pg/1 C-Squoxin and s t i r r e d vigor-ously. At the end of 24 hours the sediment was f i l t e r e d using a Whatman no. 1 q u a l i t a t i v e f i l t e r and rinsed with clean water. The sediment was added to 800 ml culture water and allowed to s e t t l e . H. azteca were added to the culture and allowed to be i n d i r e c t physical contact with the s e d i -ment. Another group of H. azteca was placed i n a 2 00 ml glass j a r three-quarters f i l l e d with water c a r e f u l l y de-canted from the culture. Two sheets of cotton gauze were placed over the j a r opening with an e l a s t i c band. The jar was h a s t i l y inverted into the culture and allowed to f l o a t . The amphipods i n the j a r therefore had contact with the 18 water but were kept from i n t e r a c t i n g with the sediment. After 4 8 hours both groups of amphipods, the water and the sediment from 4 r e p l i c a t e cultures were analyzed for r a d i o a c t i v i t y . 2.42 Toxicant Excretion To measure the rate of excretion of Squoxin by inver-] 4 tebrates, the organisms were f i r s t exposed to 500 pg/1 ' C-Squoxin i n 800 ml of water for 24 ± 4 hours. They were removed from the treatment, rinsed, and a sample taken to determine the i n i t i a l body load. At t h i s point d i s p o s i t i o n of the d i f f e r e n t invertebrate groups diverged and each must be considered separately. Fourth in s t a r Chaoborus t r i v i t t a t u s larvae were placed i n clean water and ten individuals sampled every 24 hours. A sample of individuals which had pupated during treatment and those which had pupated 24 hours a f t e r t r e a t -ment were assayed for i n i t i a l pupal residues. Those remaining were placed i n separate containers f i t t e d with a gauze screen to await the emergence of the imago. The adults, c l i n g i n g to the gauze screen, were aspirated out of the culture, promptly frozen to f a c i l i t a t e weighing, and assayed for any Squoxin residues remaining. Daphnia pulex were placed i n d i v i d u a l l y i n containers of 50 ml of water or 50 ml of water containing 1.0 X 10^ C h l o r e l l a sp. c e l l s / m l . A l l the containers were checked every 24 hours. Those individuals that molted were assayed 19 f o r t o t a l r a d i o a c t i v i t y as were a sample of i n d i v i d u a l s t h a t had not molted. T h i s procedure was continued f o r 96 hours. H y a l e l l a a z t e c a were p l a c e d i n 8 0 0 ml of c l e a n water or, i n another t e s t , 5 i n d i v i d u a l s were p l a c e d i n a s e r i e s o f j a r s c o n t a i n i n g 50 ml of water w i t h 10 ml of wet sediment from Loon Lake (U.B.C. Research F o r e s t ) . Twenty i n d i v i d u a l s from each c o n d i t i o n were sampled every 24 hours. 20 Chapter 3 RESULTS AND DISCUSSION 3.1 DEGRADATION 3.10 E f f e c t of Water Chemistry The degradation of Squoxin proceeded at a faster rate i n waters having a higher pH and greater concentration , of dissolved inorganic compounds. Recoverable Squoxin in water from Loon Lake, a productive lake north of Cache Creek, B.C., declined from 92 percent at 12 hours to 23 percent at 4 8 hours. Scarcely 2 percent or 2 ug/l remained i n the sample at 96 hours (Figure 2). In water taken from Pla c i d Lake, a small coastal mountain bog lake i n the U.B.C. Research Forest, the rate was much slower. There was no detectable degradation during the f i r s t 12 hours a f t e r treatment, but at 48 hours 6 2 percent of the Squoxin from the o r i g i n a l treatment remained. When the tes t was terminated at 96 hours, 21 percent, or 10 times the concentration i n the Loon Lake sample, remained undegraded (Figure 2). Concentration of Squoxin i n d i s t i l l e d water did not f a l l below 90 percent of the i n i t i a l value u n t i l 96 hours a f t e r treatment when i t eventually declined to 89 percent (Figure 2). Capilano River water exhibited a degradation pattern si m i l a r to that of d i s t i l l e d water (Figure 3). The FIGURE 2. Degradation of Squoxin in distilled water, Loon Lake water and Placid Lake water. Mean and range of samples indicate for both lake waters ; mean and standard deviation of 5 samples indicated for distilled water. 22 concentration of Squoxin did not f a l l below 90 percent u n t i l 48 hours aft e r treatment, when the recoverable Squoxin was 86 percent of the o r i g i n a l concentration. This l e v e l of Squoxin remained stable; 48 hours l a t e r , at 96 hours, 84 percent of the o r i g i n a l compound remained undegraded. Squoxin degraded faster i n another coastal stream, Musqueam Creek, which drains the University Endowment Lands. Only 56 percent of the o r i g i n a l treatment was detected at 12 hours and t h i s declined slowly to 44 percent at 48 hours. Like P l a c i d Lake, the concentration dropped to 2 0 percent at 96 hours when the tes t was terminated (Figure 3). The chemical c h a r a c t e r i s t i c s of these waters are displayed i n Table I. Degradation of Squoxin was most severe i n the water having the highest values for t o t a l a l k a l i n i t y , conductivity and pH, and least severe i n water TABLE I. Chemistry of waters used in degradation studies. A l l tests performed on subsamples of waters used in gradation analysis. Water Total A l k a l i n i t y Conductivity pH Color (as mg CaC0 3/l) (uS at 25°C) (Pt Units) Placid Lake 59 25 6.25 Loon Lake 286 425 8.45 Capilano 4 13.5 5.80 10 River Musqueam Creek 11 58 7.60 80 Camosun Bog 13 140 6.60 >100 11r F I G U R E 3. Degradation of Squoxin in C a p i l a n o R i v e r water, M u s q u e a m C r e e k water, and C a m o s u n Bog water. M e a n and standard deviation of 4 samples. e x h i b i t i n g the lowest values for these parameters. Although the waters i n these samples might d i f f e r i n other physical and chemical c h a r a c t e r i s t i c s , the inference i s that degrada-tion w i l l proceed much faster i n water having a high pH and/or high a l k a l i n i t y . T e r r i e r e , Kiigemagi and Chan, (1970) found that by adjusting the pH of spring water upward with NaOH the degrada-tion of Squoxin could be accelerated. Two and one-half times more Squoxin was recovered after 5 days from spring water of pH 6.2 than from the same water adjusted to pH 8.2. Burnard et a^. (1974a) reported no appreciable e f f e c t on degradation rate by the addition of 10 mg/1 CaCl 2- MacPhee and Cheng (1974), however, noted a s i g n i f i c a n t c o r r e l a t i o n of degradation rate and calcium concentration, as CaCl 2 and CaC0 3, of 10 - 100 mg/1. The only other metal ion known to cause any appreciable degradation i s ir o n , as FeCl^/ which caused a 50 percent degradation i n 24 hours (Burnard, K i i g e -magi and Terriere, 1974b). Squoxin i s capable of forming chelates with the a l k a l i metals, which could lead to some degradation in the aquatic environment. These complexes are assumed to be formed by a replacement reaction of water of hydration involving a d i r e c t i n t e r a c t i o n between cation and ligand and are not simply an ion-association product (Skytte Jensen, 1964) . 25 Squoxin i s re a d i l y oxidized to 10-14 d i f f e r e n t degradation products (Burnard, pers. comm.). The speed at which th i s occurs and the number of products formed i s dependent on the number and type of potential o x i d i z i n g agents i n the environment. A i r oxidation, p a r t i c u l a r l y i n running water, and photo-oxidation by sunlight appear to dominate these reactions. Sunlight alone w i l l reduce the recoverable Squoxin i n d i s t i l l e d water by two-thirds i n one day while aeration i n the dark reduces i t by one-half during an equivalent time period (Terriere, et_ al_. , 1970) . Squoxin has demonstrated l i t t l e tendency to adsorb to inorganic suspended s o l i d s . Terriere et a l . (1970) observed a s l i g h t depression of extraction of Squoxin from water containing 5 0 mg/1 of F l o r i s i l (activated magnesium s i l i c a t e ) , but there was no appreciable degradation due to 50 mg/1 of Attaclay or magnesium oxide. 3.11 E f f e c t of Dissolved Organic Compounds Dissolved organic compounds leached from plant material, s o i l and humus accelerated the rate of degrada-tion of Squoxin. These compounds are poorly defined poly-e l e c t r o l y t e species loosely c l a s s i f i e d as humic and f u l v i c acids. Although l i t t l e physical and chemical data are available on these compounds, they play a major role i n s o i l and water chemistry by acting as buffers, ion-exchangers, surfactants, sorbents, and chelating agents (Kononova, 1966). 26 Degradation of Squoxin i n the dark brown water of Camosun Bog i n the University Endowment Lands was rapid. Only 68 percent of a Squoxin treatment was recovered 6 hours afte r treatment; 30 percent remained at 2 4 hours, and by 72 hours none of the o r i g i n a l compound was detected (Figure 3). The degradation rate of Squoxin i n Capilano River, Musqueam Creek and Camosun Bog waters (Figure 3) demonstrated a good rela t i o n s h i p with t h e i r respective conductivity (Table I) and color, measured on a Hellige Aqua Tester (Table I) and as percent transmittance on a scanning spectro-photometer (Figure 4). As conductivity can, i n part, be attributable to the concentration of the dissolved organic compounds, the evidence strongly suggests that some degrada-ti o n can be ascribed to the dissolved organic compounds. The techniques of Sephadex column chromatography and Amicon u l t r a f i l t r a t i o n were employed to assess the degree of int e r a c t i o n of Squoxin with dissolved organic carbon com-pounds. Both methods allow for the molecular size separation of complex mixtures of compounds i n aqueous solution. Sephadex chromatography showed a d e f i n i t e associa-14 ti o n of C-Squoxm with organic color i n a Burns Bog water concentrate (Figure 5). The peak concentration of Squoxin coincided with the dark brown high molecular weight materials, which were eluted from the column f i r s t . A second peak coincided roughly with yellow compounds of lower molecular weight. 27 FIGURE 4. Spectral scan of waters i n F i g . 3 A. D i s t i l l e d water B. Capilano River water C. Musqueam Creek water D. Camosun Bog water WAVELENGTH, nm 29 3 0 r 2 * MILLILITERS THROUGH COLUMN FIGURE 5. Sephadex column chromatography of a concentrated sample of Burns Bog water allowed to react with 1 4C-Squoxin for 1 hour at 20°C. Elutant analyzed for absorbance at 4 00 nM and ng Squoxin. 