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Comparison of the responses of benthic and planktonic communities to enrichment with inorganic fertilizers Cameron, Roderick L. 1973

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9 § ^ A COMPARISON OF THE RESPONSES OF BENTHIC AND PLANKTONIC COMMUNITIES TO ENRICHMENT WITH INORGANIC FERTILIZERS by RODERICK L. CAMERON B.Sc., U n i v e r s i t y of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1973 . ANIM At RESOURCE ECOLOGY LIBRARY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER 8, B. C. CANADA In p resent ing 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree 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 fo r reference and study. I f u r t h e r agree tha t permiss ion for e x t e n s i v e copying 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 granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8. Canada Date DEL nm i ABSTRACT A comparison was made of the responses of benthic and planktonic components of natural water-sediment systems enriched with inorganic f e r t i l i z e r s . Three le v e l s of f e r -t i l i z a t i o n were applied to a series of enclosures placed in a shallow ( 1 m depth) area of Marion Lake, B r i t i s h Col-umbia and community r e s p i r a t i o n , primary production, stand-ing a l g a l crop and the d i s t r i b u t i o n of added phosphorus were measured in both the sediment and the water column over a period of two years. A loss of added phosphorus from the water column cor-responded to an increase measured i n the sediment. Uptake and release of phosphorus by the sediment was proportional to i t s concentration i n the water column, i n d i c a t i n g a water-sediment equilibrium. In an undisturbed system, how-ever, there was a net movement of phosphorus into the sedi-ment . A sustained high l e v e l of planktonic primary production persisted throughout the period of f e r t i l i z a t i o n . However, an i n i t i a l increase in t o t a l benthic primary production re-turned to p r e - f e r t i l i z a t i o n levels following the e s t a b l i s h -ment of an increased standing crop of e p i p e l i c algae. At th i s point, benthic primary production appeared to be a func-t i o n of grazing pressure, responding to increased grazing but not to addit i o n a l f e r t i l i z a t i o n . When grazing was experimen-t a l l y increased by concentrating grazers in an experimental area, i i b e n t h i c p r i m a r y p r o d u c t i o n i n c r e a s e d . F e r t i l i z e d s e d i m e n t a p p e a r e d to have a much g r e a t e r a b i l i t y t o w i t h s t a n d i n -c r e a s e d g r a z i n g p r e s s u r e t h a n n o r m a l s e d i m e n t , m a i n t a i n i n g h i g h l e v e l s of p r i m a r y p r o d u c t i o n even a t f i v e t i m e s n o r m a l g r a z e r d e n s i t y . But d e s p i t e t h i s p o t e n t i a l , measurements of g r a z e r and b a c t e r i a p o p u l a t i o n s w i t h i n t h e e x p e r i m e n t a l e n c l o s u r e s showed no r e s p o n s e t o t h e g r e a t e r s t a n d i n g c r o p of e p i p e l i c a l g a e . As a r e s u l t , s u s t a i n e d i n c r e a s e i n b e n t h i c p r i m a r y p r o d u c t i o n c o u l d be i n d u c e d o n l y e x p e r i m e n t - , a l l y . I t was c o n c l u d e d t h a t t h e b e n t h i c community was more s t a b l e i n i t s r e s p o n s e t o e n r i c h m e n t t h a n t h a t of t h e p l a n k -t o n and by a b s o r b i n g n u t r i e n t s f r o m t h e w a t e r column s e r v e d to dampen t h e e f f e c t s of t h e more p r o n o u n c e d f l u c t u a t i o n s of t h e p l a n k t o n i c community. i i i TABLE OF CONTENTS P a g e INTRODUCTION 1 METHODS 3 RESULTS 14 Distribution of Added Phosphorus 174 Response of Algae 2$ Response by Grazers 4'3 DISCUSSION LITERATURE CITED $3 APPENDIX I 6 2 Some problems in the use of antibiotics to measure respiration in lake sediments. LIST OF FIGURES Page Fig. 1 Contour map of Marion Lake, B. C. indicating position of experimental enclosures. F i g . 2 S;etr&ene_d3Uop.eni:ng£ t&i^toacsjhjufrtejSiSt&nthe 7 enclosure w a l l s . Fig. 3 Oxygen levels in the experimental enclosures 16 prior to production estimation. (June, 1970) Fig. 4 Ortho phosphate measurements in the water of 19 the large enclosures. Fig. 5 Ortho phosphate concentrations in the water 21 of the two enclosures over time following the addition of two different amounts of f e r t i l i z e r . Fig. 6 Loss of phosphorus from water column as a 23 function of i n i t i a l concentration. Fig. 7 Phosphorus measured in the sediments of the 26 experimental enclosures. Fig. 8 Planktonic primary production in the experi- 29 mental enclosures following f e r t i l i z a t i o n . (July, 1970) Fig. 9 Benthic algal standing crop in the experi- 32 mental enclosures. Fig. 10 Benthic primary production in the experi- 34 mental enclosures. (July, 1970) Fig. 11 Benthic primary production in the experi- 36 mental enclosures. (September, 1969) Fig. 12 Relation of benthic algal standing crop to production of individual algal unit. V Page F i g . 13 Response of benthic primary production to 42 increased grazing by H y a l e l l a . F i g . 14 Benthic community r e s p i r a t i o n i n experi- 46 mental enclosures. (July, 1970) F i g . 15 B a c t e r i a l pla^e count in the experimental 48 enclosures 10 d i l u t i o n . (August, 1970) v i ACKNOWLEDGEMENTS F u n d i n g of my r e s e a r c h , p a r t of t h e M a r i o n Lake P r o j e c t , was p r o v i d e d by t h e C a n a d i a n I n t e r n a t i o n a l B i o l o g i c a l Programme, as w e l l as by s c h o l a r s h i p s f r o m t h e N a t i o n a l R e s e a r c h C o u n c i l , and f o r t h e s e I am g r a t e f u l . I am i n d e b t e d to my s u p e r v i s o r , Dr. Ian E. E f f o r d , f o r t h e s u p p o r t , a d v i c e and encouragement he e x t e n d e d d u r i n g t h e c o u r s e of my r e s e a r c h . My t h a n k s a r e a l s o due t o M e s s r s . Kim H y a t t . a n d K a n j i Tsumura f o r time s p e n t a s s i s t i n g me i n t h e f i e l d . D r. Z d z i s ^ a w K a j a k p r o v i d e d v a l u a b l e s u g g e s t i o n s and a s s i s t e d by r e v i e w i n g my r e s u l t s . 