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Seasonal changes in hydrographic and chemical properties of Indian Arm and their effect on the calanoid… Whitfield, Paul Harold 1974

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SEASONAL CHANGES IN HYDROGRAPHIC AND CHEMICAL PROPERTIES OF INDIAN ARM AND THEIR EFFECT ON THE CALANOID COPEPOD EUCHAETA JAPONICA bby Paul Harold W h i t f i e l d B.Sc. (Honours), University of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology and I n s t i t u t e of Oceanography We accept t h i s thesis as conforming to the required sijajndard THE UNIVERSrTY OF BRITISH COLUMBIA Jul y , 1974 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 degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that the 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 tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that 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 thout my w r i t t e n p e r m i s s i o n . Department o f Z 0 0 L 0 G Y The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date JULY 17, 1974. i ABSTRACT This study examines seasonal changes i n the r e l a t i o n s h i p between a test organism and changes i n the hydrographic and chemical properties of Indian Arm, a coastal f j o r d . There i s a close r e l a t i o n s h i p between changes i n the hydrographic properties of the water and changes i n the metal com-plexing a b i l i t y of water i n the i n l e t , as determined with the test organism. The r e l a t i o n s h i p between the organism and the a v a i l a b i l i t y of metals changes with time; the complexing a b i l i t y of natural water increases at the time of the major i n t r u s i o n of water from the S t r a i t of Georgia into Indian Arm, and then decreases. The addition of a v a r i e t y of metals under experi-mental conditions a f f e c t s the r e l a t i o n s h i p between the organism and the complexing a b i l i t y of the water. Ad d i t i o n a l studies examine the e f f e c t of material extracted from sediment samples on the toxic e f f e c t of copper enrichment. The a b i l i t y of the extracted material to reduce the t o x i c e f f e c t changes and i s rel a t e d to the seasonal produ c t i v i t y i n the surface waters of the i n l e t . i i TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES i v LIST OF FIGURES v ACKNOWLEDGEMENTS v i i INTRODUCTION 1 ( i ) The chemistry of the complexing of metals i n seawater 2 ( i i ) Sediment extracts 6 ( i i i ) Measuring complexing a b i l i t y of natural water 6 MATERIALS AND METHODS ...... 9 (i) F i e l d studies 9 ( i i ) Analysis of f i e l d samples 14 ( i i i ) Laboratory studies 17 (iv) S t a t i s t i c a l a n alysis 20 RESULTS 24 (i) Hydrographic properties 24 ( i i ) Chemical properties 24 ( i i i ) Abundance of l i f e h i s t o r y stages 33 (iv) Laboratory studies 35 (a) Copper stressed s e r i e s 35 (b) Sediment extract s e r i e s 40 (c) U l t r a - v i o l e t s e r i e s 40 (d) Metal additions 46 (e) Measurements on water before and a f t e r u l t r a -v i o l e t treatment 46 (f) Chloroform extractions 48 i i i Page DISCUSSION 52 ( i ) F i e l d data 52 (ii ) . L a b o r a t o r y data 56 SUMMARY 70 REFERENCES 72 APPENDIX 77 i v LIST OF TABLES Table Page 1. Sampling depths and sampling dates f o r the c o l l e c t i o n of information i n the f ield,aandvjwater na_nd animals,"fori ..bc~ laboratory experiments 12 2. Si g n i f i c a n c e and sign of the c o r r e l a t i o n c o e f f i c i e n t between prefeeding naupliar stages and the other properties measure i n the f i e l d 38 3. Percent s u r v i v a l values i n the u l t r a - v i o l e t s e r i e s , with and without metal additions. Values are the percent of approximately s i x t y animals that survive u n t i l the t h i r d naupliar stage. Also shown are the EDTA equivalences which were determined, and the approximate slope of the l i n e which r e l a t e s s u r v i v a l of the organism to the EDTA concentration 44 4.~ Levels (concentrations) of various chemical constituents of the culture water, before and a f t e r u l t r a - v i o l e t i r r a d i a t i o n 49 55. Results from the chloroform extraction experiments, and the EDTA equivalence determined with the bioassay 51 V LIST OF FIGURES Figure Page 1. Generalized chemical reactions which are believed to influence the chemistry of t r a n s i t i o n metals i n seawater 4 2. Indian Arm showing s t a t i o n locations 10 3. Diagrammatic representation of the u l t r a - v i o l e t appa-ratus 21 4. Percent s u r v i v a l from the egg through to the t h i r d nauplius as a function of the duration of treatment of the culture water with u l t r a - v i o l e t l i g h t 22 5. D i s t r i b u t i o n of temperature through the water column at Ind-2 during the study period 25 6. D i s t r i b u t i o n of s a l i n i t y through the water column at Ind-2 during the study period 26 7. D i s t r i b u t i o n of density through the water column at Ind-2 during the study period 27 8. DiDistributionfof'dissolvedyoxygehhthroiighhtheawater column at Ind-2 during the f i e l d sampling period 28 9. Temperature s a l i n i t y envelopes of water below 10 meters f o r the months sampled during the period of the study 29 10. D i s t r i b u t i o n of dissolved manganese through the water column at Ind-2 during the f i e l d sampling period 31 11. D i s t r i b u t i o n of dissolved copper through the water column at Ind-2 during the f i e l d sampling period 32 12. D i s t r i b u t i o n and abundance of prefeeding naupliar stages through the water column at Ind-2 during the f i e l d sampling period 34 13. D i s t r i b u t i o n and abundance of t o t a l naupliar stages through the water column at Ind-2 during the f i e l d sampling period 36 14. D i s t r i b u t i o n of (a) prefeeding, and (b) t o t a l naupliar stages through the length of Indian Arm i n February, 1973 37 v i Figure Page 15. Tree printed from c o r r e l a t i o n matrix (scaled 0-100). Clustering by average distance method 39 16. The percent s u r v i v a l of the organism from the egg through the t h i r d nauplius plotted against the con-centration of EDTA which was added to water with 5.4 iig/1 of added copper, and the method of obtaining an EDTA equivalence f o r the toxic e f f e c t of the copper enrichment 41 17. EDTA equivalences of the sediment extracts as a function of time, and the EDTA equivalence of the t o x i c e f f e c t of the copper enrichment as a function of time 42 18. The percent s u r v i v a l of the organism from the egg through the t h i r d nauplius as a function of the EDTA concentration i n water which has been treated with u l t r a - v i o l e t l i g h t , and the method for obtaining an EDTA equivalence f o r untreated water 43 19. EDTA equivalence as determined i n u l t r a - v i o l e t treated water as a function of time 47 20. The change i n percent s u r v i v a l plotted against the change i n the organic carbon content of the water which resulted from the u l t r a - v i o l e t treatment 50 21. EDTA equivalence of the sediment extracts corrected for v a r i a t i o n i n the toxi c e f f e c t of the copper enrichment, as a function of time 60 22. EDTA equivalence of the sediment extracts (trans-formed means) as a function of time and net production subjected to a four month s h i f t 62 v i i ACKNOWLEDGEMENTS I would l i k e to o f f e r sincere thanks to a l l those people who provided t h e i r time and expertise to various aspects of t h i s study. Dr. A.G. Lewis supervised t h i s study, and provided guidance and encouragement during i t s course. I would also l i k e to thank my research committee members, Dr. P.A. Dehnel, Dr. E.V. G r i l l , and Dr. M.A. Barnes f o r t h e i r help during the preparation of t h i s manuscript. Special thanks must go to several people who contributed t h e i r time i n the c o l l e c t i o n and analysis of f i e l d samples; Mr. G.A. Gardner, Mr. A. Ramnarine, and Mr. F.A. Whitney. I also would l i k e to thank the Members of the I n s t i t u t e of Oceano-graphy, and the O f f i c e r s and Crew of the C.S.S. Vector f o r t h e i r help i n the c o l l e c t i o n of samples at sea. I would l i k e to thank the International Copper Research Association, Inc. f o r providing a fellowship f o r my support. INTRODUCTION The a b i l i t y of n a t u r a l l y occuring organic material to reduce the toxi c e f f e c t s of some metals has been suggested by several authors (Barber and Ryther, 1969; Barber et a l . , 1971; Tabata and Nishikawa, 1969; Lewis et a l . , 1972; 1973). Further, i t has been suggested that organic material i s responsible f o r maintaining a c e r t a i n l e v e l of metal i n s o l u t i o n and preventing i t from being removed by p r e c i p i t a t i o n (Johnson, 1964). Several authors have suggested that the predominant form of c e r t a i n metals i n natural waters may be an organic complex (Slowey et a l . , 1967; Williams, 1968; Slowey and Hood, 1971) although there i s considerable disagreement ( Z i r i n o and Healy, 1970; Z i r i n o and Yamamoto, 1972). I t has been suggested that the action of some organic material present i n natural waters a l t e r s the a v a i l a b i l i t y of a metal or metals (Barber and Ryther, 1969; Steeman-Nielsen and Wuim-Anderson, 1970; Lewis et a l . , 1971; 1972; 1973). Barber and Ryther (1969) suggested that the conditioning of newly upwelled water f o r phytoplankton growth occurs as the r e s u l t of increases i n the organic content of that water. Lewis et a l . (1971) showed that the addition of a synthetic chelating agent increased s u r v i v a l of a test organism during c e r t a i n periods of the year i n nearshore waters and more frequently i n near-oceanic waters, which suggested a r e l a t i o n s h i p s i m i l a r to that described by Barber and Ryther (1969). Lewis et a l . (1972; 1973) showed that c e r t a i n n a t u r a l l y occurring organics could act to reduce the t o x i c e f f e c t of increased copper concentration. Davey et a l . (1973), used a bioassay and measured the a b i l i t y of natural waters to complex amounts of copper above that which was already present. There are c e r t a i n drawbacks to t h i s type of work, however, as 2 copper w i l l displace many other metals from complexes with organic molecules, obscuring the f i n e d e t a i l of the system and possibly giving erroneously high values. High values may r e s u l t from the exchange of copper f o r the complexed metal. I f the metal which i s displaced has l i t t l e or no b i o l o g i c a l e f f e c t , i t w i l l not be adequately measured by the bioassay. This s i t u a t i o n also holds f o r chemical analyses. Although a l l of these studies i n d i c a t e that organic material i s important to the organisms because of i t s a b i l i t y to a l t e r the a v a i l a b i l i t y of metals, they f a i l to accomplish two things. F i r s t , they do not work at natural l e v e l s of eit h e r the metal or the organic material, which i s neces-sary to prevent a l t e r a t i o n s of the e f f e c t on the organism which may occur as the r e s u l t of metal i n t e r a c t i o n s and concentration e f f e c t s . Second, they do not in d i c a t e those i n t e r a c t i o n s which may a f f e c t the equilibrium system i n natural waters. The present study demonstrates some of the features of the r e l a t i o n s h i p between an organism and the metals which are present i n nat u r a l waters, and shows that organic material can a l t e r t h i s r e l a t i o n s h i p . ( i ) The chemistry of the complexing of metals i n seawater In the preceding paragraphs, the term metal a v a i l a b i l i t y has been used. A v a i l a b i l i t y , i n the context of t h i s t h e s i s , indicates the condition or q u a l i t y of a metal being present i n a form which the organism recognizes as that metal. This i n f e r s that metals can e x i s t i n forms which are not recognized by the organism, so that the organism i s not dependent upon the t o t a l metal present but only upon that f r a c t i o n which i s a v a i l a b l e to i t (Lewis and W h i t f i e l d , 1974). In the natural environment, chemical reactions of the general forms 3 shown i n Figure 1 are thought to occur. Each of these has some importance i n the chemistry of transition metals i n the natural environment (Lewis and Whitfield, 1974). One of the problems which is at present unanswered i s the relat ive biological importance of each of these components. Essentially every biogenous organic molecule has the potential to act as an electron donor molecule, some having stronger a f f i n i t i e s for specific metals. Elec-tron donors i n organic molecules include the sulphydryl, carboxyl, carbonyl, and amino groups. The formation constant (k^) of stable complexes of metals with organic molecules i s generally much greater than that for inorganic ligands (Albert, 1950; 1952). Although the concentration of inorganic ligands far exceeds that of the organic ligands, the difference i n the s t a b i l i t y of the formation constants i s suff ic ient ly great to more than counteract the concentration difference (Spencer, 1958; Marchand, 1974). The term complex describes the end product of a physico-chemical interaction between an electron donor which forms a ligand for an electron acceptor, which is the heavy metal. There are problems associated with the use of the term complex with respect to i t s biological implications because an organic molecule may affect the a v a i l a b i l i t y of a metal to an organism i n three ways: (1) For metals which tend to form precipitates in water (e .g . , iron and manganese) the organic molecule may act to increase the metal's so lubi l i ty by binding the metal i n a complex, hence increasing the total amount of metal which can remain in solution. As the level of free, metal i n solution de-creases, the complex w i l l become less stable, releasing the metal from the complex as the result of the chemical 4 Figure 1. Generalized chemical reactions which are believed to influence the chemistry of transition metals i n seawater. Me m - a metal with an ionic charge of+-m, L q - an electron donor (ligand) with an ionic charge of - q , B - sol id part icle (e .g . , clay par t i c le ) , and A - an exchangeable metal. .. +m Me + n f ^ -- Me(L)+m' -nq inorganic complex n fl +m Me + [MeL n] + m" -nq complexation M e + m + Bg .