30 Most of the Squoxin, 99.7 percent, sorbed to the Sephadex gel and was slowly eluted from the column. This sorption reaction proved to be quite strong. After three successive washings of the column with acetone, s i g n i f i c a n t amounts of Squoxin could s t i l l be eluted. The only recourse, given the compound's intr a c t a b l e chemical c h a r a c t e r i s t i c s , was to separate the high molecular weight colored compounds from the smaller inorganic and organic molecules i n a water sample. Degradation i n each, as well as the contribution of each, could then be measured. Camosun Bog water was f i l t e r e d through an Amicon u l t r a f i l t r a t i o n unit which was capable of selecting against a l l molecules having a nominal molecular weight greater than 20,000. After two f i l t r a t i o n s the retentate consisted of organic molecules greater than 20,000 molecular weight dissolved i n glass double d i s t i l l e d water. The f i l t r a t e contained organic and inorganic molecules of less than 20,000 molecular weight. The concentration of dissolved organic carbon i n the f i l t r a t e was 3.7 mg/1, while that i n the retentate was a minimum of 13.4 mg/1. Squoxin degraded quickly i n both samples (Fig. 6). The high molecular weight compounds i n the retentate caused a rapid degradation to 2 0 percent of the i n i t i a l concentra-t i o n within 4 8 hours. Concentration of Squoxin declined at a faster rate i n the f i l t r a t e . Less than 65 percent of the i n i t i a l concentration of Squoxin could be recovered 6 hours after treatment. More than 90 percent of the Squoxin FIGURE 6. Degradation of Squoxin in filtrate and retentate of Camosun Bog water filtered on an Amicon ultrafiltration unit fitted with a 20,000 nominal molecular weight filter. Vertical b a r s indicate range of 2 samples for retentate. 32 was recovered from the retentate i n an equivalent time period.. Both degradation curves converge at 72 hours with 20 percent of the Squoxin remaining. While degradation of Squoxin occurs because of the presence of dissolved organic compounds, the impact of these compounds on the maintenance of a toxic concentration during a treatment i n t e r v a l i s ambiguous. Degradation of Squoxin i n natural waters i s a r e s u l t of competitive interactions between a number of pote n t i a l degradative agents. Viewed i n t h i s way, the concentration of high molecular weight com-pounds would have l i t t l e e f f e c t on a treatment, since other degradative pathways proceed at a faster rate. The presence of high concentrations of dissolved organic compounds may r e s u l t ; i n enhanced s t a b i l i t y of Squoxin by l i m i t i n g the penetration of sunlight i n water. 3.12 E f f e c t of Bacteria Freshwater bacteria maintained i n the presence of 14 C-Squoxxn for 2 hours f a i l e d to show any a b i l i t y to degrade the compound by using i t as a carbon source (Table I I ) . The uptake of Squoxin into the c e l l s or onto the c e l l surface was equal i n both l i v e and H 2 S 0 4 - k i l l e d bacteria i n d i c a t i n g the compound was only passively adsorbed. 14 No C0 2 was respired by the l i v e samples. The bacteria therefore were unable to quickly u t i l i z e Squoxin as a carbon source or were using the natural substrate i n the water i n preference to Squoxin. 33 The entire question of microbial degradation of Squoxin i s a complex and important one. F a i l u r e to mineralize Squoxin over an experimental period of 2 hours perhaps has l i t t l e meaning. Many intractable compounds i n the environment are degraded by bacteria through enzyme induction, s h i f t s i n population gene pools, or a l t e r a t i o n of species composition. These degradation reactions may, however, take a long time or re s u l t i n compounds of equivalent t o x i c i t y . 14 TABLE I I . Uptake and mineralization of C-Squoxin by fresh-water bacteria i n Trout Lake (Vancouver, B.C.) Squoxin Concentration (pg/D CPM i n pa r t i c u l a t e fraction" Bacteria Bacteria+ H„SO„ 20 1671 1725 40 3390 3998 60 4440 5928 80 7227 8049 100 9160 9511 200 16834 16398 No C0 2 was respired during the course of the t e s t (2 hours). The types of bacteria i n freshwater depend on the mineral and organic content of the water, the s o i l s with which they are i n contact, surface p o l l u t i o n and many other factors (Ware and Roan, 1970). The heterogeneity 34 of these populations alone would require an enormous research e f f o r t to unravel the degradative c a p a b i l i t y of a suitable cross-section of freshwater microorganisms. 3.2 TOXICITY 3.20 Bacteria 14 A 100 ug/l Squoxin treatment i n h i b i t e d C-glucose uptake and re s p i r a t i o n by freshwater bacteria (Table I I I ) . Non'-parametric analysis of variance (Kruskal-Wallace test) demonstrated the mean uptake rates among the three t e s t conditions to be s i g n i f i c a n t l y d i f f e r e n t (a = .05) at both 14 enrichment l e v e l s of 60 and 100 pg C-glucose/1. At the 14 intermediate enrichment l e v e l of 60 pg C-glucose/1, the ethanol solvent dropped the uptake rate of the glucose by 16 percent. Relative to the ethanol control, the Squoxin alone depressed the uptake rate by an additional 19 percent. 14 At a higher enrichment l e v e l of 100 pg C-glucose/1, 26 percent i n h i b i t i o n of uptake was recorded for the ethanol while an additional 9 percent was due to the Squoxin. Based on these two observations, roughly 60 percent of the i n h i b i -t i o n of uptake of glucose by a Squoxin-ethanol solution can be attributed to the ethanol solvent. Each treatment contained 0.1 ml (81.6 mg) 95 per-cent ethanol, but only 1.0 pg Squoxin. On a weight basis, Squoxin i s more than 4 orders of magnitude more i n h i b i t o r y of b a c t e r i a l heterotrophic a c t i v i t y than ethanol at the 35 TABLE I I I . E f f e c t o f Squoxin on h e t e r o t r o p h i c a c t i v i t y of freshwater b a c t e r i a . Ten m i l l i l i t e r s o f Trout Lake (Vancouver) water were t r e a t e d with 100 u g / l Squoxin i n 0.1 ml 95% e t h a n o l . Samples were incubated f o r 2 hours i n a shaker bath at 15°C. 14 C-Glucose L e v e l UPTAKE (pg/l/hr) (pg / D N a t u r a l Ethanol Squoxin+ C o n t r o l C o n t r o l E t h a n o l 60 0. 96 0.67 0.70 11 0.96 0.74 0.65 11 1.17 1.16 0.69 11 1.23 1.05 0.75 mean= 1.0 8 mean= 0.91 mean= 0.70 % I n h i b i t i o n = 16 35 100 1.79 1.78 1.50 11 2.29 1.65 1.57 II 1.73 1.78 1.48 II 3.42 1.68 1.50 me an= 2.31 mean= 1.72 mean= 1.51 % I n h i b i t i o n 26 35 i n t e r m e d i a t e enrichment l e v e l and 6500 times more i n h i b i -t o r y at the hig h l e v e l . Squoxin i n a 95 percent e t h a n o l s o l v e n t depressed the maximal r a t e a t which glucose c o u l d be u t i l i z e d by b a c t e r i a when compared to a n a t u r a l c o n t r o l ( F i g . 7). A Lineweaver-Burk t r a n s f o r m a t i o n was performed on these data to convert the Michaelis-Menton k i n e t i c s curve (a h y p e r b o l i c 36 FIGURE 7. E f f e c t of Squoxin on uptake and u t i l i z a t i o n of l^c-glucose by freshwater bacteria from Trout Lake (Vancouver, B.C.). Lineweaver-Burk transformation of data; regression f i t by eye. 37 function) to a l i n e a r r e l a t i o n s h i p . The slope of the l i n e i s equal to the r e c i p r o c a l of the maximal v e l o c i t y of uptake by b a c t e r i a . The i n t e r c e p t i s equal t o the turnover time of the n a t u r a l substrate i n the sample. The turnover time i s the time i t takes the microbes to remove a l l the glucose at i t s n a t u r a l c o n c e n t r a t i o n . I t i s increased here by n e a r l y 12 hours. This means that n a t u r a l h e t e r o t r o p h i c a c t i v i t y i s slowed down to about h a l f i t s usual l e v e l . The drop i n the maximal v e l o c i t y i n d i c a t e s how the microbes are a f f e c t e d at s a t u r a t i o n l e v e l s of g l u -cose. The Squoxin plus ethanol depress t h i s r a t e by 33 percent. The c o n t r o l s used i n these determinations were unf o r t u n a t e l y given no ethanol treatment. Based on the previous work (Table I I I ) , the e f f e c t on turnover time and maximal v e l o c i t y due t o Squoxin alone would be l e s s than one-half the above mentioned values. The most reasonable e x p l a n a t i o n f o r Squoxin's e f f e c t on b a c t e r i a i s i t s r a p i d adsorption t o the c e l l membrane which would block t r a n s p o r t s i t e s . No degradation of Squoxin by b a c t e r i a was observed during a s i m i l a r p e r i o d (Table I I ) , so t h a t there could be no competition 14 between Squoxin and C-glucose f o r uptake. S i m i l a r l y , an equal amount of Squoxin was taken up by both l i v e and dead b a c t e r i a , i n d i c a t i n g an i n a c t i v e surface adsorption of the compound r a t h e r than an enzyme-mediated process. F i n a l l y , Squoxin i n aqueous s o l u t i o n forms s t i c k y f i l m s on glassware 38 and on organisms being treated. For example, at high doses of Squoxin a Chaoborus t r i v i t t a t u s larva can be l i f t e d out of the water by merely touching the body with a glass rod. At treatment levels used i n these experiments, 100 ug/l, there could conceivably be some uncoupling of phos-phorylating r e s p i r a t i o n based on studies by Nakaue, Caldwell and Buhler (1972). The formation of high energy phosphate compounds would be in h i b i t e d , which could a f f e c t the energy intensive uptake of the glucose from solution, but not the rate at which i t i s metabolized. This hypothesis i s un-a t t r a c t i v e because (1) Nakaue et_ a_l. (1972) used three times the concentration used here; (2) intact organisms might be expected to show a lag time for t h i s e f f e c t while the c e l l u -l a r reserves at ATP formed previous to treatment were drawn upon and; (3) samples taken 30 minutes aft e r treatment demonstrated i n h i b i t i o n to be as serious as samples taken at 2 hours. 