1 INTRODUCTION Hutchinson (1957) has described as "one of the most impor-tant discoveries that have ever been made in limnology" the conclusion by Einsele (1941) that a lake "operates as a self regulatory system" capable of re-stabilizing i t s e l f following the temporary disturbance of one of its operating variables. Margalef (1968) has defined such stability as "the a b i l i t y of the system to remain reasonably similar to i t s e l f inspite of... exogenous variations through agents outside the system the system has a greater resistance to changes that are external in their origin". The contemporary importance of Einsele's dis-covery in terms of Margalefs definition of stability may easily be seen in man's disruption of natural processes in many bodies of fresh water. The most typically disturbed variable is that of inorganic nutrients. Sewage, agricultural and industrial contributions have increased the phosphorus and nitrogen content of many lakes far beyond normally encountered levels. Where existing community structure was unable to absorb such an increase new communities have developed in the place of the previous ones. These community shifts often produce extremely undesirable consequences (Fruh, 1966). Natural eutrophication is a long process of gradual nutri-ent enhancement and corresponding community adjustment. Rapid unnatural additions, however, are more similar to the "temporary 2 disruptions" described by Einsele. The response to such additions should thus provide a measure of stability in the various components of a fresh water system. Dramatic eutro-phication effects are characteristically planktonic: the for-mation of algal mats, increases in bacterial action and oxygen depletion have a l l been well documented in the water column (Vollenweider, 1968). But i t is commonly believed (Margalef, 1963) that the benthic community is a more mature and there-fore more stable one than that of the plankton. In this study a comparison was made of the responses of the benthic and planktonic components of enclosed water sediment systems en-riched to various degrees with inorganic f e r t i l i z e r s . The results indicate that such a stability difference does exist and that this confers upon the community as a whole an a b i l i t y to resist structural changes at least on a short term basis. 3 METHODS This study was made in Marion Lake, situated in the coastal mountains of south western British Columbia. This lake is small (13.3 ha.) and shallow (mean depth 2.2 m maximum depth 6 m), and the bottom consists almost entirely of deep soft sediment covered with a mat of algae and chir-onomid tubes. The small volume of the lake combined with frequently rapid run off results in what is often extremely high flushing: indeed, i t has been suggested (Dickman, 1969) that during periods of high flow, the water body more closely approximates the widening of a stream than a lake. A more complete description is given by Efford (1967). Ferti l i z a t i o n experiments were conducted in an area of uniform ( l m) depth and substratum on the east side of the lake (Fig. 1). Eight enclosures constructed of a %" pipe framework with polyethylene walls were placed in this area adjacent to two control plots as indicated. These square 2 enclosures were 16 m in area and 2 m in depth; they were pressed into the sediment to a depth of approximately .5 m. Each enclosure thus enclosed a mean volume of 16 m of water subject to lake level fluctuation. Screened openings in two walls of each enclosure (Fig. 2b) were fitted with shutters which allowed the regulation of current flow through the en-closures. Quantitative experiments of P0A uptake and release 4 Fig. 1 Contour map of Marion Lake, B. C. indicating posi-tion of experimental enclosures. Figures on contour lines refer to depth in metres. Enclosed numbers in this and subsequent figures refer to enclosure identification. 1 and 2 - external control areas 7 and 3 - enclosed controls 4 and 8 - low-level f e r t i l i z a t i o n 5 and 9 - mid-level f e r t i l i z a t i o n 6 and 10 - high-level f e r t i l i z a t i o n a and b - small enclosures (see text) prevailing wind outlet 1 2 6 F i g . 2 S c r e e n e d o p e n i n g s w i t h s h u t t e r s i n t h e e n c l o s u r e w a l l s . 7 8 were accomplished in a further set of enclosures. These plywood structures were 1 m square and had sliding sides which could be removed to expose the enclosed sediment to the f u l l flushing of the water current. They were lo-cated adjacent to the larger enclosures in positions marked "a" and "b" in (Fig. 1). Three levels of f e r t i l i z a t i o n were applied, in dupli-cate, to six of the large enclosures over a period of four months through the summer of 1970. The remaining two en-closures were l e f t untreated as were two adjacent unenclosed plots of sediment: these served respectively as the enclosed and unenclosed controls. As the prevailing winds produced dr i f t in a northerly direction the treatment pairs were aligned in a linear manner such that any f e r t i l i z e r leakage would only serve to enhance successively higher level en-closures. The f e r t i l i z e r used was a blend of urea, super-phosphate and potash: i t contained 1.5% phosphate and 14% nitrate by weight. Phosphates and nitrates were selected for enhancement not only because they are commonly considered to be the most important chemical factors in algal growth (Hutchinson 1967, p. 310) but also because of their low natural levels and previous stimulatory history (Dickman, 1968) in Marion Lake. Commercial agricultural f e r t i l i z e r s such as that used also include a wide spectrum of trace elements not found in reagent quality chemicals. Application 9 was made bi-weekly during the course of the experiment and was accomplished with the use of a chemical spraying unit after the appropriate amount of f e r t i l i z e r had been dissolved in 2 - 3 gallons of lake water. The quantities of f e r t i l i z e r applied were 3 2, 160 and 800 g per enclosure for the low, mid and high levels respectively. Four days following application the shutters on the en-closures were opened to maintain an integrity of chemical and temperature conditions between the enclosed and outside water. Evidence of rapid uptake and storage of phosphorus (Hayes et al, 1952, Einsele, 1941) led to the assumption that this was more than sufficient exposure time for sedi-ment uptake. After allowing a period of time for oxygen and temperature equilibration estimates of benthic algal production and community respiration were made according to the method of Hargrave (1969). At each sampling interval six sediment cores were withdrawn from each enclosure. Pyrex cylinders 12.5 cm long were pressed into the sediment to approximately 2/3 of their length: they were then stoppered and withdrawn producing an intact sediment interface with approximately 100 ml of overlying water. Cores were stabi-lized in the dark for two hours prior to incubation to avoid continuation of oxygen production by algae removed directly from the light. Such a process, as noted by Hargrave (1969) would reduce the apparent community respiration. The over-lying water was analyzed for oxygen, and the cylinders then sealed, and incubated successively in the dark and light 10 portions of a constant temperature chamber. Cooling water for t h i s chamber was drawn from an i n l e t stream and was at a c o n s i s t e n t l y lower temperature than the lake. This was done i n order to prevent the d i s s o l u t i o n of oxygen accumu-lated during incubation i n near saturated lake water as d i s -cussed by