— - Me(B)+ m sorption Me + m + - Me(B)+sm A+m + A ion exchange P3 I 5 equilibrium which e x i s t s between the forms of the metal. In t h i s way the organic material i s acting as an agent which buffers the l e v e l of the metal which i s present i n a form other than an organic complex. (2) With some of the more toxi c metals (e.g., copper and zinc) the binding of the metal by the organic molecule a l t e r s the chemical properties of the metal to such a degree that, i n the form of an organic complex, the metal w i l l not have the same b i o l o g i c a l e f f e c t as the i o n i c form (Avakyan, 1971; Avakyan and Rabotnova, 1971). A w e l l documented example of t h i s type of mechanism i n b i o l o g i c a l systems i s the e f f e c t of metallothioneins. These proteins are produced i n the l i v e r of vertebrates and act to make a group of metals (cadmium, copper, mercury and zinc) non-toxic to organisms even when the metals are present i n very high concentrations (Olafson and Thompson, 1974). (3) The e f f e c t of the complexation of a metal may also be to change the chemical properties of the metal i n such a manner that an organism may make d i r e c t use of the metal, such as allowing the metal to pass through a c e l l membrane when i n the form.of an organic complex but not when i n an i o n i c form. Thus, i t i s possible that the organism requires some metals which i t can only u t i l i z e when i n the form of an organic complex. Organisms have been shown to be dependent upon geochemical processes to varying degrees (Barber and Ryther, 1969; Phelps et a l . , 1969; Lewis et a l . , 1971). The present study examines some geochemical r e l a t i o n s h i p s of 6 b i o l o g i c a l importance, and r e l a t e s them to hydrographic events. ( i i ) Sediment extracts Although the complexing a b i l i t y of the water i s the c o n t r o l l i n g f a c t o r , the source of organic complexing agents i n the water i s important. Water soluble material extracted from sediments has been shown to reduce the toxic e f f e c t of a copper enrichment and has been suggested to be n a t u r a l l y d i f f u s e d into the water column (Lewis et a l . , 1973). The present study monitored changes i n the a b i l i t y of sediment extract to reduce copper t o x i c i t y throughout one year, examining seasonal changes and associating them with events i n the rest of the water column. The chemical composition of the material which i s extracted i s not known but i t may be s i m i l a r to humic acids which have previously beenaextracted feromnmaririemsedimeht8s (Rashid, 1972). Trask (1939) and Gross et a l . (1972) have suggested that organic material i n sediments i s from three sources: terrigenous input, phytoplankton production, and material produced by b a c t e r i a l a c t i v i t y on eit h e r or both of the preceeding two sources. ( i i i ) Measuring complexing a b i l i t y of natural water In measuring the a b i l i t y of a sediment extract to reduce copper t o x i c i t y , both the copper stress and the a b i l i t y of the sediment extracts to reduce that stress are determined as the r e s u l t of additions to the culture water. This method cannot be used i n determining the complexing a b i l i t y of organic material i n natural water. U l t r a - v i o l e t i r r a d i a t i o n of seawater, however, causes photo-oxidation of the organic material (Beattie et a l . , 1961; Armstrong et a l . , 1966). This treatment r e s u l t s i n the elimination of organic-metal complexes ( S t r i c k l a n d , 1972). As the r e s u l t 7 of the destruction of organic material, the a v a i l a b i l i t y of the metals w i l l change (see page 3). Increases i n the concentration of the i o n i c form of p a r t i c u l a r metals can be predicted to cause a decrease i n the s u r v i v a l of the t e s t organism (Lewis et a l . , 1972). EDTA (ethylenediaminetetraacetic acid) w i l l , i f the organic material acts to complex metals i n natural water, be able to replace the active component of the organic material which has been destroyed.. The addition of EDTA i n s u f f i c i e n t amounts should restore the s u r v i v a l of the organism to that attained i n natural seawater. The amount of EDTA required to restore t h i s s u r v i v a l i s a measure of the r e l a t i o n s h i p between the test organism and the metals present i n the water. A d d i t i o n a l evidence about t h i s r e l a t i o n s h i p can be found from experiments which test the e f f e c t of increases i n the concentration of metals on the r e l a t i o n s h i p between the s u r v i v a l of the organism and the concentration of EDTA. The addition of small amounts of metals which have a high for the formation of a complex with EDTA can be predicted to a l t e r the r e l a t i o n s h i p between the concentration of EDTA and the s u r v i v a l of the organism. Large amounts of metals with lower k^ should also be able to a l t e r t h i s r e l a t i o n s h i p . Although the nature of the natural complexing agents i s not the subject of t h i s study, one attempt was made to obtain an estimate of the molecular s i z e of the material. Large molecular weight organic material has been shown to be able to reduce the tox i c e f f e c t of copper enrichment (Lewis et a l . , 1972). Khailov and Finenko (1970) used a chloroform extraction to concentrate large molecular weight organic material f or chemical analyses. Chloroform extracts have been used as an i n d i c a t i o n of organic complexed copper i n waters from the Gulf of Mexico (Slowey et a l . , 1967). If large molecular weight organic material was present i n s u f f i c i e n t 8 amounts, some indication of i t s a b i l i t y to bind metals could be obtained from differences i n the metal content of water samples as a result of chloroform extraction. In summary, this study attempts to assess further the complex relationship between an organism and some of the microchemical aspects of i t s environment. The measuring and monitoring of certain geochemical features of the natural environment are intended to provide an indication of effect on the ecology of an organism. 9 MATERIALS AND METHODS (i) F i e l d studies This study was conducted i n Indian Arm, a f j o r d type i n l e t located 15 km to the northwest of Vancouver (Figure 2). This i n l e t has been described i n r e l a t i v e l y great d e t a i l by Gilmartin (1962). The t o t a l length of Indian Arm i s 22 km, with an average width of 1 km (Gilmartin, 1962). In common with most fjords i t has a deep basin occupying a large portion of i t s length. This basin i s bounded at the south by a s i l l 26 meters deep and at the north by the d e l t a of the Indian River. Oceanographic properties of Indian Arm are con t r o l l e d by the addition of freshwater from stream and r i v e r runoff, and by the i n t r u s i o n of s a l i n e water from and the loss of brackish water to the S t r a i t of Georgia, by way of Burrard I n l e t (Gilmartin, 1962). A high l e v e l of organic production has been shown for the near-surface waters of Indian Arm (Gilmartin, 1964). Indian Arm i s less affected by freshwater input and consequently the e f f e c t s of entrainment, than high runoff i n l e t s (Pickard, 1961), so that the deep water remains r e l a t i v e l y stable. When compared with those i n l e t s which have a g l a c i a l influence or high r i v e r input, Indian Arm i s less affected by the massive amounts of sedimentary material which may be important i n sorption and ion exchange type reactions. Indian Arm i s i n an area of high mineralization, (personal communication, Anaconda Mines) and has r e l a t i v e l y high concen-tr a t i o n s of trace metals (Erickson, 1973). This i n l e t also sustains a r e l a t i v e l y large population of the test organism Euchaeta japonica (Evans, 1973). B i o l o g i c a l a v a i l a b i l i t y of metals has been studied with chemical techniques (e.g., anodic s t r i p p i n g voltammetry, Erickson, 1973) and bioassay 10 Figure 2. Indian Arm showing station locations. Also indicated are the 50 and 100 fathom bottom contours. 10a 11 techniques (Barber and Ryther, 1969; Barber et a l . , 1971; Lewis et a l . , 1972; Davey et a l . , 1973). The present study u t i l i z e s the bioassay technique with Euchaeta japonica, a holoplanktonic calanoid copepod. This organism i s found throughout the North P a c i f i c Ocean (Davis, 1949). The l i f e cycle consists of an embryo, s i x naupliar, and s i x copepodite stages, the l a s t of which i s the adult. The morphology of the developmental stages has been described by Campbell (1934). The number of eggs produced ranges from eight to twenty-four per c l u s t e r , and the number i s thought to be a function of the food supply of the adult (Evans, 1973). U n t i l the organism reaches the t h i r d naupliar stage i t feeds e n t i r e l y upon the yolk of the egg. Campbell (1934), Pandyan (1971), and Evans (1973) showed that the naupliar and f i r s t copepodite stages of Euchaeta japonica are found i n deep water (greater than 100 meters), while the l a t e r stages are found higher i n the water column. As i t i s the f i r s t three naupliar stages (prefeeding stages) which have been shown to be s e n s i t i v e to changes i n "water q u a l i t y " (Lewis and Ramnarine, 1969), the f i e l d sampling program was biased towards the deeper portion of the water column. The four stations selected f o r t h i s study are Ind-1.5 (49 20.9'N, 122°54.1'W), located midway between the s i l l and the deepest portion of the basin, i n 140 meters of water; Ind-2 (49°23.5'N, 122°52.5'W), located at the southern end of the deepest portion of the basin, i n 223 meters of water; Ind-2.5 (49°24.8'N, 122°52.2'W), located at the northern end of the deepest portion of the basin, i n 225 meters of water; and Ind-3 (49°27.3'N, 122°52.5'W), located j u s t south of the Indian River d e l t a , i n 78 meters of water (Figure 2). At each s t a t i o n , h o r i z o n t a l tows were made at s p e c i f i c depths (Table 1) with Clarke/Bumpus opening-closing samplers (Clarke and Bumpus, 1940). The Clarke/Bumpus sampler allows c o l l e c t i o n of d i s c r e t e 12 Table 1. Sampling depths and sampling dates f o r the c o l l e c t i o n of information i n the f i e l d , and water and animals for laboratory experiments. (a) Sampling Depths Depth (m) Ind-1.5 0 h 10 h b m 20 h 30 h b 50 h b 65 75 100 125 150C 175 200 (b) Sampling Dates September 14, 1972 October 12, 1972 November 7, 1972 December 5, 1972 January 16, 1973 February 13, 1973 March 13, 1973 A p r i l 16, 1973 May 15, 1973 June 16, 1973 STATIONS Ind-2 h h b m h h b h b Ind-2.5 h h b m h h b h b Ind-3 h h b m h h b h b m c h b m c h b m c h b m c h b m c h b m c h b h b h b m c h b m c* h b m c h b h b h b m c h b m c h b m c* h b m c July 18, 1973 August 15, 1973 September 12, 1973 October 17, 1973 November 7, 1973 December 5, 1973 January 22, 1974 February 19, 1974 March 15, 1974 A p r i l 3, 1974 h - indicated hydrographic samples taken (temperature, s a l i n i t y , dissolved oxygen). b - indicates b i o l o g i c a l samples taken with Clarke/Bumpus (C/B) samplers. m - indicates samples taken for analysis of dissolved metals and p a r t i -culate material. c - indicates samples taken for organic carbon determination during the period from May, 1973 to October, 1973. * - water c o l l e c t e d f o r laboratory culture studies. 13 samples and monitoring of the approximate volume of water f i l t e r e d (Yentsch and Duxbury, 1956; Regan, 1963; Tranter and Heron, 1965). The samplers were towed f o r 15 minutes at a speed s u f f i c i e n t to maintain a wire angle of 35±5° to the v e r t i c a l . This speed i s approximately two knots depending upon the wind and t i d e conditions. Samples taken from the Clarke/Bumpus nets were transferred to 4 oz. screw top j a r s i d e n t i f i e d with respect to depth of sample, cruise number, date and sample number. Samples were preserved by the immediate addition of 3-5% (V/V) of borax buffered formalin. Nets were washed between uses to prevent cross-contamination of the samples. Hydrographic data were c o l l e c t e d from a seri e s of depths (Table 1), using NIO sampling b o t t l e s (National I n s t i t u t e of Oceanography, Wormley, U.K.). Oxygen determinations were done on board ship using a modified Winkler technique ( C a r r i t t and Carpenter, 1966). Temperatures were read from reversing, thermometers (±0.01°C) mounted i n frames on the sampling b o t t l e s , and water samples were drawn for s a l i n i t y determination i n the laboratory. Surface samples were obtained with a bucket for a s a l i n i t y sample and f o r determination of temperature (±0.1°C). A bathythermograph (BT) cast was also made at each s t a t i o n to the depth of the deepest hydro-graphic sample. The trace provided from the BT was used to check temperature f l u c t u a t i o n s , and to determine the depth of the thermocline. Samples for chemical analyses were also c o l l e c t e d with the NIO b o t t l e s (because these are f o r the most part p l a s t i c ^ c o n t a m i n a t i o n problems are reduced). A two l i t e r sample was c o l l e c t e d at each of the depths s p e c i f i e d i n Table 1. This sample was then passed through a preweighed 0.45 urn M i l l i p o r e f i l t e r . This pore s i z e was chosen as i t i s the accepted l i m i t between dissolved and p a r t i c u l a t e material (Lewis and Goldberg, 1954). 