3.21 Invertebrates Invertebrate response to Squoxin treatments were variable among the 5 species tested. Simulium canadense larvae were the most seriously affected of the sample groups (Figure 8B). Complete mo r t a l i t i e s were obtained i n 4 8 hours at a concentration as low as 1000 pg/1. Complete mortality of the sample population was not observed i n bioassays containing Chaoborus t r i v i t t a t u s larvae (Figure 8A) or Hyalella azteca (Figure 9A), even at concentrations as high PERCENT RESPONSE FIGURE 8. To x i c i t y of Squoxin to Dipteran larvae. A.) Chaoborus t r i v i t t a t u s ; log-probit plo t of 96 hour observation. B.) Sim- ulium canadense; log-probit plo t of 48 hour observation. 10 20 30 4 0 50 60 70 80 90 & PERCENT RESPONSE FIGURE 9. T o x i c i t y o f Squoxin t o amphipods. A.) H y a l e l l a  a z t e c a ; l o g - p r o b i t p l o t o f 96 hour o b s e r v a t i o n . B.) Anisogam- marus r a m e l l u s ; l o g - p r o b i t p l o t of 96 hour o b s e r v a t i o n . 41 as 10,000 ug/l. Ani sogammarus ramellus was slow to respond to the toxicant, exhibiting only a 50 percent response at the highest concentration i n the series i n 48 hours. How-ever, nearly complete k i l l s were obtained at 1350 pg/1 i n 96 hours (Figure 9B). Daphnia pulex was also slow to respond. Observation at 48 hours shows less than 100 percent mortality (Figure 10B). At 96 hours, the lowest concentration e l i c i t i n g a 100 percent response i s 2400 pg/1 (Figure 10A). D. pulex larvae, 0 - 4 8 hours o l d , were more re s i s t a n t to treatment than the adults (Figure 11). The l e t h a l concentration a f f e c t i n g 50 percent of the sample population i s summarized i n Table IV. TABLE IV. To x i c i t y of Squoxin to invertebrates; summary of LC50 values. Time Organism 4 8 Hours 96 Hours Simulium canadense 60 pg/1 Chaoborus t r i v i t t a t u s r e s i s t a n t r e s i s t a n t Hyalella azteca r e s i s t a n t 3250 pg/1 Anisogammarus ramellus r e s i s t a n t 690 pg/1 Daphnia pulex (Adult) 1620 pg/1 950 pg/1 Daphnia pulex (Juvenile) 24 90 pg/1 42 10.00p 5.00 - 1.00 CD E LLI (/) O Q 0.50 0.10 0.05 Vo 20 3'6 40 S'u 6'0 7*0 86 90 §fe PERCENT RESPONSE 10.00c 5.00 ^ 1.00! CD E LU CO o o 0.50 0.10 0.05 20 3*6 40 50 60 7*6 tfi 16-PERCENT RESPONSE T 8 FIGURE 10. To x i c i t y of Squoxin to Daphnia pulex. A.) Log-probit p l o t of 96 hour observation. B.) Log-probit plo t of 48 hour observation. 4 3 10.00c (/} o O 0.10: 0.05: J I ' • • ' ' • ' ' 10 20 3040 5060 70 80 90 98 PERCENT RESPONSE F I G U R E 11. T o x i c i t y of Squoxin to Daphnia pulex l a r v a e 0 — 4 8 hours old; l o g - p r o b i t plot of 48 hour observation. 44 The recommended f i e l d treatment l e v e l of Squoxin i s 100 pg/1 because of i t s rapid degradation rate. At that l e v e l only S. canadense would be seriously affected (Figure 8B). Based on the regression l i n e , more than 80 percent of the population would be destroyed i f the 100 pg/1 concentra-ti o n were maintained for a 4 8 hour period. Squoxin was not as toxic to the invertebrate groups tested, with the exception of the b l a c k f l y larvae Simulium  canadense, as i t i s to many f i s h species. Northern squawfish, Ptychocheilus oregonensis, are by far the most sensitive species yet determined having an LC100 value of 8 pg/1 at 15.6° C (MacPhee and Ruelle, 1969). Another member of the Cyprinid family, the chiselmouth, Acrocheilus alutaceus, has a LC50 value of 65 pg/1 at 15° :C (MacPhee and Bailey, 1973). Steelhead trout, Salmo gairdneri, and coho salmon, Oncorhyn- chus kisutch, have LCO values of greater than 1000 pg/1 at 18.3° C (Crowley, 1974). The t o x i c i t y of Squoxin to invertebrates does not appear to follow phylogenetic a f f i n i t i e s . This i s i l l u s -trated by the s u s c e p t i b i l i t i e s of the two dipteran larvae, S. canadense and C. t r i v i t t a t u s . Both are i n the superfamily Culicoidea, yet they exhibit widely divergent tolerances to a Squoxin treatment. Bioassays with S. canadense show a LC50 value of 60 pg/1 i n 48 hours and indicate that a treatment of 180 pg/1 would completely devastate the population. C. t r i v i t t a t u s , on the other hand, withstands treatments of 10,000 pg/1 for 45 96 hours with less than 30 percent mortality i n the sample population. The explanation for these observed differences must be sought i n the toxicant's mode of action and the organism's adaptations to i t s habitat, i t s morphology and i t s physiology. The precise mechanism of t o x i c i t y for Squoxin has not been f u l l y investigated. Nakaue et_ al^. (1972) reported that Squoxin uncouples oxidative phosphorylation i n rat l i v e r mitochondria i n v i t r o as do many other bisphenols such as hexachlorophene. The simple phenols are i n t r i n s i c a l l y less active against mitochondrial systems than are chlorinated bisphenols. There was a d i r e c t c o r r e l a t i o n between the number of chlorine substituents of the bisphenols and uncoupling a c t i v i t y . This i s most l i k e l y a r e f l e c t i o n of a greater degree of binding to mitochondrial systems by chlorinated compounds, which leads to conformational changes of the phosphorylating enzymes. Thus 500 uM of Squoxin were required to achieve the same e f f e c t as 0.3 uM of hexachlorophene. If Squoxin i s only a r e l a t i v e l y mild uncoupler of phosphorylating r e s p i r a t i o n i t may have other, more serious effects on organisms. MacPhee (pers. comm.) asserts that the primary e f f e c t of Squoxin i s interference with oxygen transport. Squawfish l e f t i n the presence of Squoxin long enough to lose t h e i r equilibrium have l i t t l e or no red pigment i n t h e i r peritoneal or v i s c e r a l a r t e r i e s . The f i n s of small minnows exposed to Squoxin redden due to d i l a t i o n of peripheral c a p i l l a r i e s . S i m i l a r l y , the thoracic g i l l s 46 of A. ramellus, which have succumbed to a Squoxin treatment, are distended with c l o t t e d blood. The reaction of squawfish to a l e t h a l concentration of Squoxin (labored v e n t i l a t i o n , loss of equilibrium) i n d i -cated interference with the uptake, transport or u t i l i z a t i o n of oxygen. Whether th i s interference i s manifest at the sub-cellular, c e l l u l a r , tissue or organ l e v e l of integration i s unclear. The vaso-dialation observed i n squawfish suggests some e f f e c t on the autonomic nervous system. Burnard and Terriere (1974) found Squoxin tended to remain i n the brain of rainbow trout even a f t e r other tissues had been cleared of the toxicant. These e f f e c t s may only be peripheral manifestations of treatment and may not be the root ..cause of the physio-l o g i c a l c r i s e s which occurs i n susceptible organisms. The rationale i n designing the acute t o x i c i t y bio-assays was to determine, from an environmental standpoint, whether there was a relationship between the l e v e l of s u s c e p t i b i l i t y of an organism and the dissolved oxygen regimes i n the habitat to which i t was adapted. P r a c t i c a l considerations led to the choice of S. canadense and C. t r i v i t t a t u s as test organisms. They were r e l a t i v e l y c l o s e l y related, l o c a l l y available, abundant, l i v e d i n s t r i k i n g l y d i f f e r e n t habitats and have been studied to a cert a i n extent because both are considered pests. 47 Chaoborus sp. i s the only insect member of the planktonic community of lakes. Many f i e l d investigations have ascertained that these larvae can l i v e i n oxygen depleted environments for an i n d e f i n i t e period (Juday, 1908; Northcote, 1964; Stahl, 1966). Berg and Jonasson (1965) reported that Chaoborus l i v e d for days i n oxygen depleted water without any decrease i n numbers. M o r t a l i t i e s a f t e r t h i s period were ascribed to starvation; i f they are fed they may be "stored for a long time (months?)". In the b l a c k f l y family, Simulidae, l a r v a l develop-ment takes place i n r i v e r s and streams where current v e l o c i t y i s high. Larval i n f e s t a t i o n i s es p e c i a l l y high i n "adoles-cent" r i v e r s and the spillways of dams and slu i c e s where the current guarantees abundant food and oxygen (Peterson and Wolfe, 1956). S. canadense used i n these bioassays were co l l e c t e d at the mouth of Capilano Canyon i n the r i f f l e areas of the r i v e r . In both Chaoborus and Simulium the main route of uptake of dissolved oxygen from the water i s the simple d i f f u s i o n of gases through the c u t i c l e . These two larvae have r e c t a l blood g i l l s , but they are not the s i t e of p a r t i c u l a r l y active oxygen uptake under normal circumstances (Wigglesworth, 1972). The wide v a r i a b i l i t y i n t o x i c i t y of Squoxin to these two invertebrates (Figure 8A-B) becomes comprehensible within the environmental context to which they have adapted. If Squoxin does i n t e r f e r e with oxygen transport or u t i l i z a t i o n , 48 one would then hypothesize that an organism capable of l i v i n g through periods of anaerobiosis would be best able to weather a chemical severance of the available oxygen supply. Squoxin was much less toxic to the amphipod Hyalella azteca, a deposit feeder common i n the l i t t o r a l benthos, than i t was to the amphipod Anisogammarus ramellus, coll e c t e d from the i n t e r s t i c e s of stones and wood along the bottom of Musqueam Creek. Although oxygen saturation may be high at a l l times i n both habitats, Gaufin (1972) found the presence of current to be an important ecological factor i n determining the resistance of aquatic invertebrates to oxygen depletion. S i m i l a r l y , Wigglesworth (1972) reported that Chironomid larvae such as Tanytarsus brunnipes and Anatopynia nebulosa, which l i v e i n streams, show a f a l l i n oxygen consumption as soon as the oxygen content drops below 100 percent a i r saturation; whereas Chironomus longistylus and A. varia from stagnant waters maintain t h e i r normal rate of oxygen uptake u n t i l the oxygen content has f a l l e n to 15.4 percent of a i r saturation. These differences are not confined to the Chironomidae nor do they correspond with the presence or absence of hemoglobin. Daphnia pulex exhibited an intermediate resistance to Squoxin. This organism i s known to l i v e i n stagnant waters having very l i t t l e dissolved oxygen. I t i s common in sewage s t a b i l i z a t i o n ponds where i t i s reported to be most abundant when the oxygen concentration i s below 2 mg/1, 49 although t h i s may occur because organic foodstuffs are highest at t h i s time (Dinges, 1973). Squoxin tends to form flocculent precipitates at the le v e l s found harmful to Daphnia. These prec i p i t a t e s c l i n g to the carapace and appendages often immobilizing the animal, which could contribute to i t s demise. Squoxin appears to be an e f f e c t i v e respiratory toxicant, although the precise mechanism by which i t achieves t h i s e f f e c t i s poorly understood. An inverse r e l a t i o n s h i p exists between the t o x i c i t y of Squoxin and the adaptation to oxygen a v a i l a b i l i t y i n the aquatic environment as demonstrated by the invertebrates i n t h i s study. 