Nielson (1957). For t h i s reason also dark incubation always preceeded that i n the l i g h t . I n h i b i t i o n of r e s p i r a t i o n by reduced oxygen lev e l s i n sealed cores as noted by Gessner and Pannier (1958) was avoided by l i m i t i n g incubation times to 3 hrs in the dark and 2 hrs in the l i g h t . At the end of each incubation period oxygen concentrations were again measured. Gross production and community r e s p i r a t i o n was calculated from these data. T o t a l dissolved oxygen was c a l -culated at each stage with volumes corrected for losses in analysis and re-sealing of cores based upon a single volume measurement at the end of a l l incubations. The supernatant water, c o l l e c t e d i n a graduated c y l i n d e r , was retained as was the sediment for further a n a l y s i s . Similar production and r e s p i r a t i o n measures were made on the planktonic community on two occasions during the period of enrichment. These measures were made in 300 ml B.O.D. bottles incubated i n a manner i d e n t i c a l to that pre-v i o u s l y described. Dissolved oxygen was measured at a l l times with a modified Winkler technique (Fox and Wingfield, 1938). Samples were withdrawn and analyzed i n p l a s t i c syringes: for t h i s reason phosphoric acid was substituted 11 for sulphuric acid in the analysis. Although the possibility of oxygen diffusion through polyethylene was considered } the rapid analysis ( 2 - 3 min to acidification, 8 - 1 0 min to titration) makes i t unlikely that such an effect could have affected the results. .025 N phenyl arsene oxide prepared by the Hach Chemical Company was substituted for sodiun thiosul-fate in the ti t r a t i o n because of its greater s t a b i l i t y . Once the supernatant water had been poured off i t was possible to extrude the sediment core by pressing the bottom stopper up the cylinder. In this manner the top 2 cm of sediment was removed and vaccuum filtered with a suction f i l -ter apparatus. 2 g samples were weighed out from the resul-ting semi-dried sediment and recombined with 10 ml of the f i l t r a t e : by this process density differences between various samples were minimized. Measurement of the algal standing crop was based upon chlorophyll analysis (Vallentyne, 1955). This type of estimate has been c r i t i c i z e d extensively because of the v a r i a b i l i t y of chlorophyll content in algal cells and the inclusion of detrital chlorophyll in the analysis (Edmond-son and Edmondson, 1947, Margalef, 1954, summary Strickland, 1960). Hargrave (1969) has, however, established a relation of chlorophyll measured in this manner to algal biomass in the Marion tatkisej sediments: similarly differences in indicated chloro-phyll corresponded to c e l l counts conducted throughout the course of the experiment. Chemical phosphate analyses were conducted to monitor 1 2 the form and quantity of the added phosphate. Ortho phos-phate was measured in the water column just prior to flush-ing and in sediment and i n t e r s t i t i a l water at the time of production estimation by the method of Strickland and Parsons (1960). Total phosphate was measured in the sediment from samples taken at the same time. These analyses were per-formed in conjunction with Can Test Laboratories, Vancouver, B. C.^ A l l the sediment measures were expressed in ppm phosphate per gram dry wt of sediment. The difference be-tween total and ortho phosphate estimates is regarded (Ohle, 1939) as representing organic phosphorus in either dissolved or sestonic form. Within the f e r t i l i z e d enclosures the herbivorous amphi-pod Hyalella azteca was monitored as an indicator of grazer response to stimulated algal production. Six sediment samples were taken from each enclosure at bi-weekly intervals and num-bers and fresh weights tabulated. While the above procedure was intended to detect a spon-taneous response of grazers to an increase in algae i t was also necessary to examine the potential support capacity of the f e r t i l i z e d areas whether or not this potential was u t i l i z e d . This was accomplished by introducing various densities of grazers into enclosed areas of both natural and enriched sedi-ments and monitoring changes in primary production. In addi-tion, Rana aurora tadpoles were introduced into polyethylene enclosures similar to those used for f e r t i l i z a t i o n experiments. Note 1: A modification of the method described in Standard Methods (19 6 5) was employed with oxidation of organics achieved by fuming with Nitric and Perchloric acids. 13 Cores were removed from t h i s area and primary production again estimated under these circumstances. Densities of Hyale11a were manipulated in shorter experiments i n the glass c y l i n d e r cores themselves: i n t h i s instance the primary production of both enriched and unenriched sediments were compared. Cylinders were incu-bated for 48 hours before production estimation under a light/dark cycle s i m i l a r to that at the lake. Preliminary experiments indicated that photosynthetic oxygen production maintained oxygen l e v e l s close to saturation under these conditions. Near the end of the experimental period bacteria counts were made from the sediments of the enclosures. Samples were removed from each enclosure with a Kajak core sampler 4 and plated at a 10 d i l u t i o n . Colony counts and i d e n t i f i -cations were subsequently performed on both these and ex-t e r n a l control samples. 1 4 RESULTS The immediate effect of placing the enclosures was stabilization of the water column in the enclosed area. Drift studies with both dyes and floating markers indicated only a slow circular movement within the enclosures except at times of high winds. Despite this apparent stagnation both temperature and dissolved oxygen concentrations were similar in both the enclosures and the outside water at the time of production estimation (Fig. 3) Bakhtina (1967) in a similar f e r t i l i z a t i o n experiment noted a direct relation between the degree of f e r t i l i z a t i o n and the dissolved oxygen levels in enriched ponds. This was attributed to oxygen pro-duction by stimulated algal growth. In this study such accumu-lations would have been dissipated by the exchange of water through the screens or released from super-saturated solution. As dissolved oxygen in Marion Lake follows the temperature-saturation curve closely (Hargrave, 1969) i t would appear that accumulation of oxygen to super-saturation is not favored by conditions in the lake (i.e. turbulance). In any case, as w i l l be demonstrated below, the only increase in oxygen con-tribution was that of the phytoplankton who were themselves be-ing flushed to an undetermined extent. DISTRIBUTION OF ADDED PHOSPHORUS Phosphorus added in the f e r t i l i z e r was removed rapidly from the water column of both the large and small enclosures. 15 F i g . 