14 This was done to determine the r e l a t i v e concentration of copper i n par-t i c u l a t e phase as compared to that i n the dissolved phase. The f i l t e r s were immediately placed i n a freezer and returned to the laboratory f o r analysis of weight of p a r t i c u l a t e material and the amount of copper asso-ciated with the p a r t i c u l a t e material. One l i t e r of the f i l t r a t e was c o l l e c t e d into an acid-cleaned polyethylene b o t t l e , which had been rinsed with approximately 300 mlv ; of the f i l t e r e d sample. The sample was then r e f r i g e r a t e d at 8°C u n t i l analysed. Samples for organic carbon determination were drawn from the NIO b o t t l e s into a 200 ml. acid-cleaned, glass stoppered b o t t l e . Samples were drawn from t h i s through a p r e f i r e d (600°C) glass f i b e r f i l t e r , mounted i n a M i l l i p o r e syringe adapter (XX3002500), into a 10 ml. glass syringe. This apparatus was rinsed twice with 7-8 ml. of the sample. A 5 ml. sample was drawn and i n j e c t e d into a 10 ml. pyrex ampoule which had been precombusted at 600° f o r four hours i n a muffle furnace. The ampoules were kept covered with precombusted aluminum f o i l at a l l times to prevent airborn contamination. Three r e p l i c a t e s of each sample were drawnjand stored frozen u n t i l a n a l y sis. ( i i ) Analysis of f i e l d samples Each C/B sample was sorted, using a Wild M5 Stereomicroscope. The naupliar stages of Euchaeta japonica and adult females bearing eggs were removed. These were then i d e n t i f i e d to stage of development and a record of the number of each of the stages was kept.. The sorted animals were then placed i n a 2 dram screwtop v i a l which was kept i n the o r i g i n a l sample j a r . A l l counts were recorded i n i t i a l l y as t o t a l numbers and l a t e r converted to 3 numbers/m with the c a l i b r a t e d C/B flowmeter recordings. Naupliar stages were grouped into prefeeding stages ( f i r s t through t h i r d nauplius), t o t a l 15 naupliar stages, and s i x t h naupliar stage (the numerically predominant stage). These groupings serve to remove some of the v a r i a b i l i t y of numbers due to the length of time spent i n each stage. The s a l i n i t y of samples was measured with an Autolab Inductively Coupled Salinometer (Model 601, MK3). Temperatures were corrected to give the temperature at the depth of the sample. Oxygen readings were converted to give dissolved oxygen concentrations, i n m l . / l . Densities were calcu-l a t e d from the temperature and s a l i n i t y of the sample and expressed as a 3 sigma-t value. Sigma-t = ( s p e c i f i c gravity - 1) x 10 . I n i t i a l l y the conversions of data were done by hand but l a t e r the raw data was converted on a PDP12 computer, with a program written by J.R. Buckley (IOUBC). The f i l t e r s which were used i n the f i e l d f o r trace metal samples were removed from the freezer, dried at 60°C for one hour and weighed. The di f f e r e n c e between the weights of the unused and the used f i l t e r s i s a measure of the p a r t i c u l a t e material which has been removed onto the f i l t e r . Subsequent to the weighing process the f i l t e r was digested i n hot n i t r i c / p e r c h l o r i c acid (1/1, V/V). A f t e r the f i l t e r was digested, hydroxylamine buffer was added to bring the pH to approximately 7.0. Five ml. of 0.0025 M bathocuproine i n ethanol was added and the bathocuproine-copper complex extracted into chloroform. The chloroform layer was then transferred i n t o a 10 cm c e l l and the absorbance at 474 nm measured with a Perkins-Elmer double beam spectrophotometer. Concentrations were determined from a stand-ard*eurve, and expressed as pg Cu/mg p a r t i c u l a t e material. Unused f i l t e r s were treated i n the same manner to determine background l e v e l s of copper. The p r e c i s i o n of the copper determination i s about ±20% of the mean. Seawater samples c o l l e c t e d f o r trace metal analysis were removed from the r e f r i g e r a t o r and placed i n a one l i t e r glass erlenmeyer f l a s k , and 16 the metals extracted into isoamylacetate with diethyldithiocarbamate. A f t e r the layers separated subsequent to the water and reagents being s t i r r e d , the upper layer (isoamylacetate) was removed. This process was repeated three times. The isoamylacetate was removed to a 100 ml. separatory funnel, the metals being back extracted into an aqueous phase with chlorine i n 0.1N HC1. The aqueous phase was c o l l e c t e d into a 25 ml. glass stoppered graduated c y l i n d e r . This was then analysed f o r zinc and manganese (when greater than 10 yg/1) on a Techtron Atomic Absorption Spectrophotometer (no. AA-4). Of the remaining aqueous material, 15 ml. was retained f o r further analysis. TTo the 15 ml. of concentrated metals was added 2 ml. of a 1.0 M t r i s buffer (pH approximately 7.0) and 1 ml. of a 2% s o l u t i o n (by weight) of diethyldithiocarbamate. The metal-diethyl-dithiocarbamates were then extracted into 5 ml. of methylisobutyl ketone (MIBK). The MIBK phase was then subjected to atomic absorption analysis for copper, n i c k e l , cadmium, and manganese (where les s than 10 pg/1). The amount of absorption obtained f o r each metal was compared to a standard curve prepared f o r each metal and a concentration f o r the o r i g i n a l sample determined ( G r i l l , unpublished). This technique has been shown to have a greater than 95% recovery f o r a l l metals analysed ( G r i l l , unpublished). Organic carbon determination was performed by the method of Menzel and Vaccaro (1964), as described i n St r i c k l a n d and Parsons (1972). Values determined by t h i s method are considered accurate to ±0.13 mg/1 ( t h i s being the standard deviation of the mean of 10 r e p l i c a t e samples). This accuracy exceeds that achieved by Slowey and Hood (1971) (±0.44 mg/1) but i s not as accurate as that suggested by St r i c k l a n d and Parsons (1972) (±0.06 mg/1). 17 ( i i i ) Laboratory studies Animals and water f o r the laboratory studies were c o l l e c t e d at Ind-2. The water was c o l l e c t e d with a 96 l i t e r f i b e r g l a s s and l u c i t e sampler (lewis et a l . , 1971). The c o l l e c t i o n depth was 200 meters, although a d d i t i o n a l samples have been c o l l e c t e d from 125 meters. The water was subjected to pressure f i l t r a t i o n through a membrane f i l t e r (0.45 urn mean pore diameter) within 2 hours of c o l l e c t i o n and stored at 8°C i n 23 l i t e r polyethylene containers. • Animals were c o l l e c t e d with a 1 meter diameter c o n i c a l net (mesh aperture approximately 0.7 mm square) towed v e r t i c a l l y to the surface from near the bottom. The plankton was sorted and egg cl u s t e r s as w e l l as females bearing eggs were removed and transported to the labora-tory i n a 3 l i t e r thermos f l a s k f i l l e d with f i l t e r e d seawater c o l l e c t e d from 200 meters. Egg cl u s t e r s were sorted i n the laboratory, and those con-taini n g eggs of the same dark blue colour as eggs present i n the oviducts of the females, were retained f o r use i n the culture experiments. This choice ensures the use of embryos during an early stages of development. The prefeeding stages of the organism were maintained i n 1 l i t e r polypropylene erlenmeyer f l a s k s containing 600 ml. of f i l t e r e d seawater. The water used was i n a serie s of treated and enriched states, as discussed elsewhere. The fl a s k s were kept at 8°C in d a f c o n t r o l l e d environment chamber (Sherer G i l e t t Co. Model E2). Water was changed every t h i r d day and the condition of the embryos and n a u p l i i was recorded. Dead organisms, as w e l l as those which had reached the t h i r d naupliar stage (" the f i r s t feeding stage), were removed at the time of the water change. The.1 l i t e r erlenmeyer f l a s k s were f i l l e d with 600-700 ml. of 0.1 N HCl i n d i s t i l l e d water and stored between experiments. At a l l times 18 these f l a s k s were kept covered with Parafilm, ensuring that the f l a s k s were not contaminated. The e f f e c t s of various treatments and enrichments of the culture water were measured by comparison of the percent of the organisms used per te s t which survive u n t i l the t h i r d naupliar stage. Four egg c l u s t e r s (one i n each of four flasks) were used for each test i n a monthly s e r i e s . Under experimental conditions t h i s number of r e p l i c a t e s allows examination of the e f f e c t of the treatment of the media, while maintaining a standard error of the mean of les s than 5%. This estimate of error was obtained by running 12 r e p l i c a t e s and c a l c u l a t i n g a mean s u r v i v a l of a l l possible combinations of increasing numbers of f l a s k s u n t i l the 5% l e v e l had been reached. However, because of the small number of r e p l i c a t e s , the standard deviation of each mean i s r e l a t i v e l y l arge. In evaluating the r e s u l t s a difference between means greater than 10% was considered to be s t a t i s t i c a l l y s i g n i -f i c a n t . The test materials used to enrich the seawater were prepared i n a manner such that a 1 or 2 percent mixture ( i . e . , 1 or 2 ml. of enrichment media to 100 ml. of seawater) of the stock s o l u t i o n would y i e l d the desired concentration i n the culture media. A l l metal solutions used were prepared from the chloride s a l t of the metal, rather than the sulphate, as there i s some evidence that the sulphate anion changes the e f f e c t of c e r t a i n metals. A l l metal concentrations used are expressed as yg/1 of culture media. Metal additions were made on the basis of doubling or t r i p l i n g the concentration of the metal which was believed to be present, based on . previous measurements. For zinc the additions were eit h e r 4 or 8 yg/1. For manganese the additions were 50 or 100 yg/1. Additions of copper were made at several d i f f e r e n t concentrations. A copper addition of 5.4 yg/1 was 19 used i n the copper stress s e r i e s and i n determining the complexing a b i l i t y of material extracted from sediments. Copper concentrations of 1, 2, and 5 yg/1 were used i n some of the u l t r a - v i o l e t experiments. EDTA (ethylenediaminetetraacetic acid) enrichment media were prepared by d i s s o l v i n g the disodium s a l t of EDTA i n deionized water to obtain the desired concentration. The f i n a l c ulture concentrations i n the copper stress s e r i e s were 0.125 and 0.250 uM, while 0.125, 0.250, and 0.500 yM were used i n the u l t r a - v i o l e t s e r i e s . Sediment extracts were prepared from 125 ml. of sediment taken from a sample c o l l e c t e d with a Shipek grab sampler. This sediment was mixed with 800 ml. of f i l t e r e d seawater c o l l e c t e d at Ind-2 from 200 meters. This was then shaken p e r i o d i c a l l y during the f i r s t two hours of the extraction, and l e f t to stand for a further twenty-two hours. A f t e r t h i s time the supernatant was f i l t e r e d through a 0.45 ym M i l l i p o r e f i l t e r and then stored at 8°C. This material was used i n enriching culture water (previously enriched with 5.4 yg/1 of copper) at a 2% d i l u t i o n . The high wattage lamp used for i r r a d i a t i n g seawater samples i s operated under a nitrogen environment within a glass immersion w e l l . The immersion w e l l i s double walled with a co n t r o l l e d water flow passing between them to allow transfer of heat away from the lamp. Controls f o r the com-ponents of the system are e l e c t r i c a l l y interlocked with the power supply to the lamp. In addition to the e l e c t r i c a l supply to the lamp, the e l e c t r i c a l components include switches and solenoids which extinguish the lamp i n the event of f a i l u r e i n one of the associated systems, thus reducing the danger involved i n the operation of such a lamp. Also included i n the e l e c t r i c a l system i s a timing clock which allows control of the duration of sample treatment. The lamp i s suspended with a 50 1 chromatographic chamber 20 covered on the outside with aluminum f o i l . This apparatus i s shown schematically i n Figure 3. This system i s contained i n an environmental chamber which i s kept at 8°C. Further d e s c r i p t i o n of t h i s apparatus i s given asi anpApp'endix. The necessary duration of u l t r a - v i o l e t treatment of the culture water was determined from the r e s u l t s shown i n Figure 4. A f t e r four hours, the changes induced by the u l t r a - v i o l e t l i g h t are e s s e n t i a l l y complete, as indicated by the change i n the slope of the l i n e . On the basis of these r e s u l t s i t was believed that a s u f f i c i e n t duration of treatment would be s i x hours. This allows for some d e t e r i o r a t i o n of the e f f i c i e n c y of the lamp, and changes i n the amount of material which the lamp i s acting upon. Measurements of dissolved organic carbon, before and a f t e r the treatment of water with u l t r a - v i o l e t l i g h t , provides further support f o r the use of t h i s duration of exposure. Chloroform extractions were performed by the method of Khailov and Finenko (1970) on one l i t e r samples of seawater used i n culture experi-ments. The metal content of the samples a f t e r extraction was analysed with the method described elsewhere i n t h i s paper. (iv) S t a t i s t i c a l analysis Several s t a t i s t i c a l treatments were used to examine f i e l d measure-ments. These included c o r r e l a t i o n a n a l y s i s , c l u s t e r analysis and regression analysis. Analysis resulted from the use of s t a t i s t i c a l packages made avai l a b l e by the UBC Computing Center, for use with the IBM 360/70 and l a t e r with the IBM 370/168 computers. A l l of the f i l e d data c o l l e c t e d was subjected to a c o r r e l a t i o n analysis (UBC CORN). This program computes cor r e l a t i o n s between pairs of Figure 3: Diagrammatic representation of the u l t r a - v i o l e t apparatus Key to Figure 3 Water Cooling System 1. Water i n l e t valve 2. F i l t e r (Cuno type, 5 microns) 3. Water pressure reducing valve 4. Solenoid valve 5. In l e t pressure gauge 6. Well i n l e t 7. Well o u t l e t 8. Outlet pressure gauge 9. Pressure switch 10. Drain E l e c t r i c a l System 11. Off-On switch 12. Timing clock 13. C i r c u i t breaker 14. P i l o t l i g h t 15. 220 v o l t s t a b i l i z e d b a l l a s t 16. 12 inch 1200 watt mercury arc lamp Nitrogen System 17. Nitrogen tank regulator 18. Outlet needle valve 19. Erlenmeyer f l a s k with water A d d i t i o n a l Parts 20. Vycor immersion tubes 21. 50 l i t e r treatment chamber 22. Aluminum f o i l r e f l e c t o r 23. Well head assembly 24. Plate glass covers Water l i n e s E l e c t r i c a l l i n e s Nitrogen l i n e s 21a 22 Figure 4: Percent survival from the egg through to the third nauplius as a function of the duration of treatment of the culture water with ul t ra -viole t l i g h t . Each point represents the mean survival determined from approximately 60 animals. The curve i s f i t t e d by eye. 22a HOURS o f ULTRA-VIOLET TREATMENT 23 v ariables and performs s i g n i f i c a n c e tests on the c o r r e l a t i o n c o e f f i c i e n t s . This program was used also to treat several subsets of the data. The data were divided into subsets by several means. F i r s t , the e n t i r e data s e r i e s was treated. Next, a l l samples where the naupliar stages had been found were analysed ( i . e . , a l l samples taken from deeper than 75 meters). The data were divided again into subsets by stations and then by periods of the year as determined from Figure 9C. The assumption behind c o r r e l a t i o n a n a l y s i s , that the pairs of v a r i a b l e s are l i n e a r l y r e l a t e d , was tested using the UBC STRIP-SIMREG routine to check for l i n e a r i t y between v a r i a b l e s . Cluster analysis was done with UBC BMDP1M, on a data subset which consisted of a l l observations where a l l of the 15 variables had been measured. This program groups va r i a b l e s into c l u s t e r s on the basis of s i m i l a r i t y , i n t h i s case from a c o r r e l a t i o n matrix. The program prog-r e s s i v e l y groups pairs of v a r i a b l e s from high c o r r e l a t i o n s to low c o r r e l a t i o n s . 