3.3 BIOCONCENTRATION POTENTIAL 3.30 General Experiments concerned with the uptake of Squoxin, i t s excretion, and i t s p a r t i t i o n i n g among the elements of 14 simple laboratory microcosms were done with the C-labelled compound. The concentration of the toxicant was followed by determination of the r a d i o a c t i v i t y i n a sample. This allows the detection of extremely small quantities, but does not provide any information on t h e i r q u a l i t a t i v e •. composition. As discussed previously, Squoxin degrades quickly in aqueous solution to at least 11 degradation products. Since l i t t l e information exists on the i d e n t i t y or t o x i c i t y 50 of these degradation products, they were assumed to have negative e f f e c t s on the biota and are referred to i n the text as Squoxin residues. Squoxin residues are most l i k e l y bisphenolic compounds, which may be quinones, ethers, ketones, or other oxidation products. The only product known not to occur (or i n amounts less than 2 percent) i s 2-naphthol (Terriere pers. comm.). This product would r e s u l t from hydrolysis 14 of the methylene bridge where the C-label i s located. Excretion products of aquatic invertebrates could be conjugates of glucose (Menzer, 1973) or sulfate (Matsu-mura, 19 75). Conjugates are formed by the addition of a water soluble substituent to the generally hydrophobic pesticide molecule. This renders the foreign compound amenable to f i l t r a t i o n and elimination by the excretory system. Sulfate conjugation i s a common reaction of phenols and naphthols i n most species of animals including insects. Conjugation of glucose with the hydroxyl groups of almost any compound i s known to occur i n insects. Both accumulation and elimination processes of pesticide chemicals follow f i r s t - o r d e r concentration k i n e t i c s . The speed of elimination of pesticides from the body afte r termination of continuous exposure to a pesticide can be expressed as: dC/dT=kC where C i s the concentration and k the f i r s t - o r d e r reaction constant. Integration of t h i s —kT equation gives: C=CQe where C i s the concentration of the pesticide in body tissue and C^ i s the pesticide 51 concentration at the s t a r t of the termination experiment (Matsumura, 1975). The regression equations derived for toxicant excretion by invertebrates i n t h i s study take the form of the second equation above. The slope of the regression l i n e i s equal to the f i r s t - o r d e r reaction constant, k. Thus, the analysis of co-variance comparing the slopes of the regression l i n e s for 2 d i f f e r e n t invertebrates or conditions compares these reaction constants. The same i s true of the analysis of co-variance of the uptake plots in the microcosm data. 3.31 Microcosms The behaviour of Squoxin i n aquatic microcosms was useful i n assessing the l e v e l of inte r a c t i o n with both animate and inanimate organic matter. The rate and degree of sorption of pesticide compounds to suspended s o l i d s , sediment, and organisms i n the aquatic environment i n f l u -ences t h e i r degree of bioconcentration. These experiments were designed to ascertain how Squoxin would p a r t i t i o n i t -s e l f among the elements of the microcosm and whether t h i s p a r t i t i o n i n g was stable over a period of time. The uptake rate and f i n a l t i s s u e l e v e l s attained by the invertebrates i n these tests were of p a r t i c u l a r i n t e r e s t , since uptake occurred both through feeding and d i r e c t adsorption from the water. Although plagued by a n a l y t i c a l 52 d i f f i c u l t i e s , the microcosms data at least provides a r e a l i s t i c estimate of the k i n e t i c s of Squoxin i n the aquatic environment. 14 C-Squoxin introduced into laboratory microcosms proved to be a l a b i l e compound. The feature common to a l l microcosms i s the fr a c t i o n of Squoxin residue present i n the f i l t e r a b l e portion 2 hours after treatment (Figs. 12-15). The Squoxin residues then move st e a d i l y into the water phase. The a n a l y t i c a l technique used to sample these microcosms does not allow a q u a l i t a t i v e assessment of the physical state of the toxicant f i l t e r e d from solution. This l i m i t s the information gained from these data. The Squoxin residues detected i n the f i l t e r a b l e f r a c t i o n are either i n insoluble micro-crystalline form, adsorbed to suspended so l i d s or adsorbed to the m i l l i p o r e f i l t e r . In the culture containing Daphnia, C h l o r e l l a sp. and water, the Squoxin residues s o l u b i l i z e d at a uniform rate throughout the sampling period. This rate i s greatest i n Culture 2 (Fig. 13) which has the intermediate con-centration of algae. The rate at which Squoxin residues become soluble i s more or less equal in Cultures 1 and 3 (Figs. 12 and 14), the two extreme concentrations of algae. The maximum range of re p l i c a t e samples for a l l C h l o r e l l a cultures deviated only 3.4 percent from the mean for the f i l t e r a b l e material and only 3.3 percent for the water. 100r HOURS FIGURE 12. Partitioning of Squoxin in laboratory microcosms containing 200 ml Capilano River water, 10 Daphnia pulex, and Chlorella sp. at a concentration of 2.5 X 1 0 4 cells/ml (Culture 100r - I <n | •» —a I 50 IW" 150 HOURS F I G U R E 13. P a r t i t i o n i n g of Squoxin in l a b o r a t o r y m i c r o c o s m s containing 200 m l Ca p i l a n o R i v e r water, 10 Daphnia pulex, and C h l o r e l l a sp. at a co n c e n t r a t i o n of 1.0 X 1 0 b c e l l s / m l ( C u l t u r e F I G U R E 14. P a r t i t i o n i n g of Squoxin in l a b o r a t o r y m i c r o c o s m s containing 200 m l C a p i l a n o R i v e r water, 10 D aphnia pulex, and C h l o r e l l a sp. at a c o n c e n t r a t i o n of 2.0 X 10 b c e l l s / m l ( C u l t u r e 3). 56 The v a r i a t i o n i n the apparent degree of s o l u b i l i z a t i o n of Squoxin residues i n the three cultures i s most l i k e l y a r e f l e c t i o n of the v a r i a b i l i t y of the t o t a l amount of Squoxin recovered versus the t o t a l amount added: 77-114 percent. The microcosm containing organic sediment, Hya l e l l a, and water (Fig. 15) reveals the same smooth r i s e i n the amount of Squoxin residues becoming water soluble as i n the Daphnia microcosms. Unlike the Daphnia microcosms, the f i l t e r a b l e portion declines rapidly to 20 percent of the t o t a l 72 hours aft e r the addition of Squoxin and remains at t h i s l e v e l throughout the remaining sampling period. The sediment curve during the f i r s t 72 hours i s a mirror image of the suspended s o l i d curve. After 7 2 hours however, Squoxin residues began to move out of the sediment and become soluble, a trend which continues u n t i l the termina-t i o n of the t e s t . The recovery of r a d i o a c t i v i t y i n the Hy a l e l l a microcosms ranged from 82 percent i n i t i a l l y down to 32 percent. The low values are c h i e f l y a t t r i b u t a b l e to d i f f i c u l t i e s i n determining the amount of Squoxin present in the sediment. The sediment samples were dried and an indeterminate amount of the pesticide was l o s t through c o - d i s t i l l a t i o n with water. The sediment p a r t i c l e s them-selves caused severe quenching during s c i n t i l l a t i o n counting and the e f f i c i e n c y of counting was depressed to very low l e v e l s . Assuming a l l measurement error to be attributed to the sediment detection problems and d i v i d i n g the FIGURE 1 5 . Partitioning of Squoxin in laboratory microcosms containing 400 milliliters Capilano R iver water, 10 Hyalella azteca and 10 mil l i l i t e r s wet sediment. ~~ 58 concentration i n the sediment by the f r a c t i o n detected at each time, the sediment curve retains the same shape. Uptake of Squoxin residues by D. pulex was almost instantaneous i n the culture containing the highest con-centration of algae (Fig. 16 C). Daphnia i n Cultures 1 and 2 exhibited a period of rapid uptake during the f i r s t 24 hours. This i n i t i a l phase of rapid uptake was followed by a period of moderate increase i n body load; hence two regression l i n e s have been f i t to these data (Figs. 16A, B). An analysis of co-variance compared the slopes of the l i n e s of the uptake p l o t s . The slope was not s i g n i f i -cantly d i f f e r e n t for the Daphnia i n Cultures 1 and 2 (a = .05; F = 1.32 with a p r o b a b i l i t y of 0.29) during the f i r s t 24 hours a f t e r treatment. There was a s i g n i f i c a n t difference in the slopes of uptake i n Cultures 1 and 3 during the same time i n t e r v a l (a = .05; F = 20.24 with a p r o b a b i l i t y of 0.01). Over the period of moderate increase i n uptake, 33 to 144 hours, the slopes of the uptake plots were not s i g n i f i c a n t l y d i f f e r e n t for Cultures 1 and 2 (a = .05; F = 0.069 with a p r o b a b i l i t y of 0.80) or for Cultures 1 and 3 (a = .05; F = 0.76 with a p r o b a b i l i t y of 0.40). Uptake of Squoxin residues by Hyalella azteca i n microcosms containing organic sediment (Fig. 17) matched those of the Daphnia i n Cultures 1 and 2. The analysis of co-variance of these data showed the uptake rate by Hyalella in the f i r s t 24 hours to be not s i g n i f i c a n t l y d i f f e r e n t from that of the Daphnia i n Culture 1 (a = .05; F = 3.7 5 9 1000 500 1-W O) E z 100 X o o 50 (/} CO c © 1000C WT. 500 I-UJ $ CO E z X 100 o o CO 50 CO c _1 24 48 72 96 120 144 H O U R S 24 4 8 72 96 H O U R S 120 144 1 0 0 0 r 500 t -LU CO E z X 100 o z> o w 50 CO c © _L 2 4 48 72 96 120 144 168 H O U R S FIGURE 16. Uptake of Squoxin residues by Daphnia pulex i n microcosms containing d i f f e r e n t concentrations of C h l o r e l l a sp. Regression l i n e s f i t by method of least squares. A.) 2.5 X 1 0 4 c e l l s / m l . B.) 1.0 X 1 0 5 c e l l s / m l . C.) 2.0 X 1 0 5 c e l l s / m l . 