3 Oxygen lev e l s i n the experimental enclosures p r i o r to production estimation (June, 1970) co - | m z O CO m z CD -I p a H CD-I % SATURATION O2 85 9 5 105 J 17 In the case of the small enclosures which had no openings none of this loss could be attributed to leakage of the con-tained water. Ortho phosphate measured a week after addition to the large enclosures was at a consistently low level except in the most highly enriched areas (Fig. 4). A phosphate series over time was made in the smaller enclosures (Fig. 5). The re-sulting curve resembled uptake observed by other workers such as Hayes et al (1952), Einsele (1941), Hutchinson and Bowen (1947). A phosphorus equilibrium appeared to exist within the sediment and water column of the enclosures. The extent of phosphorus removal varied with its i n i t i a l concentration in the water column (Fig. 6): at higher concentrations a larger amount of phosphorus was lost from the water. Even at the extremely high levels of addition in the small enclosures a quantitative difference in uptake was noted between the two levels (Fig. 5). Conversely a release of phosphorus to the water could be effected upon reducing i t s concentration in the water by flushing the enclosures (Fig. 5). Azad (&'3Barcto.arcdsfenchasneitde m?dns-trated that algae w i l l not release stored phosphorus once i t has been taken up. This would suggest that the sediment retention process has a strong inorganic or physical component which can respond to changes in chemical concentrations of the overlying water. The existence of such an equilibrium is not a new concept: Olsen (1964) has described an equation for the phosphorus equilibrium of oxidized sediments which accounts for both uptake and release from the benthos. 18 Fig. 4 Ortho phosphate measurements in the water of the large enclosures, (mg/l) a. Concentration of i n i t i a l addition b. Concentration 4', days following addition 1 - E x t e r n a l c o n t r o l area 3 - Enclosed c o n t r o l area 4 - Low l e v e l f e r t i l i z a t i o n 5 - Mid l e v e l f e r t i l i z a t i o n 6 - High l e v e l f e r t i l i z a t i o n 20 i g . 5 Ortho phosphate concentrations i n the water of the two small enclosures over time following the addition of two d i f f e r e n t amounts of f e r t i l i z e r , (enclosures flushed on day 12) 2> 22 F i g . 6 Loss of phosphorus from water column d.n aaiiWur-o'day pW-JPSal^Js Ga^^u>§S*tifo®"« of i n i t i a l c o n c e n t r a t i o n . 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - M i d l e v e l f e r t i l i z a t i o n 6 and 10 - H i g h l e v e l f e r t i l i z a t i o n 24 The decrease in phosphorus measured in the water corre-sponded to an increase in that measured in the sediment: this relation was similar to that noted by Einsele (1941) in the Schleinsee. Here i t appeared that the phosphate fraction was being removed from the water column in a manner that was not directly proportional to the concentration of available phos-phate (Fig. 7). This observation was, however, partly an artifact of the sampling method which included in the analysis an undetermined amount of the sediments pre-fertilization his-tory. If quantities in the enclosed controls are used as a correction factor for other enclosures the measured sediment phosphorus is more closely proportional to the amount added. In any case phytoplankton in the enclosures would retain phos-phorus in particulate form in the water column. This retention would be proportional to the standing crop and the larger stand-ing crops of phytoplankton in more highly f e r t i l i z e d enclosures could in this way account for the balance of added phosphorus. The ratio of ortho: organic phosphorus was not constant through the various levels of enrichment: with greater f e r t i -lization an increasing proportion of the added phosphorus was measured as inorganic phosphate (Fig. 7). If this is indeed the case i t would appear that an increasing proportion of the added phosphorus remains unused by the algae at higher levels of addition: that i s , that the phosphorus was in excess of the algal uptake capacity. Rigler (1968) has suggested that 25 F i g . 7 Phosphorus measured i n the sediments of the experi-mental enclosures ( /g dry wt sediment). The upper histogram represents t o t a l PO^ while the lower re-presents ortho PO^ 1 and 2 - E x t e r n a l c o n t r o l a r e a s 7 and 3 - E n c l o s e d c o n t r o l s 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - Mid l e v e l f e r t i l i z a t i o n 6 and 10 - H i g h l e v e l f e r t i l i z a t i o n a and b - s m a l l e n c l o s u r e s ( s e e t e x t ) 2<* CO. C M > CC 00 Q CL CL CL' I \ I T 1 r I i 1 —I r 1 2 3 7 4 8 5 9 6 10 ENCLOSURES 27 the type of analysis (molybdenum blue) used overestimates ortho phosphate measurement by recruitment of organic phos-phate through hydrolysis. If soluble reactive phosphorus (Strickland and Parsons, 1960) measures are overestimated to the degree suggested (10-100X) the significance of the observed excesses would be considerably reduced. RESPONSE OF ALGAE Algal response was measured in terms of changes in both primary production and standing crop. Differences in the parameters were noted even between external and enclosed con-tr o l areas. Primary phytoplankton production increased in the control (unfertilized) enclosures (Fig. 8 ) over that of the outside water. Dickman (1969) made similar observations in his enclosures and attributed the increase to a reduction in phytoplankton cropping by rapid current flow. An i n i t i a l increase in epibenthic algal production was also noted in the control enclosures (Fig. 8 ) : this increase led, as in the case of increase phytoplankton production, to a higher standing crop in the enclosures than on the outside sediment. A most dramatic response in a l l enclosures was the proliferation of epiphytic algae on the walls of the enclosures themselves. This growth reached such portions that oxygen production on the enclosure walls approached that of the sediment surface on an area basis: indeed, the addition of increased surface area in the form of the enclosures was one of the greatest factors in algal growth. The standing crop of epipelic algae measured as chlorophyll 28 F i g . 