24 RESULTS (i ) Hydrographic properties Figures 5-8 summarize tabulated data published i n IOUBC Data Reports (1972; 1973; 1974), and are included here to provide evidence of the occurrence of i n t r u s i o n s . Ind-2 data was selected f or presentation because i t i s the s t a t i o n where animals and water were c o l l e c t e d f o r laboratory experiments. The other stations exhibit patterns which are s i m i l a r to those shown. Figure 5 shows the d i s t r i b u t i o n of temperature through the water column over the period of t h i s study. S i m i l a r l y , Figure 6 presents s a l i n i t y data; Figure 7, density data; and Figure 8, dissolved oxygen data. Figure 9 shows temperature s a l i n i t y envelopes for three periods of the year. In-trusions of water into the i n l e t from the S t r a i t of Georgia occurred at two times during the f i r s t year. Between the cruises of December, 1972 and January, 1973 there was a large i n t r u s i o n of water which had a higher temperature, higher s a l i n i t y , and higher dissolved oxygen than that of the deep water of Indian Arm. P r i o r to the July, 1973 cruise there was a small i n t r u s i o n of very dense water which caused the deeper portion of the water column to become more homogenous with respect to temperature, s a l i n i t y , and dissolved oxygen. Another i n t r u s i o n may have occurred at mid-depth i n January, 1974, but adequate hydrographic data are not a v a i l a b l e f or v e r i -f i c a t i o n of t h i s . Such int r u s i o n s have previously been noted i n t h i s i n l e t (Gilmartin, 1962). ( i i ) Chemical properties The samples taken for chemical analyses were biased towards the deep water as t h i s i s where the organism i s most abundant (Evans, 1973). Figure 25 Figure 5. D i s t r i b u t i o n of temperature (°C) through the water column at Ind-2 during the study period. Note: In figures 5-8 the sampling depths are given i n Table 1, and the sampling was done at monthly i n t e r v a l s . Arrows i n d i c a t e time of i n t r u s i o n . 26 F i g u r e 6. D i s t r i b u t i o n o f s a l i n i t y ( ° / 0 o ) t h r o u g h t h e w a t e r c o l u m n a t I n d - 2 d u r i n g t h e s t u d y p e r i o d . 27 Figure 7. D i s t r i b u t i o n of density (sigma-t) through the water column at Ind-2 during the study period. 28 Figure 8. Distribution of dissolved oxygen (ml. / l ) through the water column at Ind-2 during the f i e l d sampling period. 29 Figure 9. Temperature sa l ini ty envelopes of water below 10 meters for the months sampled during the period of the study. January 1973 February 1973 March 1973 A p r i l 1973 January 1974 February 1974 March 1974 May 1973 June 1973 July 1973 August 1973 3. September 1972 October 1972 November 1972 December 1972 September 1973 October 1973 November 1973 December 1973 29a 30 10 shows the d i s t r i b u t i o n of dissolved manganese through the water column at Ind-2 with time. Large changes i n the manganese d i s t r i b u t i o n occurred at two times during the year. During most of the year the manganese con-centration i n the deepest water was 6 to 10 times as great as the concen-t r a t i o n at 10 meters. At the time of the i n t r u s i o n s , manganese l e v e l s decreased i n the deep water (from 10 to 12 times) from the preceding months (e.g., January, 1973 and Ju l y , 1973), and the v e r t i c a l gradient of manganese was reduced (deep water concentrations are approximately 2 to 3 times as great as 10 meter concentration). Copper, z i n c , cadmium and n i c k e l had s i m i l a r d i s t r i b u t i o n s . The concentration of a l l of these metals remained r e l a t i v e l y constant with the exception of the changes at the time of the large i n t r u s i o n i n January. Zinc l e v e l s p r i o r to the i n t r u s i o n ranged from 10 to 15 yg/1. A f t e r the i n t r u s i o n the l e v e l of zinc ranged from 2 to 7 yg/1. The d i s t r i b u t i o n of dissolved copper through the water column i s shown as a function of time i n Figure 11. The l e v e l s of copper present i n the deep water were higher a f t e r the i n t r u s i o n than they were before i t . A f t e r the i n t r u s i o n , the l e v e l of dissolved copper declined s l i g h t l y . N i c k e l ranged i n concentration from 0.8 to 1.4 yg/1, and cadmium from 0.07 to 0.18 yg/1. The i n t r u s i o n had l i t t l e e f f e c t on the concentration of these metals. The l e v e l s of n i c k e l and cadmium determined are near the minimum l e v e l detectable by the method used. Some of the v a r i a t i o n which the data demonstrate may be a t t r i b u t a b l e to t h i s . The p a r t i c u l a t e content of the deep water nearly doubled at the time of the January i n t r u s i o n . A large increase was also noted i n July. The average weight of p a r t i c u l a t e material p r i o r to January was 4.69 mg/1 while i n January i t increased to 8.61 mg/1. The l e v e l then declined u n t i l 31 Figure 10. D i s t r i b u t i o n of dissolved manganese (yg/1) through the water column at Ind-2 during the f i e l d sampling period. Note: In Figures 10 and 11 the sampling depths are as given i n Table 1, and sampling was done at monthly i n t e r v a l s . Arrows indicate time of i n t r u s i o n . 32 Figure 11. Distribution of dissolved copper (yg/1) through the water column at Ind-2 during the f i e l d sampling period. 33 June (mean weight of p a r t i c u l a t e material i s 5.50 mg/1) then, i n J u l y , again increased (6.50 mg/1) and then decreased through the months following. Since copper l e v e l s associated with the p a r t i c u l a t e material remained r e l a t i v e l y constant during the year, no f i g u r e i s provided. Copper a s s o c i -ated with p a r t i c u l a t e material represented le s s than 5% of the t o t a l copper measured. The l e v e l s (yg/mg p a r t i c u l a t e material) increased s l i g h t l y with the increase i n p a r t i c u l a t e material i n January. Organic carbon values are also not shown gr a p h i c a l l y . Only deep water values were obtained f o r the period from May, 1973 u n t i l October, 1973. There was a decrease i n organic carbon l e v e l s with time, from 2.69 mg/1 (mean of a l l May samples) to 1.66 mg/1 (mean of October samples), except for a small increase i n July. ( i i i ) Abundance of the l i f e h i s t o r y stages The change i n numbers of the prefeeding naupliar stages ( f i r s t through t h i r d nauplius) throughout the sampling period at Ind-2 i s shown i n Figure 12. P r i o r to January, 1973 the prefeeding naupliar stages were present i n r e l a t i v e l y high numbers (greater than two per cubic meter) i n the deep water. During and a f t e r January the number dropped to les s than one per cubic meter. The number of animals then increased. Numbers greater than two per cubic meter were again reached by May and June. During J u l y the animals became more evenly d i s t r i b u t e d through the deeper portion of the water column. A f t e r J u l y the number of organisms i n these stages i n -creased i n the deepest water which was sampled, as the naupliar stages tend to become concentrated at t h i s depth. The t o t a l number of naupliar stages present i n the samples also was examined. Changes which were noted f o r the prefeeding naupliar stages were 34 Figure 12. D i s t r i b u t i o n and abundance of prefeeding naupliar stages (organisms per cubic meter) through the water column at Ind-2 during the f i e l d sampling period. Note: In Figures 12-14 the sampling depths used are as given i n Table 1. Sampling was done at monthly i n t e r v a l s . Arrows indi c a t e time of i n t r u s i o n . 1972 S 0 N D 1973 J F «ftwiH|i|HM.I...R.,,J .. - j -* . , , Rj^ 0 N 2 Prefeeding Nauplii 35 again evident (Figure 13). Figure 14 shows the d i s t r i b u t i o n of the naupliar stages of Euchaeta japonica through the length of Indian Arm during February, 1973. In t h i s f i g u r e i t i s evident that both the prefeeding (Figure 14a) and the t o t a l naupliar stages (Figure 14b) tend to be concentrated towards the mouth of the i n l e t , being higher i n the water column near the mouth than they are further up the i n l e t . This pattern i s t y p i c a l of that which ex i s t s throughout the year, although the absolute numbers fl u c t u a t e . The s i g n i f i c a n c e and sign of the c o r r e l a t i o n c o e f f i c i e n t s between the number of prefeeding naupliar stages and the other variables measured are given i n Table 2. The c o r r e l a t i o n c o e f f i c i e n t s are not shown because of unequal sample s i z e s . Although there are several s i g n i f i c a n t c o r r e l a t i o n s shown i n Table 2, prefeeding naupliar stages are only l i n e a r l y r e l a t e d to t o t a l naupliar stages and the number of organisms i n the s i x t h naupliar stage. High c o r r e l a t i o n s between prefeeding naupliar stages/: and :.the p h y s i c a l . and chemical properties of the water do not r e s u l t from l i n e a r r e l a t i o h s h i p s . The r e s u l t s of c l u s t e r analysis are shown i n Figure 15. At the 50% s i m i -l a r i t y l e v e l , three clusters are formed, i n d i c a t i n g that there are three groups of s i m i l a r l y related v a r i a b l e s . (iv) Laboratory studies (a) Copper stressed ser i e s +2 The addition of 5.4 yg/1 of Guppeto f i l t e r e d seawater, c o l l e c t e d monthly from 200 meters, caused a decrease i n s u r v i v a l of Euchaeta japonica through the prefeeding stages. This to x i c e f f e c t could be reduced by the addition of EDTA (a synthetic chelating agent). The to x i c e f f e c t which resulted from the addition of t h i s amount of copper was not constant, but was generally a 30-40% decrease i n s u r v i v a l . The "EDTA equivalence" ( i . e . , 36 Figure 13. Distribution and abundance of total ( a l l six) naupliar stages (organisms per cubic meter) through the water column at Ind-2 during the f i e l d sampling period. DEPTH (meters) 37 Figure 14. D i s t r i b u t i o n of (a) prefeeding, and (b) t o t a l naupliar stages through the length of Indian Arm i n February, 1973. 37a STATION NUMBER 1-5 2 2 .5 3 STATION NUMBER 1 5 2 2 .5 3 Table 2. Significance and sign of the correlation coefficient between prefeeding naupliar stages and the other properties measured i n the f i e l d . Characteristic A l l Data A l l Data below 75m. Ind-1.51 DATA Ind-21 SUBSET Ind-2.5 1 Ind-3 1 Prelnt? Postlnt? Inter? Temperature -/O.OOO -/0.020 -/0.011 -/O.OOO -/0.011 -/O.OOO +/0.645 -/O.OOO Salinity +/0.000 +/0.000 +/0.003 +/0.000 +/0.002 ; +/0.000 +/0.00 +/0.000 Sigma-t +/0.887 +/0.000 +/0.002 +70.000 -/0.765 "/0.857 +/0..000 +/0.000 Total naupli i +/0.000 +/0.000 +/0.000 +/0.000 +/0.000 +/0.000 +/0.000 +/0.000 Sixth nauplius +/0.000 +/0.000 +/0.000 +/0.000 +/0.000 +/0.000 +70.000 +/0.000 Mn +70.000 +/0.000 +/0.008 +/0.000 +/0.000 4 +/0.000 +/0.043 +/0.000 Zn +/0.188 +/0.192 -/0.887 +/0.370 +/0.785 +/0.229 +/0.156 +/0.122 Cu -/0.249 -/0.268 -/0.628 -/0.459 -/0.435 -/0.463 -/0.352 -/0.363 Ni +/0.865 -/0.473 -/0.691 r/0.201 - /Q.65§ ---ff>.. -70:202 -70.349 -70; 395 Cd -/0.887 -/0.510 +70.429 -/0.635 -/0.416 -/0.209 -/0.831 ' +/0.589 Particulates -/0.122 -/0.071 -/0.268 -/0.248 -/0.604 -/0.774 -/0.059 -/0.486 Particulate Cu +/0.629 +/0.873 , +/0.134 +/0.521 +/0.887 -/0.845 +/0.002 -/0.471 Organic carbon -/0.874 -/0.780 -/0.297 +/0.139 -/0.549 +/0.924 ******* -/0.801 Oxygen -/O.OOO -/0.303 -/O.OOO -/0.001 -/0.046 -/O.OOO -/0.034 -/0.002 +,- sign of the correlation coefficient . no naupliar stages were found at this station. * * * * * * * n o organic carbon samples were taken in this period. ^Data Subsets on the basis of stations. 2Data Subsets on the basis of period of the year (see Text for details) . Significance i s noted at the 10% levle at 0.100, and at 5% at 0.050 or less . 39 Figure 15. Tree printed from c o r r e l a t i o n matrix (scaled 0-100). Clustering by average distance method. Data are-iall observations which have values for the 15 c h a r a c t e r i s t i c s , The abbreviations used are: organic C - organic carbon part. Cu - p a r t i c u l a t e associated copper a - sigma-t Mn - manganese PFN - prefeeding naupliar stages NTOTAL - t o t a l naupliar stages No6 - s i x t h naupliar stage Zn - zinc Cu - copper Ni - n i c k e l Cd - cadmium pa r t i c u l a t e s - p a r t i c u l a t e material TEMPERATURE OXYGEN organic C part.Cu SALINITY Ot Mn PFN N TOTAL N 6 Zn Cu Ni Cd particulates vo 40 the amount of EDTA required to negate the t o x i c e f f e c t of the copper enrichment) i s obtained from the intercept of the s u r v i v a l value obtained i n unenriched water with the l i n e drawn drawn through the values obtained for s u r v i v a l i n the copper and EDTA enriched water, as shown i n Figure 16. The values of the intercept are shown as a function of time i n Figure 17e. (b) Sediment extract se r i e s Sediment extracts prepared from samples taken at the four stations over the period of one year have been shown to be able to reduce the toxi c e f f e c t of copper enrichment. The a b i l i t y of the extracted material to reduce the t o x i c e f f e c t changed with time, and was often d i f f e r e n t at each of the st a t i o n s . The a b i l i t y tended to be greatest i n l a t e summer and f a l l , and l e a s t i n the l a t e winter (Figure 17a-d). The a b i l i t y of the sediment extract to reduce the toxi c e f f e c t i s expressed as the amount of EDTA which causes the same reduction i n t o x i c i t y (Lewis et a l . , 1973). At times negative values f o r the EDTA equivalence were obtained. These resulted from a detrimental e f f e c t r e s u l t i n g from the addition of the material extracted from sediments, and values were obtained by extrapolation of the EDTA concentration l i n e . (c) U l t r a - v i o l e t s e r i e s As the concentration of EDTA added to u l t r a - v i o l e t treated water increased, the s u r v i v a l of Euchaeta japonica through the prefeeding stages ' also increased as shown i n Figure 18. The values plotted i n Figure 18 are the means of the r e s u l t s shown i n Table 3. During the year the slope and intercept of t h i s l i n e changes. The standard deviations of s u r v i v a l at low 41 Figure 16. The percent survival of the organism from the egg through the third nauplius plotted against the concentration of EDTA which was added to water with 5.4 yg/1 of added copper (•). Also shown i s the percent survival of the organism in unenriched water (o), and the method of obtaining an EDTA equivalence is shown by the arrows. Values shown are the means and standard deviations for the period of study. The means are based on 12 monthly experiments, each of which involved approximately 60 animals. 41a 42 Figure 17. (a-d) EDTA equivalence of the sediment extracts as a function of time,aarid (e) t o x i c e f f e c t of a copper addition expressed as the amount of EDTA"required to negate the e f f e c t of the copper addition as a function of time. 42a 1972 1973 S O N D J F M A M J J A 1 • • q II t s-a. IND 1.5 S X b. IND 2 S X c. IND 2.5 S X d. IND 3 S X e. IND 2 - 2 0 0 43 Figure 18. Percent s u r v i v a l of the organism from the egg through the t h i r d nauplius as a function of the EDTA concentration i n water which has been treated with u l t r a - v i o l e t l i g h t (•). Also shown i s the percent s u r v i v a l obtained i n untreated water (o), and the method of obtaining an EDTA equivalence i s indicated by the arrows. Values shown are the means of the twelve months of the study (approximately 700 animals i n t o t a l f o r each of the means) and standard deviations. 