24 48 72 96 120 144 168 192 HOURS FIGURE 17. Uptake of Squoxin residues by Hyalella azteca in a laboratory microcosm containing organic sediment. Regression lines fit by method of least squares. 61 with a p r o b a b i l i t y of 0.10) or of Culture 2 (a .05; F = 5.4 with a pr o b a b i l i t y of 0.06). S i m i l a r l y the uptake rate during the period of moderate uptake was not s i g n i f i c a n t l y d i f f e r e n t from that during the same period for the Daphnia i n Culture 1 (a = .05; F = 0.01 with a p r o b a b i l i t y of 0.91). The microcosm data underscore the ubiquity of Squoxin residues i n an aqueous micro-environment. Unlike many pesticides which are adsorbed strongly to the surfaces of organic material, Squoxin residues desorb r e a d i l y from these constituents. At least part of the movement of Squoxin residues from the f i l t e r a b l e component i n the Daphnia microcosms represents the desorption of the pesticide from C h l o r e l l a . 14 Ch l o r e l l a sp. treated with C-Squoxin, centrifuged from suspension, and then resuspended i n clean water, show a p a r t i t i o n i n g e f f e c t between the c e l l s and the water within 24 hours. The concentration of Squoxin residues are then equally divided between c e l l s and water. Sodergren (1968) found the opposite to be true with DDT [1,1,1-trichloro-2,2-bis-(4-chlorphenyl) ethane]. C h l o r e l l a sp. accumulated DDT within 15 seconds of exposure. Transferring these c e l l s to clean water he found no DDT was l o s t to the media. Squoxin residues are also "leaked" into the sur-rounding water from the sediment to which they have previously sorbed. These organic matter/water re l a t i o n s may be the basis of observations by Terriere (pers. comm.) of persistance of Squoxin i n a pond 14 days a f t e r treatment 62 when laboratory studies indicated a much shorter residual l i f e . MacPhee and Cheng (19 74) observed that measurable concentrations of Squoxin i n water containing phytoplankton persisted for 72 hours longer than i t persisted i n the controls. Consideration of the size of the Daphnia i n the three cultures provides at least a p a r t i a l explanation of the anomalous uptake pattern shown by the organisms i n Culture 3. Daphnia i n Cultures 1 and 2 had i n d i v i d u a l mean weights of 0.9 and 1.0 mg respectively, while Daphnia i n Culture 3 had i n d i v i d u a l mean weights of 0.3 mg. The Daphnia i n Culture 3 therefore had a greater surface area to weight r a t i o r e l a -t i v e to those i n the other two cultures. Tissue concentration, calculated on a weight basis, might then increase propor-t i o n a l l y faster i n the smaller animals. The processing of the more abundant contaminated C h l o r e l l a c e l l s might also have contributed to t h i s rapid uptake by Daphnia i n Culture 3. However, McMahon and Rigler (1963) reported that the frequency of movement of the thora-c i c and mandibular appendages of Daphnia magna progressively decreased with increasing concentration of C h l o r e l l a vulgaris 5 above 10 c e l l s / m l . This suggests a slowing of the f i l t e r -ing rate with increasing algae concentration beyond a certain threshold; an e f f e c t documented for Daphnia pulex by Kryutchkova and Sladecek (1969). Collected c e l l s were also p e r i o d i c a l l y rejected. Perhaps Squoxin residues were gained by desorption from the additional c e l l s processed even though they might not have been consumed. 63 The uptake curve for H. azteca can be r a t i o n a l i z e d because the sediment/water in t e r a c t i o n was described quanti-t a t i v e l y by the a n a l y t i c a l technique. Examination of the amount of Squoxin adsorbing to the sediment shows a steep increase between 4 and 33 hours si m i l a r to the uptake p l o t for H y a l e l l a . The organism appears to act simply as an adsorption s i t e i n the culture rather than exhi b i t i n g a lag time for uptake by ingestion of contaminated sediment. In a l l four of the microcosms, invertebrates showed a "plateau" phase of uptake of Squoxin residues which were remarkably s i m i l a r both i n rate and l e v e l of uptake. This was either a saturation of a l l susceptible tissues or a balance of uptake and excretion. Unlike most l i p o p h i l i c , water insoluble pesticide chemicals, Squoxin residues displayed a p r o c l i v i t y to desorb from organic matter i n the water. It i s not known whether the movement of Squoxin from sediment and phytoplankton back into the water represents the enhanced s o l u b i l i t y of degrad-ation products or desorption of the parent compound. Squoxin residues showed no sign of desorption from inverte-brates i n the microcosms as i t did from inanimate sediment p a r t i c l e s . 3.32 Uptake Through Feeding Only a small amount of Squoxin was transported by the consumption of contaminated food items. The deposit-feeding amphipod, Hyalella azteca, i n the presence of 64 contaminated organic sediment accumulated 7.40 ng Squoxin residues/mg wet weight 1 (Table V). H. azteca i n the same culture vessel, but confined to a f l o a t i n g cage, exhibited Squoxin residues of 1.7 ng/mg wet weight. This l e v e l was 2 3 percent of the concentration in the grazing amphipods. The concentration of Squoxin residues in the water was 0.3 percent of the concentration i n the sediment. TABLE V. Uptake of C-Squoxin by Hyalella azteca from contaminated organic sediment 1. "Grazers" had di r e c t physical contact with sediment; "caged" animals were placed i n f l o a t i n g mesh containers i n the same experimental vessel. Sample sizes are i n parentheses. Sample Mean ng Squoxin/ mg wet weight SD Grazer 7.40 (221) 0.85 Caged 1.72 (56) 0.86 Sediment 5,559,650.00 (4) 801,980.00 Water 18,720.00 (4) 3,450.00 50 ml wet sediment was added to 150 ml water and treated with 1 4C-Squoxin at a rate of 1000 ug/l for 24 hours. The sediment was then f i l t e r e d and placed i n clean water. Squoxin residue uptake i n H. azteca occurs by d i r e c t sorption from the water and sediment material as well as assimilation with food p a r t i c l e s . Sorption from the water ng/mg = mg/kg = ppm 65 alone accounted for nearly one-quarter of the t o t a l uptake. Even t h i s amount i s underestimated as the mesh cage of the control group did not allow a free and continuous exchange of water because of a surface tension e f f e c t of the screen-ing material. The amount of Squoxin residues taken up through d i r e c t sorption from the sediment cannot be separated from that ingested and assimilated. The t o t a l amount by both these routes seems small considering the overwhelming p a r t i -tioning of the Squoxin residues i n the sediment versus the water, 300:1. H. azteca exposed to ambient water concentra-14 tions of 500 ug/l C-Squoxin exhibited i n i t i a l residues of more than 300 ng/mg wet weight (Figure 20). During the course of the test, the experimental group spent most i f not a l l the time burrowing i n and sorting the sediment i n the chamber as i s normal for H. azteca. Hargrave (1970) estimated that a 700 ug i n d i v i d u a l at 15°C produces 18 to 20 pg (dry weight) of feces per hour. This means that the gut i s emptied and r e f i l l e d twice every hour, but l i t t l e i s assimilated. This mode of l i f e suggests that the main route of uptake i n H. azteca i s through adsorption of the Squoxin residues rather than digestion and assimilation. Epifanio (1973) studying the uptake of d i e l d r i n (1, 2, 3, 4, 10, 10-hexachloro-6, 7-epoxy-l,4,4a,5,6,7,8, 8a-octahydro-exo-l,4-endo-5,8-dimethanonaphthalene) by crab larvae found that i f equal concentrations were available 66 to the zoeae i n food and seawater> the animals would accumulate;'} the pesticide about 8000 times as f a s t from the water. In t h i s case, the crab larvae were planktonic and t h e i r food was contaminated Artemia s a l i n a , but i t does i l l u s t r a t e the necessity of considering the habitat and mode of l i f e when considering uptake of pes t i c i d e s . Because of the fast f i l t e r i n g rate of the larvae, the actual amount of d i e l d r i n to which i t was exposed was much higher by way of the water. Chaoborus t r i v i t t a t u s larvae preying on contaminated Daphnia pulex retained 5 percent of the t o t a l a c t i v i t y i n -gested (Table VI). During the feeding period small sub-samples showed that levels retained from t o t a l ingested residues varied from 10-25 percent. TABLE VI. Retention of Squoxin by Chaoborus t r i v i t t a t u s fed contaminated Daphnia pulex every 6 days. D. pulex was treated with 50 0 jug l^c-Squoxin/l water for 24 hours p r i o r to each feeding. Sample sizes given i n parentheses. No. Daphnia ingested Hours after l a s t feeding Mean ng Squoxin SD Percent retained^ Estimated tissue cone, (ng/mg wet wt.) 1 10 72 50.2 (19) 16. 7 6.2 5.32 10 144 39.4 (12) 20. 3 4.9 4.18 10 288 37.4 (20) 33. 3 4.6 3.97 Mean ng Squoxin i n contaminated D. pulex was 8 0.9 ± 2.9 ng/ i n d i v i d u a l at the time of feeding. 2 Mean weight of i n d i v i d u a l C. t r i v i t t a t u s larva - 9.4 ± 1.7 mg; derived from a subsample (n = 100) of laboratory population at time of assay. 67 Although the percentage of t o t a l ingested Squoxin residues retained by C. t r i v i t t a t u s i s small, these residues appear to be more stable than those derived from a contact dose of the compound. At 144 hours after the l a s t feeding, C. t r i v i t t a t u s exhibited estimated tissue concentrations of 4.18 ng/mg wet weight. Animals exposed to an ambient water treatment and then placed i n clean water for 150 hours had tissue concentrations of 0.8 ng/mg (Table VII) or 5 times lower. Squoxin residues gained through ingestion might be more amenable to storage i n adipose tissue. This would tend to alienate them from the metabolic processes of de-t o x i f i c a t i o n . 