8 Planktonic primary production i n the experimental enclosures following f e r t i l i z a t i o n ( J u l y , 1970) 1 and 2 - E x t e r n a l c o n t r o l s 7 and 3 - I n t e r n a l c o n t r o l s 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - Mid l e v e l f e r t i l i z a t i o n 6 and 10 - H i g h l e v e l f e r t i l i z a t i o n 2<* ~ i 1 • 1 1 1 1 0 2 0 4 0 F E R T I L I Z E R C 9 . / M 2 ) 30 increased in a near linear manner with increased enrichment (Fig. 9). Measured chlorophyll in the most highly enriched enclosures was 10X that of the control areas and measures in each of the treatment pairs were, with the exception of the mid level, closely similar. While no actual measurements were made of phytoplankton standing crop i t was visually evi-dent that enrichment produced an increase here as well. At high levels of f e r t i l i z a t i o n plankton blooms formed which lasted almost the entire duration of the experiment in spite of the intermittent flushing of the enclosures. Primary production in the water column increased with increasing levels of f e r t i l i z a t i o n (Fig. 8). This is a response observed many times in similar studies (Dickman, 1968, Batkina, 1967) as an accompaniment to an increase standing crop. Vinberg (1965) in a discussion of pond fer-t i l i z a t i o n , states (p. 213) "the i n i t i a l effect of a r t i f i c i a l f e r t i l i z e r s in effectively f e r t i l i z e d ponds is to stimulate the vigorous development of phytoplankton". Epipelic primary production, however, did not demonstrate a sustained increase as a response to f e r t i l i z a t i o n (Fig. 10): a stimulated pro-duction was noted only in the course of a pilot experiment conducted in the f a l l of 1969 when i n i t i a l f e r t i l i z a t i o n was accomplished (Fig. 11). When the program resumed in May 1970 the epibenthic algae had assumed the variation in standing crop noted in the i n i t i a l measurements (Fig. 9) and 0^ production had returned to pre-fertilization levels in a l l enclosures. 31 F i g . 9 Benthic a l g a l standing crop in the experimental enclosures ( c h l o r o p h y l l units /g dry wt sediment) July, 1970 1 and 2 - E x t e r n a l c o n t r o l a r e a s 7 and 3 - E n c l o s e d c o n t r o l s 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - Mid l e v e l f e r t i l i z a t i o n 6 and 10 - h i g h l e v e l f e r t i l i z a t i o n a and b - s m a l l e n c l o s u r e s ( s e e t e x t ) F E R T I L I Z E R C g . / M 2 J 33 F i g . 10 Benthic primary production in the experimental enclosures (July, 1970) 1 and 2 - E x t e r n a l c o n t r o l a r e a s 7 and 3 - E n c l o s e d c o n t r o l s 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - Mid l e v e l f e r t i l i z a t i o n 6 and 10 - H i g h l e v e l f e r t i l i z a t i o n a and b - s m a l l e n c l o s u r e s ( s e e t e x t ) 35 Fig. 11 Benthic primary production in the experimental enclosures (September, 1969) 1 - E x t e r n a l c o n t r o l area 3 - Enclosed c o n t r o l area 4 - Low l e v e l f e r t i l i z a t i o n 5 - Mid l e v e l f e r t i l i z a t i o n 6 - High l e v e l f e r t i l i z a t i o n 37 This variation in standing crop continued to exist for the duration of observation (September, 1970). It can only be assumed that each particular standing crop developed during the winter and spring (1969-70) through the stimulation of f e r t i l i z e r added during the course of the pilot experiments in the f a l l of 1969. An indication of this likelihood may be seen in the proportionally stimulated oxygen production measured at this time. The significance of primary production and standing crop measures may be more clearly seen by examining the relation of these two factors under various conditions. While phytoplanktonic primary production increased as a function of increased standing crop that of the epipelic algae appeared to remain constant through a wide range of standing crop (Fig. 12). In terms of a single algal unit this means that the production of a phytoplankter remains constant while that of an epipelic alga decreases with an increase in standing crop (Fig. 12). This inverse response is not limited to nutri-ent induced standing crop changes. Where grazing was a r t i f i -c i a l l y increased by unnaturally high concentrations of grazing tadpoles the production of individual algae in the reduced algal population was increased (Fig. 12): the total production on an area basis was the same under these circumstances as that in the undisturbed sediments. In a similar experiment by Dickman (1968) heavy grazing by hatching tadpoles produced a similar result in epiplytic algae: under these intense 3 8 F i g . 12 Relation of benthic a l g a l standing crop to production of i n d i v i d u a l a l g a l u n i t . Note: "G" r e f e r s to observations made in enclosures with high tadpole density. 1 and 2 - E x t e r n a l c o n t r o l a r e a s 7 and 3 - E n c l o s e d c o n t r o l s 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - Mid l e v e l f e r t i l i z a t i o n 6 and 10 - H i g h l e v e l f e r t i l i z a t i o n a and b - s m a l l e n c l o s u r e s ( s e e t e x t ) 40 grazing pressures, algae species present were those with the most rapid rate of growth. Only in the H y a l e l l a g r azing experiments did total primary production as measured in a cylinder core drop below natural levels (Fig. 13). But even in this case the reduction was due not to a drop in individual 0^ production but rather to the drastically re-duced algal crop. The response of enriched sediment to heavy grazing was quite different from that of the natural community. As a l -ready indicated the drastic reduction by grazers of the nat-ural algal population resulted in a drop in primary production. In enriched sediments, however, the heavier algal standing crop responded to grazing with an increase in total production (Fig. 13). Assuming that the production capacity of any in-dividual alga is dependent only upon the avail a b i l i t y of growth requirements i t seems quite obvious that the total production potential is much greater in a situation of high standing crop. The algae removed by grazers in the enriched sediment represent a much smaller proportion of the total community than in the natural situation. This removal apparently provides the sur-vivors with sufficient stimulation that individual production is able to increase. In this way a larger standing crop appears to possess a higher production potential which may be expressed only when increased grazing gives the opportunity for growth. 4 1 Fig. 13 Response of benthic primary production to increased grazing by Hyalella Closed circles represent unenriched sediments Closed triangles represent enriched sediments from enclosure 6 (high f e r t i l i z a t i o n l e v e l ) D E N S I T Y H Y A L E L L A 43 RESPONSE BY GRAZERS The next question is quite obviously that of whether this production potential is being u t i l i z e d . Evidence from several directions would indicate that i t is not. The f i r s t indication may be seen in the consistancy of primary production through the various enclosures. As was demonstrated in the Hyale11a grazing experiments the immedi-ate response of enriched sediments to increased grazing was an increase in primary production. The fact that algal pro-duction did not increase in the f e r t i l i z e d enclosures suggests that removal of epipelic algae was similar in a l l areas. Measures of community respiration were made at the same time as primary production estimation. The respiration of the benthic community makes demands upon the dissolved oxygen in the overlying water: these demands are proportional to the metabolism of the community as a whole. As may be seen in (Fig. K4), there was very l i t t l e increase in community oxygen uptake with an increase in f e r t i l i z a t i o n . Small increases are likely attributable to the larger algae community in the highly enriched enclosures: Hargrave (1969) has stated that algae account for 25% of the total benthic respiration although this proportion, estimated by difference from the remainder of the community, is likely to be overestimated as a result of an underestimation of the bacterial contribution (see Appendix 1). Hyale11a azteca was examined directly in an attempt to 44 F i g . 