43a 44 Table 3. Percent s u r v i v a l values i n the u l t r a - v i o l e t s e r i e s , with and without metal additions. Values are the percent of approximately s i x t y animals that survive u n t i l the t h i r d naupliar stage. Also shown are the EDTA equivalences which were determined, and the approximate slope of the l i n e which re l a t e s s u r v i v a l of the organism to the EDTA concentration. Month Water Metal  x Addition' Untreated Ultra-violet Ultra-violet 0.125 uM EDTA May 2-200 none 73 34 48 June 2-200 none 64 27 36 July 2-200 none 54 21 37 August 2-200 none 63 22 36 September 2-200 none 52 34 40 2-200 Mn 100' 11 20 October 2-200 none 70 46 54 2-200 Zn 8 43 34 2-200 Mn 100 18 50 2-125 none 75 63 51^ November 2-200 none 71 34 50 2-200 Zn 8 43 44 2-200 Mn 50 55 58 2-125 none 80 50 '"•51 2-125 Mn 50 83 55 December 2-200 none 67 53 51 2-200 Mn 100 60 37 a) n r-l W i cd O u m 4-» C N rH • S3 o 55 49 51 58 48 46 61 45 57 51 , 73 49 54 6>7 65 56 59 <v u c <i CD 4-1 H rH CU Q c8 rH W 4-1 > O cd •H •H a 6 > 3 - •H CU cd O O 0) M O U Cu 4 J m ft o H a rH • Cu rH O 3 -\=> o < ! C/3 76.84 7171 75 75 76 63 77 48 71 62 83 61 59 83 75 83 80 .84 .88 1.08 1.06 .84 .86 .62 .04 .42 .00 .98 .34 .02 .66 .40 .60 .86 .45 .47 .30 .38 .38 .38 .19 .33 .00 .45 .70*' .36 .27 continued 45 ... continued ... December 2-125 none January 2-200 none 2-200 Zn 4 2-200 Mn 50 2-200 Mn 50 Zn 4 February 2-200 none 2-200 Cu 1 Mn 100 Cu 1 2-200 2-200 none 79 83 54 55 80 81 69 2-200 Mn 100 23 23 March 74 2-200 Cu 1 69 2-200 Mn 50 52 2-200 Cu 2 60 A p r i l 2-200 none 84 2-200 Cu 1 71 2-200 Cu 2- 61 2-200 Cu 5 46 May 2-200 none 69 2-200 Cu 1 67 2-200 Cu 2 60 2-200 Cu 3 54 2-200 Cu 5 43 50 41 65 46 53 38 40 11 30 56 39 57 47 46 32 82 42 33 36 70 58 32 47 42 55 58 45 1 Metal additions are yg/1. 2 No intercept. 3 Value obtained by extrapolation. 58 57 70 66 53 60 59 45 39 65 59 76 48 66 57 64 65 66 62 49 60 50 74 78 60 60 58 86 59 58 59 80 67 79 53 86 74 46 71 79 74 33 73 63 .48 •74 .00 .28 .10 .96 .38 .94 .58 .48 .56 .44 .12 .80 .84 -.72 .70 .92 .76 -.74 .30 .62 ,54* .56* .11 .50 .58* .09 .41 .45 .60* .48 .46 .28 .05 .37 .38 .13 .18 46 EDTA concentrations are very much larger than those at high EDTA concen-t r a t i o n s . EDTA equivalence of untreated water (open c i r c l e ) was determined from the intercept of the s u r v i v a l i n untreated water with the l i n e r e l a t i n g s u r v i v a l to EDTA concentration, as shown by the arrows i n Figure 18 ( i n t h i s case the mean EDTA equivalence i s approximately 0.4 uM). The EDTA equiva-lences determined i n t h i s manner are shown as a function of time i n Figure 19. During the f i r s t four months the values were e r r a t i c , and became stable i n the following four months (September to December). During January the EDTA equivalence of the water reached a maximum l e v e l and then decreased through the subsequent months. (d) Metal additions Metals were added to u l t r a - v i o l e t treated and untreated seawater. The r e s u l t s from these ser i e s along with those f o r corresponding water with no metal additions are given i n Table 3. The EDTA equivalence obtained i n water with metal additions e i t h e r d i d not-.changeoorddecreased. • The r e l a t i o n -ship between s u r v i v a l and increasing EDTA changes with some metals and not with others, depending both on the metal used and i t s concentration. The slope of the l i n e which r e l a t e s the s u r v i v a l of the organism through the prefeeding stages to the concentration of EDTA was d i f f e r e n t i n water to which metals had been added (Table 3). Variations i n slope are dependant both upon the metal used and i t s concentration. For example, i n March, 1973, neither 1 yg/1 copper or 50 yg/1 manganese additions a l t e r e d the slope, while 2 yg/1 copper reduced the slope. (e) Measurements on water before and a f t e r u l t r a - v i o l e t treatment A ser i e s of measurements were made on water used i n the laboratory 47 Figure 19. EDTA equivalence as determined i n u l t r a - v i o l e t treated water as a function of time. 47a 48 experiments, before and a f t e r the treatment with u l t r a - v i o l e t l i g h t . These values are shown i n Table 4. Measurements of the dissolved organic carbon concentrations are compared with the diff e r e n c e i n percent s u r v i v a l of the organism between untreated and treated water (Figure 20). (f) Chloroform extractions Table 5 shows the values obtained f o r metal concentrations i n water which had been extracted with chloroform. Also included are the metal con-centrations i n water which had not been treated i n t h i s manner, and the EDTA equivalence as determined with the u l t r a - v i o l e t bioassay s e r i e s . Approximately one h a l f of the dissolved manganese was removed by the extraction i n the May sample, but not i n the June sample. The other metals were only s l i g h t l y affected by the extraction. The t o t a l amount of metals removed by the chloroform extraction does not coincide with the EDTA equiva-lence as determined by the bioassay of u l t r a - v i o l e t treated water. 49 Table 4. Levels (concentrations) of various chemical constituents of the culture water, before and after ul t ra -viole t i r radiat ion (October 1973). Constituent 200 meter water 125 meter water Before After Before After zinc (yg/1) 8.1 8.0 6.5 6.3 manganese (yg/1) 48.1 48.1 5.2 5.2 copper (yg/1) 1.2 1.2 0.8 0.8 nickel (yg/1) 1.3 1.3 1.2 1.2 cadmium (yg/1) 0.09 0.09 0.11 0.11 organic carbon (mg/1) 2.04 0.52 1.58 0.45 pH 7.81 7.80 percent survival 70 46 75 63 50 Figure 20. The change in percent survival plotted against the change in the organic carbon content of the water which resulted from the ul t ra -viole t treatment. 50a A O R G A N I C C A R B O N mg/L 51 Table 5. Results from the chloroform extraction experiments, and the EDTA equivalence determined with the bioassay. Sample Treatment Concentration (yg/1) Mnn ' Zn Cu Ni ' ' Cd Metals Removed (VM) EDTA equivalence (yM) May 200m. June 200m. none CHCl^ nn none CHC1„ 20.2 4.5 1.0 1.0 0.11 10.3 2.2 1.0 1.0 0.10 91.8 6.2 1.3 1.1 0.11 57.2 6.2 1.1 li.l)? 0.07 0.22 0.68 0.45 0.43 52 DISCUSSION (i) F i e l d data The water column i n Indian Arm i s s t r a t i f i e d with low s a l i n i t y water near the surface, and high s a l i n i t y water from the S t r a i t of Georgia, at depth. Intermediate to these two layers i s a t r a n s i t i o n zone which f l u c t u -ates i n depth and properties, depending upon the c h a r a c t e r i s t i c s of the upper and lower layers. The major perturbations to t h i s system are i n t r u -sions of water into the I n l e t from the S t r a i t of Georgia (Gilmartin, 1962). Two int r u s i o n s of high s a l i n i t y water occurred during the period of the f i e l d study. The f i r s t of these was i n January, 1973 (see Figures 5 and 6). This i n t r u s i o n resulted i n a p a r t i a l breakdown of the s t r a t i f i -c ation and was of a s u f f i c i e n t l y dense nature to replace most of the deep water i n the i n l e t . (Intrusions of t h i s type, at t h i s time of year, have been noted by Gilmartin, 1962; G i l f i l l a n , 1970; Gardner, 1972; and Evans, 1973). A second i n t r u s i o n occurred sometime i n J u l y , 1973. I t was of a r e l a t i v e l y minor nature (Lafond, personal communication) and i s more apparent from the manganese data and b i o l o g i c a l data than from temperature and s a l i n i t y values. (A January i n t r u s i o n also occurred i n 1974). The January, 1973 i n t r u s i o n i s evident on the density p r o f i l e (Figure 7). Subsequent to t h i s i n t r u s i o n there was a gradual decay of the density structure of the i n l e t . The July i n t r u s i o n enhanced the rate of decrease of the density of the deep water. The decrease i n density i s the r e s u l t of the estuarine c i r c u l a t i o n of the i n l e t . The l e v e l s of dissolved oxygen increased i n January (Figure 8) while i n J u l y the oxygen present appeared to become mixed throughout the lower 100 meters of the water column. The gradual decreases i n oxygen content of the deep water a f t e r the in t r u s i o n s 53 are presumably the r e s u l t of b i o l o g i c a l u t i l i z a t i o n . In a p p l i c a t i o n to the study of the b i o l o g i c a l a v a i l a b i l i t y of metals, the January i n t r u s i o n , being of r e l a t i v e l y large magnitude, was expected to have a large e f f e c t on the organism-metal r e l a t i o n s h i p . The July i n t r u s i o n , on the other hand, does not r e s u l t i n the displacement of the deep water of the i n l e t , and should have had a l e s s e r e f f e c t on the organism-metal r e l a t i o n -ship. During t h i s study the naupliar stages of Euchaeta j aponica were found to be concentrated towards the bottom of the water column (see Figures 12 and 13). This has also been found i n other studies (e.g., Pandyan, 1971; Evans, 1973). The naupliar stages were found i n higher numbers, and higher i n the water column, towards the mouth of the i n l e t (Figure 14). This i s possibly the r e s u l t of the nature of the water c i r c u l a t i o n i n the i n l e t . Water entering the i n l e t may move up the i n l e t at mid depth for some distance. This could r e s u l t i n an area near the mouth where there i s l i t t l e exchange of water. The d i s t r i b u t i o n of dissolved manganese exhibits a s i m i l a r pattern, and substantiates t h i s suggestion. Reductions i n numbers of both prefeeding and t o t a l naupliar stages (e.g., Figures 12 and 13) appear to be the r e s u l t of the intrusions of water into the i n l e t . In January there was a large drop i n the number of organisms present. During J u l y , however, the organisms became more evenly d i s t r i b u t e d through the water column. Similar r e s u l t s were found by Evans (1973). The r e s u l t s of the c o r r e l a t i o n analysis (Table 2) show strong p o s i t i v e c o r r e l a t i o n s between the numbers of prefeeding naupliar stages and both s a l i n i t y and density. Negative c o r r e l a t i o n s are obtained with both tempera-ture^and dissolved oxygen. Regression a n a l y s i s , however, shows that a l i n e a r r e l a t i o n s h i p between prefeeding naupliar stages and each of these properties 54 does not e x i s t . Cluster analysis suggests that the r e l a t i o n s h i p s which exi s t may be the r e s u l t of the v e r t i c a l d i s t r i b u t i o n of these properties (Figure 15). In c l u s t e r i n g of the v a r i a b l e s , temperature and dissolved oxygen are shown to be r e l a t i v e l y s i m i l a r . S a l i n i t y and density are shown to be very s i m i l a r (0.98) and become clustered with a l l of those properties which reach a maximum i n the lower portion of the water ;column.>. On the basis of Figure 9 the year was divided i n t o three d i s t i n c t periods. The f i r s t period (preintrusion) i s t y p i f i e d by having nearly uniform s a l i n i t y but a large temperature range. The second period of the year (ppstintrusion) i s t y p i f i e d by having a wide range of s a l i n i t i e s while the temperature of the water i s r e l a t i v e l y uniform. During t h i s period the water s t a b i l i t y i s e n t i r e l y contolled by the s a l i n i t y structure. The t h i r d period of the year (intermediate;)) i s the t r a n s i t i o n period between the postintrusion and p r e i n t r u s i o n patterns. V e r t i c a l v a r i a t i o n s i n both temperature and s a l i n i t y are c h a r a c t e r i s t i c of t h i s period, the s t a b i l i t y of the water column being c o n t r o l l e d by both of these features. Although these periods are referred to as being d i s t i n c t , one must keep i n mind that, except f o r the i n t r u s i o n , they form a graded s e r i e s . Chemical properties of the deep water change as a function of time and hydrographic events. There are large changes i n the concentration of dissolved manganese at two times during the study period (see Figure 10). The January i n t r u s i o n resulted i n the reduction of the high l e v e l s of manga-nese evident p r i o r to the i n t r u s i o n . A f t e r January, the manganese concen-t r a t i o n s increased to very high l e v e l s (maximum concentration 131 yg/1) u n t i l J u l y , when they again dropped. This i s the r e s u l t of the mixing of the lower 100 meters of the water column, which caused the manganese l e v e l to decrease, possibly the r e s u l t of both r e d i s t r i b u t i o n and p r e c i p i t a t i o n . Subsequent to 55 the July i n t r u s i o n , manganese l e v e l s at the deeper depths again increased. The concentrating of manganese into the deep water may be the r e s u l t of the formation of Mn (IV) oxides which are r e l a t i v e l y i n s o l u b l e . There i s also some evidence of manganese e x i s t i n g i n a complex with high molecular weight organic material from some of the laboratory studies. Large molecular weight organic material present i n seawater has been shown to be surface active ( R i l e y , 1963) and able to bind t r a n s i t i o n metals (Barsdate, 1970). When chlorform i s shaken i n seawater i t forms many small droplets, causing a large increase i n surface area. This increases the i n t e r a c t i o n with the surface active material (Khailov and Finenko, 1970). When the chloroform i s allowed to separate i t draws the surface active organic material with i t , forming a t h i r d layer which consists of the chloro-form, some organic material, and a small amount of captured water. The extraction of seawater with chloroform a l t e r e d the concentrations of metals present. The r e s u l t s , shown i n Table 5, i n d i c a t e that i n both samples approximately 50% of the manganese was removed by the extraction. No diffe r e n c e i n copper concentration was evident i n May while the d i f f e r e n c e found i n June could e a s i l y have been the r e s u l t of experimental error. Some of the zinc was removed by the extraction i n May but none i n June. There i s a discrepancy between the amount of organic bound metals which were removed by the extraction and that which was estimated by the addition of EDTA i n the bioassay. The r e s u l t s from these experiments can serve only as an i n d i c a t i o n of organic bound metals because i n s u f f i c i e n t controls were used. Copper, z i n c , cadmium, and n i c k e l were also affected by the i n t r u -sions (e.g., Figure 11). Those changes which occurred at the i n t r u s i o n were probably the r e s u l t of a d i f f e r e n c e i n the concentration of each metal i n the intruding water and the water which i t displaced. Increases i n the amount of 56 p a r t i c u l a t e material and changes i n the amount of copper associated with the p a r t i c u l a t e material may be the r e s u l t of the amount and composition of such material i n the intruding water. I t i s also possible that the intr u s i o n s caused the mixing of non-consolidated sedimentary material i n t o the water column. If t h i s i s the case, the material may ori g i n a t e e i t h e r i n Indian Arm or i n Burrard I n l e t . T h i s , however, requires further examination. The c o r r e l a t i o n analysis