3.33 Toxicant Excretion 14 Daphnia pulex, exposed to a 500 pg/1 C-Squoxin treatment, were able to excrete the toxicant given time and an uncontaminated environment. Squoxin residue con-centration i n the tissues of these crustaceans were compared in i n d i v i d u a l s that had molted aft e r treatment with those that had not. Typical curves (Figures 18A-B) show an immediate rapid f a l l i n i n i t i a l concentration in the f i r s t 24 hours i n both groups. This probably represents desorp-t i o n of the residues from the carapace rather than an active excretion process of the organism. Squoxin residues remain stable i n pre-molt individuals from 24 to 72 hours a f t e r treatment. Between 72 and 96 hours there i s a mobilization of the compound and the concentration f a l l s sharply. FIGURE 18. Excretion of Squoxin residues by Daphnia pulex. Organisms treated for 24 hours with 500 ug/l 1 4C-Squoxin. A.) Organisms transferred to i n d i v i d u a l 50 ml con-ta i n e r s of clean water a f t e r treatment. B.) Organisms transferred to i n d i v i d u a l 50 ml containers of clean water with the addition of C h l o r e l l a sp. at a concentration of 1.0 X 10 5 c e l l s / m l . 69 Individuals undergoing a molt are able to r i d them-selves of the compound at a much faster rate. Concentrations of Squoxin residues i n the tissues f a l l s t e a d i l y through time and do not exhibit the period of s t a b i l i t y of the pre-molt i n d i v i d u a l s . The increased permeability of the c u t i c l e a f t e r molting i s primarily responsible for the acceleration of the rate of d e t o x i f i c a t i o n i n the samples taken a f t e r molting. Cast exoskeletons had only 2.2 ± 0.9 ng of Squoxin residues associated with them on an in d i v i d u a l basis. S i m i l a r l y Derr and Zabik (1972) found that exuviae d i d not represent a major route of DDE [1,l-dichloro-2,2-bis(p-chlorophenyl) ethylene] residue elimination i n Chironomous tentans. Only 1.4 to 4.4 percent of the t o t a l residue of DDE was l o s t to the exuviae. Evidence suggests that as the tissue concentration of Squoxin residues f a l l to low l e v e l s , the exoskeleton increasingly acts as a sink for the t o t a l body burden. A pooled sample of 16 exoskeletons taken a f t e r the second molt following treatment showed a residue l e v e l of 3.4 ng/ ind i v i d u a l exoskeleton. This i s a substantial l e v e l when compared to the concentration remaining i n the body, 0.47 ng/mg wet weight, a f t e r undergoing the second molt. Daphnia used i n t h i s experiment had a mean weight of 0.5 mg. Expressing the res u l t s of d i f f e r e n t Squoxin tr e a t -ments as a percentage of the i n i t i a l concentration (Fig. 19) shows that the addition of C h l o r e l l a to the post-treatment HOURS FIGURE 19. Excretion of Squoxin residues by Daphnia. pulex. Data from F i g . 12 expressed as a percentage of initial tissue" concentration. 71 clean cultures had no discernible e f f e c t on the excretion rate. Residues declined to the same l e v e l without regard to the presence or absence of algae. Ecdysis was the dominant factor i n determining excretion rate, although a l l levels declined to less than 10 percent within 96 hours. The addition of organic sediment to clean water into which treated Hyalella azteca were placed had a great e f f e c t on the concentration of Squoxin residues i n the tissues of the amphipod (Fig. 20). There was a rapid decline of Squoxin residues i n the f i r s t 48 hours a f t e r treatment. Subsequent to t h i s rapid loss there was a period of slow decline up to 192 hours. Two regression l i n e s were f i t to these points. The slope of the second, "slow l i n e " , was subtracted from the f i r s t by means of back-projection (Wagner, 1975). This analysis assumes that 2 d i s t i n c t processes were occurring. The "slow l i n e " represents the biochemical d e t o x i f i c a t i o n mechanisms of the organism. The other l i n e represents the passive desorption of the p e s t i -cide residues from the carapace to the organic sediment mediated by the concentration gradient. H. azteca placed i n clean water lacking sediment exhibited only a slow excretion rate without the dramatic decline of concentration. An analysis of co-variance demon-strated the slope of t h i s l i n e was not s i g n i f i c a n t l y d i f f e r e n t from the slope of the second or "slow l i n e " exhibited by the H. azteca i n the sediment cultures (ot = .05; F = 4.4 with a p r o b a b i l i t y of 0.06). looor t-i g UJ i-UJ CO E x o ID o to co c ^WATER HOURS FIGURE 20. Excretion of Squoxin residues by Hyalella azteca. organisms treated for 24 hours with 500 u.g/1 -Squoxin in f i l t e r e d w a t e r a n d s u b s e q u e n t l y t r a n s f e r r e d t o A . ) c l e a n w a t e r o r B.) clean water with the addition of organic sediment. Regression line (A.) fit by method of least squares; (B) fit by method of least squares utilizing a back-projection technique. 73 The excretion of Squoxin residues by Chaoborus  t r i v i t t a t u s was variable among the sample population. Total body burden declined to less than 10 percent within 1 4 4 hours. Observation 24 hours l a t e r shows t h i s l e v e l to be stable. In the same manner the slope of the l i n e for the excretion of Squoxin residues by C. t r i v i t t a t u s was compared to that of H. azteca. Since C. t r i v i t t a t u s was placed i n clean water without any other addition the observed data (Fig. 2 1 ) were matched to that of the amphipod cultures which contained no sediment. The n u l l hypothesis of no s i g n i f i c a n t difference i n the slopes of the l i n e s was accepted ( a = . 0 5 ; F = 1.9 with a p r o b a b i l i t y of 0 . 1 8 ) . A portion of the treated C. t r i v i t t a t u s pupated and underwent metamorphosis to the imago. Larvae which pupated during treatment had residues twice that of the larvae i n the same treatment vessel. Animals which pupated i n clean water 24 hours aft e r treatment had residue l e v e l s 5 times lower than larvae under similar conditions. The imagos re s u l t i n g from both groups of pupae, however had tissue concentration which were v i r t u a l l y i d e n t i c a l (Table VII). Squoxin i s apparently amenable to excretion by organisms. Terriere and Burnard ( 1 9 7 5 ) found that rainbow trout were able to eliminate over 90 percent of the compound within 4 8 hours. Rats administered radio-active Squoxin were able to clear the residues from the body within 4 8 - 7 2 hours, about 90 percent of i t v i a the b i l e (Terriere pers. comm.). 74 1 0 0 i 501 x o LU I -LU CD E 10 x o O CO cn c 0.5 50 100 150 H O U R S 2 0 0 250 FIGURE 21. Excretion of Squoxin residues by Chaoborus trivittatus fourth instar larvae. Regression line fit by method of least squares. 75 TABLE V I I . Residue d e t e r m i n a t i o n i n Chaoborus t r i v i t t a t u s . F o u rth i n s t a r l a r v a e t r e a t e d at 500 ug ^C-Squoxin/1 water f o r 2 4 hours, then p l a c e d i n c l e a n , f i l t e r e d water. Residue l e v e l s monitored i n l a r v a e , pupae, and imagos. Status Hours a f t e r treatment No. i n d i v i d u a l s ng mg Squoxin/ wet wt. Larvae 0 20 9.5 24 10 12.4 48 10 2.3 72 10 5.8 96 10 2.2 120 10 5.1 150 13 0.8 168 12 0.9 Pupae; pupated d u r i n g treatment 0 10 19.5 Pupae; pupated i n c l e a n water 24 19 2.5 Imagos; pupated d u r i n g treatment; metamor-phosed i n c l e a n water __1 15 0.3 Imagos; pupated i n c l e a n water; meta-morphosed i n c l e a n water __1 50 0.2 Time of metamorphosis v a r i a b l e w i t h i n sample but a l l events subsequent t o 168 hours. 76 Aquatic invertebrates also demonstrated a b i l i t y to excrete the toxicant. At least part of the tissue concen-t r a t i o n of Squoxin residues were l o s t through desorption-d i f f u s i o n mechanisms. The rapid loss by D. pulex upon molting, by H. azteca i n contact with organic sediment and by C. t r i v i t t a t u s upon pupation i l l u s t r a t e these mechanisms. Exactly what proportion of d e t o x i f i c a t i o n i s accomplished by biochemical means i n invertebrates cannot be determined by these assays. Elucidation of the b i l e as the main route of excretion i n rainbow trout (Terriere and Burnard, 1975), however, does demonstrate the f e a s i b i l i t y of i n t r i n s i c degradative enzyme systems i n invertebrates. 3.34 The Potential for Bioconcentration The assessment of the bioconcentration pot e n t i a l of Squoxin i n aquatic ecosystems must rel y heavily on theory derived from the environmental d i s p o s i t i o n of the more per-s i s t a n t chlorinated pesticide chemicals. Squoxin residues have not been monitored i n the environment due to the lack of adequate a n a l y t i c a l techniques and the number of poorly characterized degradation products. Bioconcentration i s e s s e n t i a l l y the r e d i s t r i b u t i o n of a d i f f u s e compound i n the environment mediated by b i o l o g i -c a l processes and a t t r i b u t e s . C l a s s i c a l l y , the bioconcen-t r a t i o n of pesticide chemicals was conceptualized as being controlled by trophic r e l a t i o n s (e.g. Woodwell, Wurster, and Isaacson, 1967). Rosenberg (1975) finding the range 77 of concentration of d i e l d r i n to be similar i n both primary and secondary consumer invertebrates i n a slough, re-evaluated t h i s " t r o p h i c - l e v e l e f f e c t " . He concluded from a review of the l i t e r a t u r e on concentration of chlorinated hydrocarbon pesticides in invertebrate communities that bioconcentration i s more l i k e l y a function of habitat, mode of l i f e and exchange e q u i l i b r i a than food. It i s also affected by the size of the organism, pharmacokinetics, physical-chemical properties of the pesticide and various e x t r i n s i c factors. Rosenberg's (1975) view of bioconcentration i s a t r i p a r t i t e balance of the physical/chemical c h a r a c t e r i s t i c s of the pesti c i d e , the nature and content of the aquatic environment and b i o l o g i c a l c h a r a c t e r i s t i c s and processes. This seems to be a r a t i o n a l , balanced conceptual framework for assessing the potential of a compound to become con-centrated to a s i g n i f i c a n t degree i n the biota. Hamelink, Waybrant and B a l l (1971) aft e r examining f i e l d and laboratory data on DDT concentration by complete and "broken" food chains concluded that exchange e q u i l i b r i a regulate the b i o l o g i c a l magnification of chlorinated hydro-carbons. Exchange e q u i l i b r i a refers to the p a r t i t i o n i n g e f f e c t of pesticides among