14 Benthic community r e s p i r a t i o n in experimental enclosures (July, 1970) 1 and 2 - E x t e r n a l c o n t r o l a r e a s 7 and 3 - E n c l o s e d c o n t r o l s 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - Mid l e v e l f e r t i l i z a t i o n 6 and 10 - H i g h l e v e l f e r t i l i z a t i o n a and b - s m a l l e n c l o s u r e s ( s e e t e x t ) 46 detect a grazer response to the increased food supply. Num-bers and fresh weights of individuals removed in enclosure samples showed no s t a t i s t i c a l l y significant differences'be-tween the various enclosures: this evidence reinforces that of the respiration estimation in indicating that the grazing component of the benthic community has not responded to the algal increase. This is not an isolated observation. Vin-berg (1965) in a survey of pond fish culture, states "In many experiments in the f e r t i l i z a t i o n of ponds the stocks of benthonic food for the fish failed to show any increase" (p. 338). Batkina, (1967) states that the dynamics of chironimid larve "was the same during the season in a l l (fert i l i z e d and control) ponds and did not depend on fer-t i l i z e r dosage." (p. 11). The results of the bacterial plate counts are given in (Fig. 16). Again there is no significant difference apparent among the various enclosures. This indicates two things. First, i t would appear that the bacteria present in the sedi-ment are incapable of responding directly to an increase in inorganic nutrients. Finally, as a likely pathway for bac-t e r i a l nutrition is via algal exudates (Kuznetzov, 1968) and the release of such materials is proportional to algal pro-duction (Fogg, 1956) this observation is somewhat of a con-firmation of the similar primary production rates observed in the various enclosures. 47 Fig. 15 Bacterial plate count in the experimental enclo-4 sures 10 dilution. (August, 1970) 1 and 2 - E x t e r n a l c o n t r o l areas 7 and 3 - Enclosed c o n t r o l s 4 and 8 - Low l e v e l f e r t i l i z a t i o n 5 and 9 - Mid l e v e l f e r t i l i z a t i o n 6 and 10 - High l e v e l f e r t i l i z a t i o n a and b - small enclosures (see t e x t ) 4 9 DISCUSSION The response of an aquatic system to inorganic f e r t i -lization may involve either proportional stimulation of entire system or "the excessive activity of any group or groups of organisms (which) can lead to an ecological unbalance" (Fruh, 1966). A sustained imbalance may develop conditions under which the original community structure cannot exist. These conditions are typically the result of an increase in algal production which in turn leads to changes in both other com-munity components and in water chemistry. The maintenance of an imbalance requires that the primary production increase is a lengthy one. But nutrient induced production increases are transitory processes: they are the means to an end which is a standing crop appropriate to the new resource conditions. A higher rate of primary production can only be maintained until the standing crop has reached an equilibrium with the available resources. This is not simply a matter of nutrient supplies but of conditions which determine the disposition of algal material produced. Where the potential increase in standing crop is limited in its accumulation then high production rates may be sustained only with a proportionate increase in removal by the grazing com-ponents. The results of this study suggest that the response of benthic grazers imposes a limit on the' extent of primary production increases and maintains a proportionality between the two components. 50 Phosphorus uptake in the various enclosures demonstrated the rapid removal observed by many investigators and reviewed by Hutchinson (1967). Evidence of a sediment-water equilibrium, however, has particular significance in Marion Lake. Olsen (1964) in describing an equilibrium process in similar oxidized sediment states: "the bottom deposits must be regarded as phos-phate reservoirs from or to which phosphate w i l l be transferred whenever the equilibrium is disturbed". This certainly appeared to be the case where either f e r t i l i z e r addition or flushing disturbed the phosphorus balance of the enclosures. But within the lake i t s e l f a different problem arises: as the specific conductivity of Marion Lake water is in the range of 6 to 10 micro ohms (Dickman, 1968) the potential for leaching of sedi-ment nutrients particularly at times of rapid flow becomes apparent. Although d i f f i c u l t i e s of measurement of low phos-phorus concentrations prevented further release experiments at more r e a l i s t i c nutrient levels the possibility exists that not only phosphorus but other inorganic and organic nutrients may be lost to the benthos either by equilibration or actual mechanical flushing. Such a phenomenon may account for the increase in epipelic standing algal crop in control enclosures. Dickman (1968) noted an increase in phytoplankton under similar conditions and attributed that observation to a relaxation of cropping by current flow. An increasingly large proportion of sedimentary phosphorus was present in inorganic form at higher f e r t i l i z a t i o n levels. 51 As algal cells can remove phosphorus from the water to some 6X that required for maximum growth (Azad and Borchardt, 1970) the presence of large quantities of ortho phosphate in these enclosures suggests that addition has exceeded algal growth requirements (p. 22). The observed stabilization of production in benthic algae cannot, as a result, be explained on the basis of nutrient supply. It would appear that the expanding algal standing crop has equilibrated with the limi-tations of accumulation as discussed previously. Further evidence for this conclusion is the response of enriched sedi-ment to increased grazing: the dramatic increase in primary production in these experiments was brought about simply by removal of a portion of the algal standing crop. Limitations on the range of primary production are very different in the benthic and planktonic communities subjected to the same stimulation. There are many instances of vast phytoplankton increases resulting from nutrient stimulation (Vinberg, 1965) and in Marion Lake a ten-fold increase in p r i -mary production followed