showed a strong p o s i t i v e c o r r e l a t i o n between the concentration of manganese and the concentration of prefeeding naupliar stages (Table 2). This i s probably the r e s u l t of both being s i m i l a r l y affected by the movement of water within the i n l e t . Regression analysis showed that prefeeding naupliar stages and manganese were not l i n e a r l y r e l ated. Strong c o r r e l a t i o n s of prefeeding naupliar stages were obtained with p a r t i c u l a t e copper (positive) and p a r t i c u l a t e material (negative) during the p o s t i n t r u s i o n period. These probably resulted from the changes which occurred subsequent to the i n t r u s i o n ; that i s , the s e t t l i n g out of suspended material and the increase i n the concentration of prefeeding naupliar stages. Three d i s t i n c t c l u s t e r s of variables were formed at the 50% si m i -l a r i t y l e v e l i n Figure 15. The var i a b l e s within^.the c l u s t e r bounded by temperature and p a r t i c u l a t e copper were d i s t r i b u t e d such that the maximum values obtained were i n near surface waters. The var i a b l e s i n the c l u s t e r bounded by s a l i n i t y and s i x t h naupliar stage reached t h e i r maximum values i n the deepest portion of the water column. The var i a b l e s i n the c l u s t e r , from zinc to p a r t i c u l a t e material, were d i s t r i b u t e d more or less evenly through the water column. ( i i ) Laboratory data By running a serie s i n which EDTA i s added i n addition to either 57 the copper enrichment or the u l t r a - v i o l e t treatment i t i s possible to i n -crease the s u r v i v a l of the organism above that which occurs where the t r e a t -ment i s applied but no EDTA has been added. The amount of EDTA which i s required to increase s u r v i v a l i n the treatment to that which i s obtained i n untreated water (or with s p e c i f i c enrichments) i s a measure of the r e l a t i o n -ship between the organism and the environmental water. What th i s amount of EDTA represents depends upon the series which i s being considered. For a copper enrichment, the amount of EDTA which i s required to increase s u r v i v a l of the organism to that attained i n the unenriched condition i s a measure of the toxic e f f e c t of the copper enrichment. For the sediment extract enrich-ments i t i s an approximation of the a b i l i t y of the extracted material to reduce the toxic e f f e c t of the copper (e.g., Lewis et a l . , 1973). In the u l t r a - v i o l e t experiments i t i s a measure of the a b i l i t y of the organic material, which was destroyed, to complex metals. Experiments on the toxic e f f e c t of the copper enrichment provided i n d i r e c t evidence of changes i n the i n t e r - r e l a t i o n s h i p s of the organic and metal components i n natural systems. The t o x i c e f f e c t produced by a copper addition can be predicted to be constant. Changes i n i t s e f f e c t may have been the r e s u l t of a l t e r a t i o n s i n the r e l a t i o n s h i p between the organism and the metal-organic equilibrium i n natural water. The toxic e f f e c t of the addition of 5.4 yg/1 of copper should be eliminated by the addition of an equimolar concentration of EDTA (approxi-mately 0.11. yM). This was found to be the case during the f a l l of 1972 (preintrusion period, Figure 9), and for one other month (July, 1973). During t h i s period the to x i c e f f e c t of the copper addition was the r e s u l t of the increase i n copper concentration. However, during the period from January, 1973 u n t i l July, 1973 t h i s was not the case, as the addition of a d i s c r e t e 58 amount of copper resulted i n a t o x i c e f f e c t equivalent to that of a greater amount of copper than was used. Figure 16 shows the method used to obtain the EDTA equivalence of the copper s t r e s s . Figure 17e shows the pattern of the t o x i c e f f e c t of copper as a function of time'. The toxic e f f e c t of the copper enrichment began to increase i n January and continued to increase u n t i l May (postintrusion period, Figure 9). From May u n t i l July the t o x i c e f f e c t decreased (intermediate period, Figure 9). There i s at present no evidence for explaining t h i s occurrence other than possibly the competition of metals. However, the t o x i c e f f e c t of the copper enrichment can be considered i n the l i g h t of the u l t r a - v i o l e t experiments and the changes i n hydrographic properties. At the time of the i n t r u s i o n there i s a displacement of .the deep water of the i n l e t , r e s u l t i n g i n a d i f f e r e n t set of chemical e n t i t i e s i n deep water a f t e r the i n t r u s i o n . I t i s believed that the intruding water may have had a r e l a t i v e l y large concentration of organic material which could act to complex metals. The a b i l i t y of t h i s organic material may be such that i t formed a complex with a metal which would be displaced when exposed to excess amounts of copper. The r e s u l t of t h i s displacement had an i n c r e a s i n g l y harmful e f f e c t during the months following January. This harmful e f f e c t was reduced i n the months following May, suggesting that one of two mechanisms may be acting. F i r s t , the chemical composition and a c t i v i t y of the organic material may have changed i n a manner which prevented the enhancement mechan-ism from becoming manifest. This could be the r e s u l t of production of organic material i n near surface layers which was then transported into the deep water. Second, the l e v e l of the t o x i c component might be reduced e i t h e r by the e f f e c t s of increased entrainment associated with increases i n runoff, or by sorption onto a p a r t i c l e which s e t t l e d out of the water column. 59 The mechanism of the increase i n the t o x i c e f f e c t of a copper enrich-ment i s not known at present and needs further examination. I t i s important to note that the changes i n the toxi c e f f e c t of the copper enrichment occur simultaneously to intrusions of water into Indian Arm. It i s possible that the answer l i e s i n the chemical changes which occur following an i n t r u s i o n . The a b i l i t y of material extracted from sediments to reduce the toxi c e f f e c t s of a copper enrichment was d i f f e r e n t at each of the four locations studied. The noticeable changes as well as the differences between stations may be an i n d i c a t i o n that the material responsible for the a c t i v i t y of the extract i s organic. One of the most noticeable features i n Figure 17 i s the large v a r i a b i l i t y i n the nature of sediment extracts from Ind-3. This s t a t i o n i s r e l a t i v e l y shallow and l i e s between the s i t e s of entry of the two largest sources of freshwater and accompanying sediment load. The sediment extracts from Ind-2.5 show the l e a s t v a r i a b i l i t y of the stations examined. There i s a tendency evident at a l l of the stations with respect to the a b i l i t y of the sediment extracts to reduce the toxi c e f f e c t of copper enrichment. The a b i l i t y of the sediment extract to reduce the t o x i c e f f e c t wasllowest during the winter and highest i n the. l a t e summer. The EDTA equivalences f o r the sediment extracts have been adjusted for v a r i a t i o n i n the toxi c e f f e c t of the copper enrichment of the test water. The correction of the EDTA equivalences f o r t h i s v a r i a t i o n has been done i n two manners. The f i r s t was by addition or subtraction of the differ e n c e be-tween the observed EDTA equivalence of the copper enrichment's e f f e c t , and that t h e o r e t i c a l l y expected, from the EDTA equivalence of the sediment ex-t r a c t as shown"in Figure 21. The second was by multip l y i n g the EDTA equiva-lence of the sediment extract by a fa c t o r which i s the predicted EDTA equiva-lence of the copper enrichment divided by the observed. The f i r s t of the..:-60 Figure 21. EDTA equivalence of the sediment extracts corrected for v a r i a t i o n i n the toxic effect of the copper enrichment, as a function of time. 60a 1972 S 0 u N D 1973 J F M A M J J J L . f ' L. A 3 • C T CD < I— Q UJ IND 1.5 S X IND 2 SX IND 2.5 SX IND 3 SX 61 adjustments assumed that the change i n the e f f e c t of copper alt e r e d only the intercept of the l i n e r e l a t i n g s u r v i v a l of the organism to the concentration of EDTA, while the second assumed that the change i n e f f e c t a l t e r e d the slope of the l i n e . I t should be r e a l i z e d that both of these methods are extreme approximations, as the r e a l c o rrection f a c t o r would l i k e l y involve both height and slope adjustments. In order to examine further the nature of sediment extracts, the means f o r a l l the stations of the corrected EDTA equivalences, adjusted i n both manners described i n the above, were plotted against time (Figure 22). The patterns which the two means exhibit are very s i m i l a r which suggests that the corrections applied provided a reasonable approximation. The actual adjustment which should be applied would r e s u l t i n a l i n e which would l i e between the two shown i n Figure 22. Attempts were made to c o r r e l a t e both of the derived means with values given for net production given by Gilmartin (1964) f o r Indian Arm. If net production controls the input of organic material into the sediments, a time lag must be allowed for the movement of material from the surface waters into the sediments. When the data which Gilmartin (1964) gives were subjected to a four month s h i f t , as shown i n Figure 22, there was a p o s i t i v e and s t a t i s t i -c a l l y s i g n i f i c a n t c o r r e l a t i o n with mean EDTA equivalences of the sediment extracts (r = 0.830; p = 0.003 f o r additive adjusted means with net production, r = 0.937; p = 0.0001, for m u l t i p l i c a t i v e adjusted means with net production). The r e s u l t i s what would be expected on the b a s i s of the suggestion by Gross et a l . (1972) that organic material i n sediments i s derived from three sources; phytoplankton production, terrigenous input, and b a c t e r i a l reworking of material from the f i r s t two sources. The data show that phyto-plankton production i s the primary source of organic material i n the sediments 62 Figure 22. EDTA equivalence of the sediment extracts (transformed means) as a function of time and net production subjected to a four month s h i f t (data f o r net production from Gilmartin, 1964). 1972 1973 62a 63 of Indian Arm. The use of u l t r a - v i o l e t l i g h t to destroy organic material was noted by Armstrong et a l . (1966) as a decrease i n the e x t i n c t i o n c o e f f i c i e n t of l i g h t at 2000-2500 X. This band of wave lengths i s c h a r a c t e r i c t i c of the presence of organic material. Other workers have also suggested that t h i s was possible (e.g., Williams, 1968; Williams et a l . , 1969; S t r i c k l a n d , 1972). Hamilton and C a r l u c c i (1966) suggested treatment of culture water with u l t r a -v i o l e t l i g h t to make i t free of organic material. The apparatus used i n the present study was s i m i l a r to that used by Williams et a l . (1969), and e a r l i e r by Anderson (1947; 1955). Chemical measurements show that organic material present i n natural waters was oxidized by u l t r a - v i o l e t i r r a d i a t i o n (Table 4, Figure 19). The low l e v e l s of organic material which remained subsequent to the treatment of water may have been non-oxidized portions of the o r i g i n a l m a t e r i a l , or may have been the r e s u l t of contamination from p l a s t i c materials used i n the trans f e r and storage of the treated water. Other components of the seawater were apparently not altered by the treatment (Table 4}) . Armstrong et a l . (1966) suggested that the pH of the water may be altered i n proportion to the amount of organic material which i-ssdestroyed, as a r e s u l t of increased concentrations of carbon dioxide, with the subsequent formation of b i -carbonate ions. Those measurements which were made during t h i s study i n d i -cated that changes i n the pH were minimal. This may be the r e s u l t of aeration of the water during t r a n s f e r of the water to the culture vessels. The technique used i n t h i s thesis measured the complexing a b i l i t y of the water i n terms of the r e l a t i o n s h i p between the organism and the water i n which i t was cultured, a measurement which can be made only i n t h i s way. The use of a bioassay i n measuring complexing a b i l i t y has some l i m i t a t i o n s . 64 Some attempts were made to determine i f the estimates of complexing a b i l i t y obtained by t h i s method could be increased by the addition of small amounts of copper. Copper has a very high k^ and w i l l enter into complexes d i s -placing metals which may or may not be detrimental to the organism. I f the metals which are displaced are non-toxic or there i s a supply of free ligands then the measured complexing a b i l i t y w i l l increase. I f , however, the metal which i s displaced i s as detrimental as copper, i t w i l l remain detrimental i n u l t r a - v i o l e t treated water and the o r i g i n a l estimate of the complexing a b i l i t y of the water w i l l not be increased. The breakdown of organic material which i s bound to metal ions w i l l r e s u l t i n the release of the metal from the organic complex. This may change the r e l a t i o n s h i p between the organism and the metal. The metal may have properties which cause i t to be toxi c to the organism i n the i o n i c form (e.g., Steeman-Nielsen and Wuim-Anderson, 1970). I t s release from the com-plex, i n s u f f i c i e n t q u a n t i t i e s , may be detrimental to the organism. If the organic material i s responsible f or making the metal a v a i l a b l e to the organism, ei t h e r by increasing i t s s o l u b i l i t y or by holding the metal i n the form i n which the organism requires i t , the destruction of the organic material w i l l r e s u l t i n the metal becoming unavailable to the organism. The release of metal ions into s o l u t i o n may also a l t e r the r e l a t i o n s h i p between metals, an area which needs much further examination ( i . e . , Table 2, r e s u l t s from copper seri e s i n ' A p r i l , ••..1974, where the addition of both 1 yg/1 and 5 yg/1 of copper caused a decrease i n the intercept on the s u r v i v a l axis of the l i n e r e l a t i n g percent s u r v i v a l to EDTA concentration, while 2 yg/1 of copper resulted i n a complete r e v e r s a l of the slope). The a b i l i t y of a synthetic chelating agent to serve as a substitute for the destroyed organic material w i l l depend l a r g e l y upon the cause f o r the 65 decrease i n s u r v i v a l . In the case of water from Indian Arm, EDTA has, where no metal additions have been made, served to replace that property which was l o s t from the water