water and organic matter based on t h e i r s o l u b i l i t y i n polar and non-polar solvents. Metcalf et a l . (197 5) found that the uptake and con-centration of organic compounds was a function of t h e i r l i p i d / water p a r t i t i o n c o e f f i c i e n t s and t h e i r resistance to degrada-ti o n by enzymatic processes, e s p e c i a l l y the multifunction 78 oxidase systems. They reported an excellent c o r r e l a t i o n between the physical/chemical properties of 9 d i f f e r e n t pesticides and the degree of biomagnification i n laboratory model ecosystems. Squoxin i s probably less l i k e l y than many other pesticides to reach unacceptable lev e l s i n organisms con-sidering only i t s physical/chemical a t t r i b u t e s . The d i s t r i b u t i o n of 1.0 gram of Squoxin i n a binary solvent system of hexane/water would be 790 mg/210 mg. These values are very d i f f e r e n t from those reported for DDT: 999.99 mg/0.01 mg i n the same binary solvent system (Kenaga, 1972). These values mean that DDT strongly sorbs to an organic or l i p i d compound and powerfully r e s i s t s any movement into aqueous solution. The ready desorption of Squoxin or i t s degradation products from organic sediment i n the micro-cosms i l l u s t r a t e the difference i n these two compounds. The low s t a b i l i t y of Squoxin i n aqueous solution insures that the compound w i l l not be present i n the environ-ment for long, and^hence available for uptake by organisms, for a long period. One or more of the degradation products may have environmentally undesirable e f f e c t s . Their impact cannot be assessed u n t i l t h e i r i d e n t i t i e s are established. Hamelink et_ al_. (1971) proposed that the l e v e l of contamination of lake water and organisms might be reduced i f the binding capacity of the sediment i s increased or i f highly contaminated sediments were covered over or removed from the basin. This view i s consistent with research 79 conducted by Ter r i e r e , e_t a l . (1966). They found toxa-phene (2,2,5-endo, 6-exo,8,9,10-heptachlorobornane, one of a number of toxic components) p e r s i s t i n g i n toxic con-centrations about f i v e times longer i n the water of an oligotrophic lake than i n the water of an eutrophic lake. Squoxin's sorption/desorption from organic con-stituents w i l l probably have a great e f f e c t on i t s d i s t r i b u t i o n . In l o t i c environments, the steady desorption may serve to d i l u t e the compound or i t s degradation products by i n j e c t i n g small amounts into aqueous solution that would be rapidly flushed away. In lakes, t h i s desorption may have the opposite e f f e c t . The compound i n aqueous solution i s probably more amenable to uptake, e s p e c i a l l y by organisms l i v i n g i n or near the sediment. The sediment represents a large organic reservoir into which much of a Squoxin t r e a t -ment w i l l i n i t i a l l y p a r t i t i o n . The slow s o l u b i l i z a t i o n of residues i n the sediment into i n t e r s t i t i a l water could contribute to higher or more persistant body burdens i n benthic invertebrates. Excretion of Squoxin residues i s rapid and occurs i n a wide var i e t y of organisms. As noted previously both rainbow trout and laboratory rats were able to metabolize the compound and eliminate i t from the body, mainly by way of the b i l e . Invertebrates also demonstrated the a b i l i t y to purge t h e i r tissues of over 90 percent of the toxicant, although they required much more time than did the trout. 80 Based on Squoxin's physical/chemical properties, i t s behaviour i n microcosms, and i t s excretion rate, i t seems unlikel y i t could be b i o l o g i c a l l y magnified to a s i g n i f i c a n t degree. Squoxin residues might p e r s i s t i n the aquatic environment due to t h e i r steady desorption from organic material. The continuous rapid excretion of these residues by salmonids and aquatic invertebrates w i l l probably preclude t h e i r concentration i n the biota. 81 Chapter 4 SUMMARY AND CONCLUSIONS 4.1 SUMMARY The main conclusions derived from t h i s study are: 1. ) Degradation of Squoxin i s more severe i n water having a high pH and/dr high a l k a l i n i t y . . 2. ) Degradation of Squoxin can occur due to the pre-sence of high molecular weight humic compounds dissolved in water. The rate at which t h i s occurs i s not as great as the rate of degradation due to dissolved inorganic compounds. 3. ) Freshwater bacteria show no tendency to degrade Squoxin i n short term t e s t s . 4. ) Squoxin i n h i b i t s the uptake and u t i l i z a t i o n of glucose by freshwater bacteria i n short term t e s t s . 5. ) Squoxin was less toxic to a l l invertebrates tested than i t i s to the northern squawfish. The most sensitive species tested was the bl a c k f l y larva, Simulium canadense which had an LC50 value of 60 jag/1 i n 48 hours. It was proposed that t o x i c i t y of Squoxin to invertebrates i s inversely proportional to adaptations to habitats ex h i b i t i n g low lev e l s of oxygen a v a i l a b i l i t y . 6. ) Only small amounts of Squoxin were transferred to organisms feeding on contaminated food items when compared 82 to the dose obtained from a contact exposure. 7. ) Invertebrates exhibited an a b i l i t y to excrete Squoxin given time and an uncontaminated environment. Daphnia pulex excreted over 90 percent of the toxicant i n 96 hours while H y a l e l l a azteca required 275 hours to reach s i m i l a r l e v e l s . 8. ) Microcosm data showed Squoxin residues to adsorb to organic material. This reaction reversed i t s e l f , and the compound became increasingly soluble throughout the test period. Invertebrates i n the microcosms took up Squoxin read i l y i n the f i r s t 24 hours. Uptake then l e v e l l e d o f f and tissue concentration of Squoxin increased only at a very slow rate during the remainder of the t e s t . 9. ) As a r e s u l t of Squoxin 1s water s o l u b i l i t y , low p a r t i t i o n c o e f f i c i e n t , and the a b i l i t y of organisms to ex-crete i t , the p i s c i c i d e would not appear to be b i o l o g i c a l l y magnified to a s i g n i f i c a n t degree i n aquatic ecosystems. 4.2 PARTITIONING OF SQUOXIN Figure 22 represents an attempt to quantify the amount of Squoxin residues present i n the component of an hypothetical aquatic ecosystem 24 hours a f t e r a 500 ug/l treatment. Most of the figures were derived indepen-dently from microcosm data as well as from treatments p r i o r to monitoring the rate of excretion by invertebrates. The Squoxin residues present i n each compartment are therefore more ind i c a t i v e of the proportion present rather than an absolute value. 83 FIGURE 22. P a r t i t i o n i n g of 500 ug/l Squoxin residues i n a hypothetical l e n t i c ecosystem 2 4 hours after treatment. Numbers indicate ug Squoxin i n each environmental component except A.) tissue concentration i n trout (mg/kg) a f t e r a 50 ug/l treatment for 24 hours (Burnard and Te r r i e r e , 1974) and B.) tissue concentration (mg/kg) i n squawfish a f t e r a le t h a l dose (Ibid., 1974). 85 The values presented for trout and squawfish are tissue concentrations derived from a 24 hour treatment of 50 ug/l (Burnard and Te r r i e r e , 1974), although a l l the squawfish died within 6-12 hours a f t e r i n i t i a t i o n of the Squoxin treatment. The assessment of p a r t i t i o n i n g assumes that the treatment i s rapidly and evenly mixed throughout the system and that the treatment was applied as an ethanolic solution of 1 mg Squoxin/ml 95 percent ethanol. The number of organisms/liter were as follows: Daphnia pulex, 50; Hyalella azteca, 25; and Chaoborus t r i v i t t a t u s , 22 0. The large number of Chaoborus/liter stems from a treatment p r i o r to measuring i t s excretion rate. Unlike Hyalella and Daphnia, the treatment took place i n clean water without the addition of phytoplankton or sediment as competing substrates for adsorption of residues. 5 The organic suspended s o l i d s consisted of 1.0 X 10 C h l o r e l l a c e l l s / m l , but t h i s was augmented by inanimate fine sediment p a r t i c l e s which remained i n suspension aft e r addi-t i o n to a microcosm. There was 25 ml of wet sediment/liter i n the microcosm. Figure 22 represents the p a r t i t i o n i n g of Squoxin in a model ecosystem predicated on the foregoing assumptions and conditions. A change i n either the composition of the environment or the formulation of the p i s c i c i d e would e f f e c t i t s apportionment among ecosystem components. The value of t h i s figure i s as a guide to the environmental pathways and pools of Squoxin residues i n l e n t i c ecosystems. 86 LITERATURE CITED American Public Health Association. 1971. Standard Methods  for the Examination of Water and Wastewater. 13th E d i t i o n . Washington, D.C. pp. 874. Applegate, V.C., J.H. Howell, J.W. Moffett, B.G.H. Johnson, and M. Smith. 1961. Use of 3-triflourmethyl-4-nitrophenol as a s e l e c t i v e sea lamprey larvacide. Great Lakes Fishery Commission. Technical Report #1. pp. 35. Berg, K. and P.M. Jonasson. 1965. Oxygen consumption of profundal lake animals at low oxygen content of the water. Hydrobiologia 26: 131-143. Bray, G.A. 1960. A simple e f f i c i e n t l i q u i d s c i n t i l l a t o r for counting aqueous solutions i n a l i q u i d s c i n t i l -l a t i o n counter. A n a l y t i c a l Biochem. 1: 279-285. Brusven, M.A. and C. MacPhee. 1974. An evaluation of Squoxin on insect d r i f t . Trans. Am. Fis h . Soc. 103 (2) : 362-365. Burnard, R.J. and L.C. Te r r i e r e . 1974. The uptake of the se l e c t i v e p i s c i c i d e , Squoxin, (1,1'-methylenedi-2-naphthol) by the rainbow trout and squawfish. Oregon A g r i c u l t u r a l Experimental Station Technical Paper # 3791. Burnard, R.J., U. Kiigemagi and L.C. Te r r i e r e . 1974a. The s t a b i l i t y of Squoxin i n water. Unpublished report. Burnard, R.J., U. Kiigemagi and L.C. Terrier e . 1974b. A n a l y t i c a l methods for the detection of the p i s c i c i d e Squoxin (1,1 1-methylenedi-2-naphthol) i n water and f i s h . Unpublished report. Cartwright, J.W. 1973. Experimental use of Squoxin as "Scavenger 300" or "Sonar 300" (1,1'-methylenedi-2- naphthol, monosodium salt) i n a small B r i t i s h Columbia lake. Mimeo manuscript, B r i t i s h Columbia Fish and W i l d l i f e Branch. V i c t o r i a , B. C. Clemens, W.A. and J.A. Munro. 