the addition of f e r t i l i z e r s to an enclosed area (Dickman, 1968). The sudden availability of nutrients following spring and f a l l turnovers is believed to be a primary factor in the characteristic blooms of many dim-i c t i c lakes (Hutchinson, 1967). The benthic community, however, is more limited in the range of its response. Margalef (1963) relates this difference to a difference in maturity. He states "Planktonic communities retain always a less mature character 5 2 than benthic communities and i t is to be expected, in good agreement with observation, that fluctuations in planktonic populations are of shorter period and wider ranges" and fur-ther more that "fluctuations in less mature systems are more related to environmental changes". Limitations on the growth of an algal population are ultimately limitations on the standing crop. A change in an input such as inorganic nutrients changes the potential stand-ing crop as determined by available resources and until this potential is realized production w i l l increase. Only when cropping pressures are such that the potential standing crop cannot be reached does such a stimulation operate on the rate of production in a sustained manner. This is a situation equi-valent to Slobodkin's (i960) grazer limited population where a small number of organisms with an abundance of resources have a high individual production. An example of such a situation was seen where a r t i f i c i a l l y increased grazer density resulted in a lowered standing algal crop. Individual production here was the highest recorded in unenriched conditions. Where grazers do not limit algal production the standing crop of algae w i l l increase until i t is in proportion to its resources. At this time further production can only be a function of the replacement of materials removed regardless of the level of standing crop. This situation has been des-cribed by Slobodkin as a resource limited one where there is a high standing crop with low individual production. A situa-tion similar to this developed in the sediment of the enriched 53 enclosures. When standing crop of algae increased, total pro-duction remained constant; a function of maintenance rather than expansion. If this is indeed the case i t is obvious by the excess of nutrients present that some other factor has replaced these as the limiting resource. Margalef (1963), on the other hand, describes such a situation as being an example of the more efficient energy conversion by a more mature, complex system where "a reduced waste of energy allows maintenance of a higher biomass with the same supply of energy". This may thus be a further indication of the rela-tive maturity of the benthic community. In the plankton, however, production increases as a func-tion of enrichment and Dickman noted that "changes in standing crop and primary productivity were closely correlated (r = 0.89) ... an increase in standing crop results in an increase in p r i -mary productivity". If similar treatment can produce such different results then factors which limit the extent of algal increase must diff e r in the two communities. If sufficient resources are available the question of whether or not a high level of algal production can persist depends upon the disposition of the material produced. An accumulation is a simple solution. Where the potential standing crop as determined by nutrient resources is much greater than that of the original situation the production increase may be a lengthy one and the increased algal crop may simply accumu-late to high levels. This occurs in a phytoplankton bloom. 54 If the degree of accumulation is limited then consumption must increase along with production to sustain a high produc-tion rate. Both of these factors operate in the water column. The difference between actual and potential phytoplankton crop is often several hundred times (Hutchinson, 1967). At the same time energy is required to maintain the plankton in the water column and there is a net movement into the benthos of potential energy in the form or settling plankters, (Margalef, 1963). This energy requirement increases proportionately with the stand-ing crop and consumes a portion of the increased production. The situation on the sediment surface is quite different. Accumulation is limited by the available space and this alone may halt the expanding algae. Indeed, the introduction of a greater surface area in the form of enclosure walls increased the standing crop of the area immediately as this new space was colonized. Furthermore energy for movement is not required by sessile algae: only energy for the retention of high phos-phorus concentrations could increase as a function of enrichment but i t is generally held (Kamen, 1963) that this retention is through conversion to organic or polyphosphates rather than by an active process. The only possible increase in consumption of algal material produced in the sediment must, therefore, be ex-trinsic to the algae themselves; that is a response of the other components of the epibenthic community. It would appear that the conversion of inorganic nutrients in the enclosed systems has been an appropriate one in terms of 55 the requirements of the existing community. Partly through design and partly through chance factors which may divert such inputs from the existing trophic chain were avoided. One such factor is maintenance of oxygen levels. The low oxygen levels of stagnation are a familiar step in diverting nutrient inputs through anaerobic or near •anaerobic i n v e r t e -brates and bacteria. Batkina (1967), on the other hand, has suggested that an oversaturation of oxygen was responsible for the inhibition of invertebrate grazers in a similar enrichment study. Both such eventualities were avoided by the circulation of water through the enclosures. The excessive development of undesirable blue-green algae may also divert primarily fixed energy from usable channels: at no time did such a development occur in the enclosures probably as a result of the high N:P ratio of the f e r t i l i z e r . Yet a l l the evidence accumulated in this study indicates that the grazing component of the benthic community has not responded to the increase in algal supply with a proportionate increase in removal. This may be more a result of the duration of the experiment than of actual response of the community. A population increase would likely be by small increments resulting from increased fecundity and survival: as this study spanned only a single reproductive season for most of the benthic invertebrate grazers i t is most unrealistic to speculate on the long term response to increased nutrients. In any case the assumption of an increase in grazing pre-supposes that the grazers involved 56 are food limited: discussions with individuals examining the grazers in Marion Lake have indicated that this is not likely the case. But even i f a grazer response is taking place the fact that i t