by u l t r a - v i o l e t i r r a d i a t i o n . This i s not, however, the case i n waters where the metals are not s u f f i c i e n t l y a v a i l a b l e to the organism (Lewis, unpublished). The decrease i n s u r v i v a l which occurs as a r e s u l t of treatment of water with u l t r a - v i o l e t l i g h t i s d i r e c t l y proportional to the amount of organic material which i s destroyed (Figure 19). This indicates that there i s a d i r e c t r e l a t i o n s h i p between organism s u r v i v a l and the quantity of organic material which i s present i n n a t u r a l seawater. The EDTA equivalence of deep water, as determined with the u l t r a -v i o l e t treated s e r i e s , changes as a function of time (Figure 19). During the year there i s a r e l a t i v e l y persistent decrease i n the values obtained. The two deviations from t h i s pattern occur at the time of in t r u s i o n s of water into Indian Arm. In January, 1974 there i s an increase i n the EDTA equiva-lence from the low l e v e l of l a t e winter to the maximum which was noted during the period of study. In J u l y , 1973 there was ^a ld^ojpl inrdjheirEDTA equivalence to the lowest value which was obtained!.. The i n t r u s i o n i s l i k e l y the c o n t r o l l i n g factor at these times. The i n t r u s i o n of water into the i n l e t i n January r e s u l t s i n an exchange of the deep water f o r near surface water from the S t r a i t of Georgia. In July the i n t r u s i o n d i d not cause a d i s p l a c e -ment of the deep water from the i n l e t , but created some v e r t i c a l mixing through the deeper portion of the water column. I t i s believed that t h i s i s the cause for the decreases i n the EDTA equivalence at that time. Gilmartin (1964) shows primary production i n the i n l e t i s highest i n the early spring (e.g., Figure 22). Fogg (1958; 1966) and Lucas (1947; 1949; 1958) suggest that during periods of high p r o d u c t i v i t y there i s a release of 66 metabolites (e.g., g l y c o l l i c a c i d , Shah and Fogg, 1973) by phytoplankton which increases the organic content of the water. Unfortunately, no informa-t i o n concerning organic carbon l e v e l s i n the i n l e t are a v a i l a b l e during most of the year, although from the information which i s a v a i l a b l e , i t appears that the organic carbon content of the deep water decreased through the period from May u n t i l October. The i n t r u s i o n of near surface waters from the S t r a i t of Georgia into the i n l e t , and increased p r o d u c t i v i t y i n the surface waters of the i n l e t during spring may both contribute towards increasing the organic carbon content of the deep water. During the period i n which organic carbon l e v e l s decreased there was also a decrease i n the EDTA equivalence of the water (Figure 19). I t seems l i k e l y that both the i n t r u s i o n and increased p r o d u c t i v i t y are responsible f o r the high l e v e l s that the EDTA equivalence determined during the spring. The u l t r a - v i o l e t EDTA serie s measures the r e l a t i o n s h i p betivreen the metals and the organism as a function of the amount of complexing which i s present i n the natural water. Because of differences i n "tneibiological properties of the various metals, the organisms respond to each i n a c h a r a c t e r i s t i c manner. For example, copper i s highly t o x i c when present i n an i o n i c form (Pagenkopf et a l . , 1974), but r e l a t i v e l y innocuous when present i n an organic complex (Lewis et a l . , 1972; Lewis and W h i t f i e l d , 1974). Other metals may act i n a s i m i l a r manner or may be t o t a l l y d i s s i m i l a r . Some metals which may be bound to organic material seem to be b e n e f i c i a l when present i n an i o n i c form (e.g., i r o n , Lewis unpublished) or may have l i t t l e or no e f f e c t (e.g., manganese). Copper, z i n c , and manganese were used i n attempts at measuring t o t a l complexing a b i l i t y and i n attempts at under-standing the r e l a t i o n s h i p between the organism and various metals. 67 The concentration of EDTA which produced a s u r v i v a l i n u l t r a - v i o l e t treated water equivalent to that which occurred i n untreated water may not be a measure of a l l the complexing material present i n natural waters. EDTA enters into stable complexes on a 1:1 molar basis with the majority of t r a n s i t i o n metals (Martell and Ca l v i n , 1959), while natural organic material may be present i n as great as 6:1 (molar) organic:metal complexes depending upon the nature of both the organic material and the metal. A d d i t i o n a l l y , . i t i s possible that some organic material complexes metals but does not occupy a l l of the coordination s i t e s on the metal and thus may not change the properties of the metal s u f f i c i e n t l y to a l t e r i t s b i o l o g i c a l e f f e c t . This complexing a b i l i t y i s impossible to measure at present and w i l l l i k e l y remain so u n t i l chemical techniques are refined to allow t h i s type of re s o l u t i o n . The addition of any of the metals used (copper, z i n c , and manganese) to water which was not treated with u l t r a - v i o l e t l i g h t always caused a de-crease i n s u r v i v a l . This r e l a t i o n s h i p does not hold f o r water which has been treated with u l t r a - v i o l e t l i g h t , as manganese often increased the s u r v i v a l as did one concentration of copper (Table 3). Both manganese and zinc also a l t e r e d the r e l a t i o n s h i p of the s u r v i v a l of the organism to the concentration of EDTA (as evidenced by changes i n slope). S u r v i v a l i n u l t r a -v i o l e t treated water i s not changed by the addition of manganese when the EDTA concentration i s zero, but i s decreased at higher EDTA concentrations. This indicates that the metal i s l i k e l y non-toxic as opposed to the e f f e c t of the addition of manganese to untreated water. Further evidence of t h i s i s shown i n those experiments where manganese and copper were added together. The t o x i c e f f e c t of copper was reduced by the presence of manganese (see Table 3). Manganese i s believed to a l t e r the a v a i l a b i l i t y of a p a r t i c u l a r 68 metal by competing f o r the EDTA as i t s concentration increases. Zinc decreases the toxic e f f e c t which r e s u l t s from the u l t r a - v i o l e t treatment. The slope of the l i n e r e l a t i n g organism s u r v i v a l to EDTA concentration under a zinc addition i s e s s e n t i a l l y zero. This e f f e c t may be due to the i n t e r -action of the added zinc with the EDTA. Zinc i t s e l f has a low tox i c e f f e c t on the organism and, i n f a c t , might be s l i g h t l y b e n e f i c i a l . However, i t has a high s t a b i l i t y constant (pk g = 16.5) f o r the formation of a complex with EDTA. At low l e v e l s of EDTA, zinc acts i n a manner which does not a f f e c t the organism. As the l e v e l of EDTA increases there i s s u f f i c i e n t zinc present to allow 25% of the EDTA to e x i s t i n a complex with the added zinc . This r e l a t i o n s h i p should develop i n a l i n e a r fashion. It appears, however, that a break does occur. Possibly, as the EDTA concentration i n -creases i n i t i a l l y i t i s bound almost e n t i r e l y to the added zi n c (and other metals of higher pk g) . Above a c e r t a i n l i m i t t h i s e f f e c t changes, i n that the increase i n EDTA concentration does not bind any further z i n c . The EDTA then begins to bind metals which have lower s t a b i l i t y constants, causing the s u r v i v a l of the organism to decrease proportionally. This may be a r e s u l t of the binding of a p a r t i c u l a r metal which the organism requires. +2 The addition of Cu 1 (1 yg/1 Cu ) causes a consistent but not s i g n i -f i c a n t decrease i n s u r v i v a l over the range of EDTA concentrations which were used. This i s thought to be the r e s u l t of two features of the system. The toxic e f f e c t of the copper addition i s well known (e.g., Lewis et a l . , 1972) but i s decreased by the increase of EDTA concentration (Lewis et a l . , 1972). The e f f e c t of increasing the EDTA concentration i n treated seawater i s to increase s u r v i v a l of the organism. When considered together i t i s apparent that the l i n e s should be p a r a l l e l because the binding of copper by EDTA has the highest s t a b i l i t y constant of a l l the metals considered and i n t h i s 69 respect r e s u l t s i n a pattern s i m i l a r to that obtained with zinc. From t h i s i t may be suggested the pattern of s u r v i v a l which r e s u l t s subsequent to a metal addition i s dependant upon two d i s t i n c t f a c t o r s : the s t a b i l i t y of the complex which i s formed between the metal and the EDTA used as a standard, and; the e f f e c t of the metal i n unenriched water. This i s only a supposition at present and i s an area which should be considered f o r further research, e s p e c i a l l y i n l i g h t of the r e s u l t s obtained with increasing copper l e v e l s . The s u r v i v a l of the organism i n untreated water decreased as the r e s u l t of copper additions, and can be shown to be d i r e c t l y proportional to the concentration of copper which was added (e.g., Table 3, May r e s u l t s f or untreated water). In u l t r a - v i o l e t treated water t h i s was not the case. The +2 addition of Cu 1 (1 yg/1 Cu ) did not change the r e l a t i o n s h i p between the , s u r v i v a l of the organism and the concentration of EDTA. If one considers the r e s u l t s found for untreated water, one would expect that the s u r v i v a l decrease obtained with increasing copper concentrations would be proportional to the amount of copper which was added. However, t h i s i s not the case. +2 The addition of Cu 2 (2 pg/1 Cu ) caused a s i g n i f i c a n t increase i n s u r v i v a l at low EDTA l e v e l s and a s i g n i f i c a n t decrease i n s u r v i v a l at high l e v e l s , when compared with u l t r a - v i o l e t treated seawater (e.g., Table 3, A p r i l and May, 1974). At higher concentrations of copper (Cu 3, Cu 5), increasing EDTA concentration again resulted i n an increase i n the s u r v i v a l of the organism, but s u r v i v a l was s i g n i f i c a n t l y lower through the e n t i r e range of EDTA concentrations when compared to u l t r a - v i o l e t water which was not enriched with copper. The mechanism which i s responsible f o r these r e s u l t s i s not known, but suggests a further regulatory mechanism of organic material which i s present i n natural waters. 70 SUMMARY 1. Major changes i n the abundance of the prefeeding stages of Euchaeta  japonica and i n the concentrations of trace metals occur" simultaneous to intrusions of water into Indian Arm. 2. Subsequent to winter intrusions there i s an increase i n the complexation capacity of the organic material present i n the water. 3. The summer intrusion causes mixing of the properties measured through the deeper portion of the water column.-and results i n a decrease i n the complexing capacity of the water. 4. The toxic effect of a copper enrichment changes with time increasing from the predicted l e v e l at the time of the int r u s i o n , reaching a spring maximum, and then decreasing to the predicted l e v e l i n July. 5. The a b i l i t y of material extracted from sediments to reduce copper t o x i c i t y changes through the year being highest i n early f a l l and lowest i n the late winter. 6. The a c t i v i t y of sediment extracts i s related to the production of organic material i n the nearsurface waters. 7. Laboratory experiments and measurements show that organic material present i n natural waters i s capable of cont r o l l i n g the a v a i l a b i l i t y of trace metals to Euchaeta japonica developmental stages. These include: 71 (i) Ul t ra -viole t i rradiat ion of seawater destroys the organic material which is present, but does not alter the other components of the water, ( i i ) Decrease i n survival of the organism results i n water which has been treated with ul t ra -v iole t l i g h t , and the decrease is proportional to the amount of organic material which is destroyed, ( i i i ) Additions of several metals alter the relationship between the organism and the complexing a b i l i t y of the water i n experimental conditions. 8. The complex effect of organic material on the a v a i l a b i l i t y of metals prevents the prediction of the abundance of Euchaeta japonica prefeeding stages on the basis of one or more of the properties of the water. 72 REFERENCES Al b e r t , A. 1950. Quantitative studies of the a v i d i t y of, n a t u r a l l y occurring substances f o r trace metals. 1. Amino acids having only two i o n -i z i n g groups. Biochem. J . 47_: 531-538. Al b e r t , A. 1952. Quantitative studies of the a v i d i t y of n a t u r a l l y occurring substances f o r trace metals. 2. Amino acids having three i o n i z i n g groups. Biochem. J. 50:691-697. Anderson, W.T. 1947. Photosensitization i n c h l o r i n a t i o n . Ind. Eng. Chem. 39:844-846. Anderson, W.T. 1955. Lamp equipment for photochemical processes: employs high i n t e n s i t y mercury vapour arc. Chem. Eng. Prog. 51_:571-572. Armstrong, F.A.J., P.M. Williams, and J.D.H. Str i c k l a n d . 1966. Photo-oxidation of organic material i n seawater by u l t r a - v i o l e t r a d i a t i o n , a n a l y t i c a l and other a p p l i c a t i o n s . Nature 211:481-483. Avakyan, A.Z. 1971. Comparitive t o x i c i t y of free ions and complexes of copper and amino acids to Canida u t i l i s . M i c r o b i o l . 40:363-368. Avakyan, Z.A. and I.L. Rabotnova. 1971. Comparitive t o x i c i t y of free ions and complexes of copper with organic acids f or Canida u t i l i s . M i c r o b i o l . 40:262-266. Barber, R.T. and J.H. Ryfcher. 1969. Organic chelators: Factors a f f e c t i n g primary production i n the Cromwell Current upwelling. J. Exp. Mar. B i o l . E c o l . 3:191-199. Barber, R.T., R.C. Dugdale, J . J . Maclssac, and R.L. Smith. 1971. Variations i n phytoplankton growth associated with the source and conditioning of upwelling water. Invest. Pesq. 35:171-193. Barsdate, R.J. 1970. T r a n s i t i o n metal binding by large molecules i n high l a t i t u d e waters. In: D.W. Hood, edt. Symposium on Organic Matter i n Natural Waters. Univ. Alaska. Occ. Publ. No. 1. pp 485-496. Beatt i e , J . , C. Bricker, and D. Garvin. 1961. Ph o t o l y t i c determination of trace amounts of organic material i n water. Anal. Chem. 30:1890-1892. Campbell, M.H. 1934. The l i f e h i s t o r y and post embryonic development of the copepods Calanus tonus and Euchaeta japonica Marukawa. J. Mar. B i o l . Bd. Can. l_:l-65. C a r r i t t , D.E. and J.H. Carpenter. 1966. oComparisora^andsevaluaticm of currently employed modifications i n the Winkler method f or determining dissolved oxygen i n seawater.: a NASCO Report. J . Mar. Res. 24:286-318. Clarke, G.L. and D.F. Bumpus. 