1934. The food of the squaw-f i s h . Fish. Res. Board Can. Pac. Progr. Rep. 19: 3- 4. Crowley, G.J. 19 74. A review of the l i t e r a t u r e on the use of Squoxin i n f i s h e r i e s . USNTIS, PB no. 235 456. 29 pp. 87 Derr, S.K. and M.J. Zabik. 1972. B i o l o g i c a l l y active compounds in the aquatic environment: the uptake and d i s t r i b u t i o n of [1,l-dichloro-2,2-bis(p-chlorophenyl)ethylene], DDE by Chironomus tentans Fabricius (Diptera : Chironomidae). Trans. Am. Fish. Soc. 101: 323-9. Dinges, R. 1973. Ecology of Daphnia i n S t a b i l i z a t i o n Ponds. Texas State Dept. of Health, Div i s i o n of Wastewater Technology and Surveillance. pp. 155. Epifanio, C.E. 1973. D i e l d r i n uptake by larvae of the crab Leptodius floridanus. Marine B i o l . 19: 320-2. Finney, D.J. 1971. Probit Analysis. Cambridge University Press. 3rd e d i t i o n . pp. 333. Foerster, R.E. and W.E. Ricker. 1942. The e f f e c t of reduc-ti o n of predaceous f i s h on su r v i v a l of young sockeye salmon at Cultus Lake. J. Fish. Res. Board Can. 5(4): 315-336. Gaufin, A.R. 19 73. Water Quality Requirements of Aquatic Insects. Project 18050 FLS EPA. Washington, D.C. EPA-660/3-73-004. pp. 89. Hamelink, J.L., R.C. Waybrant, and R.C. B a l l . 1971. A proposal: exchange e q u i l i b r i a control the degree chlorinated hydrocarbons are b i o l o g i c a l l y magni-f i e d i n l e n t i c environments. Trans. Am. Fish. Soc. 100: 207-14. Hamilton, J.A.R., L.O. Rothfus, M.W. Erho, and J.D. Remington. 1970. Use of a hydroelectric reservoir for the rearing of coho salmon (Oncorhynchus kisutch). Washington State Dept. of F i s h e r i e s , Research B u l l . #9. pp. 65. Hargrave, B.T. 1970. The u t i l i z a t i o n of benthic microflora by Hyalella azteca (Amphipoda). Jour. Animal Ecol. 39: 427-37. Johnson, J.M. 1972. Tolerance of juvenile sockeye salmon and zooplankton to the sele c t i v e squawfish t o x i -cant 1,1 1-methylenedi-2-naphthol. Prog. Fish Cult. 34 (3) : 122-5. Juday, C. 1908. Some aquatic invertebrates that l i v e under anaerobic conditions. Trans. Wis. Acad. S c i . Arts Lett. 16: 10-16. 88 Keating, J.F. 1972. Results of 1971 f i e l d studies of Squoxin (Scavenger 300, Sonar 300), a p i s c i c i d e s e l e c t i v e to squawfish and related minnows i n Idaho. Idaho Fish and Game Dept. mimeo report. Boise, Idaho. Keating, J.F., U. Kiigemagi, L.C. Terriere and R.L. Swan. 19 72. Recent developments in the t e s t i n g of Squoxin, a p i s c i c i d e s e l e c t i v e l y l e t h a l to squaw-f i s h . Proc. 52nd Annual Conf. West. Assoc. State Game and Fish Commissioners, Portland, Oregon. Kenaga, E.E. 19 72. Guidelines for environmental studies of p e s t i c i d e s : determination of bioconcentration p o t e n t i a l . Residue Reviews 44: 74-113. Kiigemagi, U., R.J. Burnard and L.C. T e r r i e r e . 1975. A n a l y t i c a l methods for the detection of the p i s c i c i d e 1,1 1-methylenedi-2-naphthol (Squoxin) i n f i s h and water. Jour. Agric. Food Chem. 2 3(4): 717-20. Kononova, M.M. 1966. S o i l Organic Matter, 2nd e d i t i o n , Pergamon Press, Oxford. Kryutchkova, N.M. and V. Sladecek. 1969. Quantitative relations of the feeding and growth of Daphnia  pulex obtusa (Kurz) Scourfield. Hydrobiologia 33 (1) : 47-64. Lennon, R.E. 1970. Control of freshwater f i s h with chemicals. Proc. of the 4th Vertebrate Pest Conference. pp. 124-137. McMahon, J.W. and F.H. Rigler. 1963. Mechanisms regulating the feeding rate of Daphnia magna Straus. Can. Jour. Zool. 41: 321-32. MacPhee, C. and G.C. Bailey. 197 3. T o x i c i t y of Squoxin to f i s h species other than squawfish. Idaho Fish and Game Dept. Federal Aid i n Fish and W i l d l i f e Restoration Job Progress Report, Project F-64-R-2. MacPhee, C. and F. Cheng. 1974. Factors a f f e c t i n g degrada-ti o n of Squoxin and t o x i c i t y of Squoxin to six species of f i s h . Idaho Fish and Game Dept. Federal Aid i n Fish and W i l d l i f e Restoration, Project F-64-R-3. MacPhee, C. and R. Ruelle. 1969. A chemical s e l e c t i v e l y l e t h a l to squawfish (Ptychocheilus oregonensis and P. umpquae). Trans. Am. F i s h . Soc. 4: 676-84. 89 Matsuraura, F. 19 75. Toxicology of Insecticides. Plenum Press, New York. pp. 5 03. Menzer, R.E. 1973. B i o l o g i c a l oxidation and conjugation of pesticide chemicals. Residue Reviews 48: 79-116. Metcalf, R.L., J.R. Sanborn, P. Lu, and D. Nye. 1975. Laboratory model ecosystem studies of the degrada-t i o n and fate of radio-labeled t r i - , t e t r a - , and pentachlorobiphenyl compared with DDE. Archives Environ. Contam. Toxicol. 3(2): 151-65. Nakaue, H.S., Caldwell R.S., and Buhler D.R. 1972. B i s -phenols: uncouplers of phosphorylating r e s p i r a t i o n . Biochem. Pharmacol. 21(16): 2273-7. Northcote, T.G. 1964. Use of a high-frequency echo sounder to record d i s t r i b u t i o n and migration of Chaoborus larvae. Limnol. and Oceanog. 9: 87-91. Peterson, D.G. and L.S. Wolfe. 1956. The biology and control of black f l i e s (Diptera: Simulidae) i n Canada. Proc. 10th Int. Congr. Ent., Montreal, Canada. 3: 551-64. Ricker, W.E. 1941. The consumption of young sockeye salmon by predaceous f i s h . J. Fish. Res. Board Can. 5(3): 293-313. Rosenberg, D.M. 1975. Food chain concentration of c h l o r i n -ated hydrocarbon pesticides i n invertebrate commun-i t i e s : a re-evaluation. Quaestiones Entomologicae 11: 97-110. Skytte Jensen, B. 1964. Solvent extraction of metal chelates. I I I . A potentiometric investigation of a l k a l i - i o n extraction by bis(2-hydroxy-l-naphthyl) methane. Acta Chem. Scand. 18(3): 739-49. 14 Sodergren, A. 196 8. Uptake and accumulation of C-DDT by C h l o r e l l a sp. (Chlorophyceae). Oikos 19: 126-38. Stahl, J.B. 1966. The ecology of Chaoborus i n Myers Lake, Indiana. Limnol. Oceanog. 11: 177-83. Staley, G.S. and K.J. H a l l . 1974. Squoxin: a review of research and applications. Fisheries Technical C i r c u l a r #13. B r i t i s h Columbia F i s h and W i l d l i f e Branch. V i c t o r i a , B.C. pp. 13. 90 Steigenberger, L.W. and P.A. Larkin. 1974. Feeding a c t i v i t y and rates of digestion of northern squawfish (Ptychocheilus oregonensis). J. Fi s h . Res. Board Can. 31: 411-20. Terr i e r e , L.C. and R.J. Burnard. 1975. Uptake, tissue d i s t r i b u t i o n , and clearance of the selec t i v e p i s c i c i d e 1,1 1-methylenedi-2-naphthol (Squoxin) by the rainbow trout and the squawfish. Jour. Agric. Food Chem. 23(4): 714-17. Terriere, L . C , U. Kiigemagi and E. Chan. 1970. The s t a b i l i t y of 1,1 1-methylenedi-2-naphthol (Squaxon) i n water. Oregon State University Dept. of Agriculture Chem. Unpublished report. Terriere, L.C, U. Kiigemagi, A.R. Gerlach and R.L. Borovicka. 1966. The persistence of toxaphene i n lake water and i t s uptake by aquatic plants and animals. J. Agric. and Food Chem. 14: 66-9. Thompson, R.B. 1959. Food of the squawfish, Ptychocheilus  oregonensis (Richardson) of the lower Columbia River. U.S. Dept. Interior, Fish and W i l d l i f e Service, Fishery B u l l e t i n No. 158: 43-58. Wagner, J.G. 1975. Fundamentals of C l i n i c a l Pharmaco- ki n e t i c s . Drug Intelligence Publications, Hamilton, I l l i n o i s , pp. 461. Ware, G.W. and Roan, C C 1970. Interaction of pesticides with aquatic microorganisms and plankton. Residue Reviews 33: 15-45. Wigglesworth, V.B. 1972. The Pr i n c i p l e s of Insect  Physiology. 7th e d i t i o n , Chapman and H a l l , London. pp. 827. Woodwell, G.M., C F . Wurster, J r . , and P.A. Isaacson. 1967. DDT residues i n an east coast estuary: a case for b i o l o g i c a l concentration of a persistant p e s t i c i d e . Science 156: 821-4. 91 APPENDIX 1. Comparison of Rf values obtained by thin-layer chromato-graphy of synthesized l^c-Squoxin with commercially prepared Squoxin and 2-napthol. S i l i c a gel thin-layer plates containing flourescent indicator (Eastman Kodak #13181) activated at 105°C for 30 minutes. Plates developed i n the solvent system, trichloromethane : methyl ethyl ketone : diethylamine, 2 0 : 10 : 1, for 1 hour 45 minutes. Sample Rf X 100 Squoxin (Aldrich Chem. Co.) 29, 31 C-Squoxin (product from synthesis #1) 30 C-Squoxin (product from synthesis #2) 28 2-Naphthol 52, 49 92 APPENDIX 2 Co.l©rimetric Method for the Measurement of Squoxin* Reagents: 1. Squoxin standard - Stock solution, lmg/ml i n 95% ethanol. Store in a r e f r i g e r a t o r . This solution i s stable for several weeks. Use for working standards by d i l u t i n g with d i s t i l l e d water to provide solutions containing 10 ug/ml. 2. Diazo blue reagent (van Asperen, 1962) - This i s prepared by mixing two solutions, one containing 1% diazo blue and the other containing 5% sodium l a u r y l s u l f a t e . Two parts of the diazo blue solution are mixed with 5 parts of sodium l a u r y l s u l f a t e . This reagent i s unstable and should be prepared every two days and stored i n a r e f r i g e r a t o r . 3. Buffer- pH 8.0 phosphate. Dissolve 27.8 g NaH2P04 i n 1 l i t e r of water (solution A) and 53.65 g Na2HPC>4 . 7 ,H20 per l i t e r (solution B). Mix 5.3 ml of solution A and 94.7 ml of solution B and d i l u t e to 200 ml to obtain a pH 8.0 solution. 4. Carbon te t r a c h l o r i d e . 5. HC1, IN Equipment and supplies 1. Spectrophotometer - Any instrument capable of readings i n the 552 mu range i s suitable. 2. Glassware - Ordinary laboratory beakers, f l a s k s , funnels, cylinders and pipettes. Procedure 1. C a l i b r a t i o n curve - Prepare standards containing 2-12 ug Squoxin. Add buffer to make a t o t a l volume of 4.2 ml, then add 0.8 ml of the diazo blue reagent, measure the color within 10 minutes. Prepare a standard curve from the data. 2. Analysis of samples - A 500 ml sample w i l l provide a s e n s i t i v i t y of about two ppb. Add 1 ml of 1 N HC1 to insure a c i d i t y , then shake with 25 ml of CCI4. Remove the lower layer and re-extract with a second 25 ml portion and combine the extracts. Take a suitable aliquot (5-2 0 mis) for analysis and remove the CCI4 by evaporation under an a i r 93 j e t . Add 0.2 ml ethanol, 4.0 mis of buffer, and 0.8 ml of diazo blue reagent. Measure the color as above and compare with the standard curve. *Terriere et a l . , 1970. 

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