1940. The plankton sampler - an instrument f o r quantitative plankton i n v e s t i g a t i o n s . Limnol. Ocean. Spec. Publ. No. 5. 73 Davey, E.W., M.J. Morgan, and S.J. Erickson. 1973. A b i o l o g i c a l measurement of the copper complexation capacity of seawater. Limnol. Ocean. 18: 993-997. Davis, C G . 1949. The pelagic copepoda of the Northeastern P a c i f i c Ocean. Univ. Wash. Publ. B i o l . 14:1-118. Erickson, P.E. 1973. The voltammetric determination of copper and lead i n seawater: Applications to Indian Arm and Burrard I n l e t . {M.Sc.Thesis, Chem., Univ. B r i t . Col. 84pp. Evans, M.S. 1973. The d i s t r i b u t i o n a l ecology of the calanoid copepod Paraeuchaeta elongata E s t e r l y . Ph.D. Thesis, Zool., Univ. B r i t . Col. 112pp. Fogg, G.E. 1958. E x t r a c e l l u l a r products of phytoplankton and the estimate of primary production. Rapp. Pv. Cons. Int. Explor. Mer. 144:56-60. Fogg, G.E. 1966. The e x t r a c e l l u a l a r products of algae. OOcean.'-'iMar. B i o l . Ann. Rev. 4:195-212. • Gardner, G.A. 1972. The d i s t r i b u t i o n of the l i f e h i s t o r y stages of Calanus  plumchrus Marukawa (Copepoda:Calanoida) i n the S t r a i t of Georgia. M.Sc. Thesis, Zool., Univ. B r i t . Col. 55pp. G i l f i l l a n , E.S. 1970. The e f f e c t of changes i n temperature, s a l i n i t y , and undefined properties of seawater on the r e s p i r a t i o n of Euphausia  p a c i f i c a Hansen (Crustacea) i n r e l a t i o n to the species ecology. Ph.D. Thesis, Zool., Univ. B r i t . Col. 126pp. Gilmartin, M. 1962. Annual c y c l i c a l changes i n the phy s i c a l oceanography of a B r i t i s h Columbia f j o r d . J. F i s h . Res. Bd. Can. 19:921-974. Gilmar t i n , M. 1964. The primary production of a B r i t i s h Columbia f j o r d . J . F i s h . Res. Bd. Can. 21:505-538. Gross, M.G., A.G. Carey, G.A. Fowler, and L.D. Kulm. 1972. D i s t r i b u t i o n of organic carbon i n surface sediment, Northeast P a c i f i c Ocean. In: A.T. Pruter and D.L. Alverson, edt. The Columbia River Estuary and Adjacent Ocean Waters. Univ. Wash. Press. Seattle, pp. 481-483. I n s t i t u t e of Oceanography. 1972. U n i v e r s i t y of B r i t i s h Columbia. Data Report No. 34. 96pp. I n s t i t u t e of Oceanography. 1973. Un i v e r s i t y of B r i t i s h Columbia. Data Report. In Press. I n s t i t u t e of Oceanography. 1974. U n i v e r s i t y of B r i t i s h Columbia. Data Report. In Preparation. Johnson, R. 1964. Seawater, the natural medium of phytoplankton. I I . Trace metals and chelation, and general discussion. J . Mar. B i o l . Assn. U.K. 44:87-109. 74 K h a i l o v , K.M. a n d Z.Z. F i n e n k o . 1 9 7 0 . O r g a n i c m a c r o m o l e c u l a r c o m p o u n d s d i s s o l v e d i n s e a w a t e r a n d t h e i r i n c l u s i o n i n t o f o o d c h a i n s . I n : J . H . S t e e l e , e d t . M a r i n e F o o d C h a i n s , p p . 6-18. L e w i s , A.G. a n d A. R a m n a r i n e . 1 9 6 9 . Some c h e m i c a l f a c t o r s a f f e c t i n g t h e e a r l y d e v e l o p m e n t a l s t a g e s o f E u c h a e t a j a p o n i c a M a r u k a w a . J . F i s h . R e s . B d . C a n . 2 ( 6 : 1 3 4 7 - 1 3 6 2 . L e w i s , A.G. a n d P.H. W h i t f i e l d . 1 9 7 4 . T h e b i o l o g i c a l i m p o r t a n c e o f c o p p e r i n t h e s e a . A l i t e r a t u r e r e v i e w . I N C R A C o n t r a c t R e p o r t # 2 2 3 . 1 3 2 p p . L e w i s , A.G., A. R a m n a r i n e , a n d M.S. E v a n s . 1 9 7 1 . N a t u r a l c h e l a t o r s - a n i n d i c a t i o n o f a c t i v i t y w i t h t h e c a l a n o i d c o p e p o d E u c h a e t a j a p o n i c a . M a r . B i o l . 1 1 : 1 - 4 . L e w i s , A.G., P.H. W h i t f i e l d , a n d A. R a m n a r i n e . 1 9 7 2 . Some p a r t i c u l a t e a n d s o l u b l e a g e n t s a f f e c t i n g t h e r e l a t i o n s h i p b e t w e e n m e t a l t o x i c i t y a n d o r g a n i s m s u r v i v a l i n t h e c a l a n o i d c o p e p o d E u c h a e t a j a p o n i c a . M a r . B i o l . 1 7 : 2 1 5 - 2 2 1 . L e w i s , A.G., P.H. W h i t f i e l d , a n d A. R a m n a r i n e . 1 9 7 3 . R e d u c t i o n o f c o p p e r t o x i c i t y t o a m a r i n e c o p e p o d b y s e d i m e n t e x t r a c t . L i m n o l . O c e a n . 1 8 : 3 2 4 - 3 2 7 . L e w i s , G . J . a n d E.D. G o l d b e r g . 1 9 5 4 . I r o n i n m a r i n e w a t e r s . J . M a r . R e s . 1 3 : 1 8 3 - 1 9 7 . L u c a s , C E . 1 9 4 7 . T h e e c o l o g i c a l e f f e c t s o f e x t e r n a l m e t a b o l i t e s . B i o l . R e v . 2 2 : 2 7 0 - 2 9 5 . L u c a s , C E . 1 9 4 9 . E x t e r n a l m e t a b o l i t e s a n d e c o l o g i c a l a d a p t a t i o n . Symp. S o c . E x p . B i o l . 3 _ : 3 3 6 - 3 5 6 . L u c a s , C E . 1 9 5 8 . E x t e r n a l m e t a b o l i t e s a n d p r o d u c t i v i t y . R a p p . P v . C o n s . I n t . E x p l o r . M e r . 1 4 4 : 1 5 5 - 1 5 8 . M a r c h a n d , M. 1 9 7 4 . C o n s i d e r a t i o n s s u r l e s f o r m e s p h y s i c o - c h e m i q u e s d u c o b a l t , m a n g a n e s e , z i n c , c h r o m e e t f e r d a n s u n e e a u d e m e r e n r i c h i e o u n o n d e m a t i e r e o r g a n i q u e . J . C o n s . I n t . E x p l o r . M e r . 3 5 : 1 3 0 - 1 4 2 . M a r t e l l , A . E . a n d M. C a l v i n . 1 9 5 9 . C h e m i s t r y o f t h e M e t a l C h e l a t e C o m p o u n d s . P r e n t i c e H a l l . N . J . 6 1 3 p p . M e n z e l , D.W. a n d R . F . V a c c a r o . 1 9 6 4 . T h e m e a s u r e m e n t o f d i s s o l v e d o r g a n i c a n d p a r t i c u l a t e c a r b o n i n s e a w a t e r . L i m n o l . O c e a n . 9_:138-142. O l a f s o n , R.W. a n d J . A . J . T h o m p s o n . 1 9 7 4 . I s o l a t i o n o f h e a v y m e t a l b i n d i n g p r o t e i n s f r o m m a r i n e v e r t e b r a t e s . M a r . B i o l , i n p r e s s . P a g e n k o p f , C K . , R . C R u s s o , a n d R.V. T h u r s t o n . 1 9 7 4 . E f f e c t o f c o m p l e x a t i o n o n t o x i c i t y o f c o p p e r t o f i s h e s . J . F i s h . R e s . B d . C a n . 3 1 : 4 6 2 - 4 6 5 . P a n d y a n , A . S . 1 9 7 1 . F o o d a n d t r o p h i c r e l a t i o n s h i p s o f t h e d e v e l o p m e n t a l 75 stages of Euchaeta japonica Marukawa and Calanus plumchrus Marukawa. Ph.D. Thesis. Zool., Univ. B r i t . Col. 195pp. Phelps, D.K., R.J. Santiago, D. Luciano, and N. B r i z a r r y . 1969. Trace element composition of insore and offshore benthic populations. Proc. Sec. Nat. Symp. Radioecology. pp. 774. Pickard, G.L.S 1961. Oceanographic features of i n l e t s i n the B r i t i s h Columbia mainland coast. J . F i s h . Res. Bd. Can. 18:907-999. Rashid, M.A. 1972. Quinone content of humic compounds i s o l a t e d from the marine environment. S o i l S c i . 113:181-188. Regan, L. 1963. F i e l d t r i a l s with the Clarke-Bumpus plankton sampler. IOUBC Manus. Rept. No. 16. 28pp. R i l e y , G.A. 1963. Organic aggregates i n seawater and the dynamics of t h e i r u t i l i z a t i o n . Limnol. Ocean. 8^:372-381. Shah, N.M. and G.E. Fogg. 1973. The determination of g l y c o l l i c acid i n seawater. J. Mar. B i o l . Assn. U.K. 53:321-378. Slowey, J.F. and D.W. Hood. 1971. Copper, manganese, and zinc concentrations i n Gulf of Mexico water. Geochim. Cosmochim. Acta. 35:121-138. Slowey, J.F., L.M. J e f f r e y , and D.W. Hood. 1967. Evidence f or organic complexed copper i n seawater. Nature 214:377-378. Spencer, C.P. 1958. The chemistry of EDTA i n seawater. J . Mar. B i o l . Assn. U.K. 37_:127-144. Steeman-Neilsen, E. and S. Wuim-Anderson. 1970. Copper ions as poison i n the sea and freshwater. Mar. B i o l , (K93-97. Str i c k l a n d , J.D.H. 1972. Research on the marine planktonic food web at the I n s t i t u t e of Marine Resources: A review of the past seven years of work. Ocean. Mar. B i o l . Ann. Rev. 10:349-414. St r i c k l a n d , J.D.H. and T.R. Parsons. 1972. A P r a c t i c a l Handbook of Seawater  Analysis. F i s h . Res. Bd. Can. B u l l . 167 (Second Edition) 310pp. Tabata, K. and K. Nisikawa. 1969. Studies on the t o x i c i t y of heavy metals to aquatic organisms and the factors which decrease the t o x i c i t y . V. A t r i a l to decrease the t o x i c i t y of heavy metal ions by the addition of complexing agents. B u l l . Tokai Reg. Rish. Res. Lab. 58:225-264. Tranter, D.J. and A.C. Heron. 1965. F i l t r a t i o n c h a r a c t e r i s t i c s of Clarke-Bumpus samplers. Aust. J. Mar. Fresh. Res. 16:281-291. Trask, P.D. 1939. Organic content of recent marine sediments. In: P.D. Trask, edt. Recent Marine Sediments, pp 428-453. Williams, P.M. 1968. The as s o c i a t i o n of copper with dissolved organic material i n seawater. Limnol. Ocean. 14:156-158. 76 W i l l i a m s , P.M. 1 9 6 8 . S t a b l e c a r b o n i s o t o p e s i n t h e d i s s o l v e d o r g a n i c m a t t e r o f t h e s e a . N a t u r e 2 1 9 : 1 5 2 - 1 5 3 . W i l l i a m s , P.M., H. O s e c h g e r , a n d P. K i n n e y . 1 9 6 9 . N a t u r a l r a d i o c a r b o n a c t i v i t y o f t h e d i s s o l v e d o r g a n i c c a r b o n i n t h e N o r t h e a s t P a c i f i c O c e a n . N a t u r e 2 2 4 : 2 5 8 - 2 6 5 . Y e n t s c h , C.S. a n d A.C. D u x b u r y . 1 9 5 6 . Some o f t h e f a c t o r s a f f e c t i n g t h e c a l i b r a t i o n n u m b e r o f t h e C l a r k e - B u m p u s q u a n t i t a t i v e p l a n k t o n s a m p l e r . L i m n o l . O c e a n . l _ : 2 6 8 - 2 7 3 . Z i r i n o , A. a n d M.L. H e a l y . 1 9 7 0 . I n o r g a n i c z i n c c o m p l e x e s i n s e a w a t e r . L i m n o l . O c e a n . 1 5 : 9 5 6 - 9 5 8 . Z i r i n o , A. a n d S. Y a m a m o t o . 1 9 7 2 . A p H - d e p e n d a n t m o d e l f o r t h e c h e m i c a l s p e c i a t i o n o f c o p p e r , z i n c , c a d m i u m , a n d l e a d i n s e a w a t e r . L i m n o l . O c e a n . 1 7 : 6 6 1 - 6 7 1 . 77 APPENDIX Description of the u l t r a - v i o l e t lamp apparatus The lamp used (Hanovia 189A-16) i s a high i n t e n s i t y , 12 inch mercury arc under medium pressure i n a clear fused quartz chamber. Clear fused quartz transmits the complete u l t r a - v i o l e t spectrum. This lamp operates at an input wattage of 1200. The following gives an i n d i c a t i o n of the output of the lamp; 13673-10140 A ( i n f r a red) 48.68 watts, 5780-4045 £ ( v i s i b l e ) 187.07 watts, 3660-3341 X (near u l t r a - v i o l e t ) 104.03 watts, 3130-2804 X (medium u l t r a - v i o l e t ) 117.01 watts, and 2753-2224 X (far u l t r a - v i o l e t ) 116.15 watts. The maximum output of t h i s lamp i s at 3660 X. The lamp has a s t a i n l e s s s t e e l tube, 5/16" O.D. extending the length of the lamp assembly. This tube c a r r i e s a flow of nitrogen gas (99% pure) to the bottom of the w e l l , purging the inner w e l l . Approximately 6 lbs/sq. i n . p o s i t i v e pressure i s maintained i n the inner well to prevent the seepage of other materials into the lamp chamber. The nitrogen provides a neutral environment, permitting the lamp to operate at high temperatures without the oxidation of the external lamp parts. The flow of nitrogen into t h i s chamber i s c o ntrolled by a regulator on the tank which supplies the nitrogen. Pressure i s maintained i n the inner w e l l with the aid of a needle valve mounted on the cover of the w e l l head assembly, A l i n e runs from t h i s valve into an erlenmeyer f l a s k containing water. This allows an i n d i c a t i o n of the amount of nitrogen which i s passing through the inner w e l l . The en t i r e w e l l head assembly i s constructed of corrosion r e s i s t a n t aluminum. The explosion proof junction box contains the high voltage termi-nals f o r the lamp connections. The w e l l head assembly supports the inner and outer immersion wells. An i n l e t and an outlet i n the w e l l head assembly 78 allow the c i r c u l a t i o n of water as a coolant. A piece of tygon tubing i s connected to the i n l e t i n the well head assembly and the other end i s anchored to a centering spring at the lower end of the inner w e l l ensuring the d e l i v e r y of water to the annular space at the base of the immersion w e l l assembly. C i r c u l a t i o n of water between the immersion tubes at a rate of 1 to 2 gallons per minute and at 35 lbs/sq. i n . transfers the heat produced by the lamp into the outlet flow. This water i s f i r s t passed through a "Cuno" f i l t e r (5 micron porosity) to help prevent the formation of deposits between the immersion wells. The cooling system i s safeguarded against f a i l u r e by the i n c l u s i o n of an automatic pressure switch arranged to extinguish the lamp i f a water pressure f a i l u r e occurs. The flow of water also serves to prevent the trans f e r of heat produced by the lamp to the sample being treated. The immersion well assembly consists of two concentric tubes of Corning #7010 Vycor, a quartz l i k e glass (96% s i l i c a ) . Vycor transmits u l t r a -v i o l e t l i g h t more r e a d i l y than does either Corex or Pyrex, allowing 92% trans-mission at 3500 R. The power supply f o r the lamp i s an o i l immersed transformer providing stable voltage i n the range of 200-240 v o l t s and 7 amps. A d d i t i o n a l portions of the e l e c t r i c a l system include the following. A pressure reducing valve prevents water pressure i n the immersion tubes from exceeding 37 lbs/sq. i n . A solenoid valve i s mounted on the water l i n e before i t enters the immersion wells, and i s interconnected to the e l e c t r i c a l supply i n a manner which allows water flow to occur independant of the operation of the lamp. A pressure switch on the water outflow l i n e responds to a pressure diff e r e n c e of 4 l b s / sq. i n . , shutting the lamp power off i n the event of a pressure drop. Two pressure gauges are mounted near the i n l e t and outlet ports on the w e l l head 79 assembly, allowing water pressure to be checked v i s u a l l y . The power supply to the lamp i s wired through a timing clock. This allows the duration of the treatment of the water to be ea s i l y controlled. The entire assembly i s mounted i n a controlled environment chamber maintained at 8°C. The u l t r a - v i o l e t lamp immersion tube assembly i s suspended i n a manner which allows i t to s i t within a 50 l i t e r chromatographic chamber. This chamber i s used for holding the seawater during treatment. The chamber i s covered with four pieces of plate glass f i t t e d i n a manner which reduces the p o s s i b i l i t y of contamination. With the immersion tube i n place the chamber holds 44 l i t e r s of seawater for treatment. Water i s transferred to and from the treatment chamber by siphoning through a 3/4" I.D. nylon tube, although a tygon tube of the same diameter was used during the l a t t e r portion of the study. Further d e t a i l s concerning the operational portions of the lamp can be obtained from Hanovia Lamp D i v i s i o n , Canrad Precision Industries, Newark, New Jersey. 

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