is not an immediate one is significant. In short term disturbances a time lag in structural change is as important as no response at a l l . This is because i t gives sufficient time for the disturbance to be corrected before permanent community adjustments take place. Admittedly some of the conditions which avoided community changes were a r t i f i c i a l l y controlled as previously indicated. But the significant point is that a sudden disproportionate input has been dealt with in a manner which has made i t available to the existing community. Furthermore, a mechanism is apparently operating in the benthic community to maintain the proportions of the various existing components: the extent of algal produc-tion is limited by the fact that i t is not being utilized by the rest of the community. Resistance to structural alteration in the face of changing nutrient conditions is evidence of st a b i l i t y as previously defined. In this respect the benthic community may be considered a more stable, structure than that of the plankton. But the nature of the relation between these two communities is such that benthic stability is conferred upon the system as a whole. This results from the fact that "a net transfer of energy exists from plank-ton to benthos" and "strong fluctuations in plankton populations represent a heavy export towards the benthos" (Margalef, 1963). It i s l i k e l y that at least a portion of the benthic chl o r o p h y l l measured in the experimental enclosures resulted from sedimentation of enhanced plankton populations. In t h i s way the wider f l u c t u a t i o n s of the plankton populations are absorbed by the benthic community. This tran s f e r provides energy to further increase the maturity and s t a b i l i t y of the benthic component at the expense of the plankton: "what the one does in excess production", states Margalef, " i s put in use by the other". In Marion Lake the planktonic community i s held in a state of immaturity through intensive cropping by current flow, (Dickman, 1969). As such i t may be expected to show a more dramatic response to changing nutrient conditions than the more mature benthic community. The r e s u l t s of t h i s study con-firm these predictions and further indicate that the benthic component can dampen phytoplankton fluctuations through sedi-mentation of nutrients and p a r t i c u l a t e matter. This energy transfer appears to enhance even further the s t a b i l i t y of the benthic community and, thus, of the entire system. 58 LITERATURE CITED Azad, H. S. and J. A. Borchardt. 1970. Variations in Phos-phorus Uptake by Algae. Environmental Sciences and Technology. 4(9):737-743. Baktina, V. I. 1968. Effect of inorganic and organic f e r t i -lizers on the development of food organisms in rearing ponds. Fisheries Research Board of Canada Translation Series No. 1152 Volume 7.. Dickman, M. D. MS. 1968. The relation of freshwater plankton productivity to species composition during induced suc-cessions. Ph. D. Thesis University of British Columbia 1968. The effect of grazing by tadpoles on the structure of a periphyton community. Ecology 49(6):1188-90. 1969. Some effects of lake renewal on phyto-plankton productivity and species composition. Limnol. and Oceanogr. 14(5):660-666. Edmondson, W. T. and Y. H. Edmondson. 1947. Measurements of production in f e r t i l i z e d salt water. J. Mar. Res. 6(3): 228. Efford, I. E. 1967. Temporal and spatial differences in phyto-plankton productivity in Marion Lake, British Columbia. J. Fish. Res. Bd. Canada 24(11):2283-2307. Einsele, W. 1941. Die umsetzung von zugefuhrtem, anorganischen Phosphat im eutrophen See und ihre Ruckwirkung auf seinen Gesamthaushalt. Z. Fisch. 39:407-488. Fruh, E. C. 1967. The overall picture of eutrophication. Journal WPCF 39(9):1449-1463. 59 Fogg, G. E. 1966. The extracellular products of algae. Ocenaogr. Mar. Biol. Ann. Rev. 4:195-212. Fox, H. M. and C. A. Wingfield. 1938. A portable apparatus for the determination of oxygen dissolved in a small volume of water. J. Exp. Bio l . 15:437-445. Gessner, F. and F. Pannier. 1958. Influence of oxygen ten-sion on respiration of phytoplankton. Limnol. and Oceanogr. 3(4):478. Hargrave, B. T. MS. 1969. Inter-relationships between a deposit-feeding amphipod and metabolism of sediment microflora. Ph. D. Thesis University of British Col-umbia . Hayes, F. R., McCarter, J. A., Cameron, M. L. and Livingstone, D. A. 1952. On the kinetics of phosphorus exchanges in lakes. J. Ecol. 40:202-216. Hutchinson, G. E. 1957. A Treatise on Limnology. Volume 1. John Wiley & Sons, Inc. 1957. A Treatise on Limnology. Volume 2. John Wiley &' Sons, Inc. Hutchinson, G. E. and V. T. Bowen. 1947. A direct demonstration of the phosphorus cycle in a small lake. Proc. Nat. Acad. Sci., Wash., 33:148-153. Kamen, M. D. 1963. Primary Processes in Photosynthesis, Advan. Biochem. Series. Academic Press, New York. p. 30 Kuznetzov, S. I. 1968. Recent studies on the role of micro-organisms in the cycling of substances in lakes. Limnol. and Oceanogr. 13(2):211-224. 60 Margalef, R. 1963. On Certain Unifying Principles in Ecology. The Amer. Nat. 97:357-371. 1963. Perspectives in Ecological Theory. The University of Chicago Press, Chicago. Nielsen, E. S. 1957. Experimental methods for measuring organic production in the sea. Paper presented at a Symposium of the International Council for the Explora-tion of the Sea. Bergen, 1957. Preprint B/No. 1. Olsen, S. 1964. Phosphate equilibrium between reduced sedi-ments and water; laboratory experiments with radioactive phosphorus. Verh. Internat. Verein. Limnol. XV:333-341. Rigler, F. H. 1968. Further observations inconsistent with the hypothesis that the molybdenum blue method measures orthophosphate in lake water. Limnol. and Oceanogr. 13(1):7-13. Ohle, W. 1 9 3 9 . Zur Vervollkommnung der hydrochemischen Analyse. III. Die Phosphorbestimmung. Angew. Chem., 51:906-911. Slobodkin, L. 1960. Ecological energy relationships at the population level. Amer. Nat. 94:213-236. Strickland, J. D. 1965. Primary Productivity in Aquatic En-vironments. Proceedings of an I.B.P. PF symposium. Pallanza, 1965. Strickland, J. D. and T. R. Parsons. 1960. A manual of sea water analysis. Bull. Fish. Res. Bd. of Canada 125:1-78. 61 Vallentyne, J. R. 1955. Sedimentary chlorophyll determin-ations as a paleobotanical method. Can. J. Botany, 33:304-313. Vinberg, G. G. and V. P. Lyakhnovich. 1965. Fertilizations of fish ponds. Fish. Res. Bd. of Canada Trasnlation Series No. 1339. Vollenweider, R. A. 1968. Scientific fundamentals of the eutrophications of lakes and flowing waters. Organiza-tion for Economic Cooperation and Development. DAS/ CSI/6868. 27:1-159. 6 2 SOME PROBLEMS IN THE USE OF ANTIBIOTICS TO MEASURE RESPIRATION IN LAKE SEDIMENTS Rod Cameron Institute of Resource Ecology University of British Columbia Canadian International Biological Contribution No: Programme 63 ABSTRACT The antibiotics, neomycin and streptomycin, failed to block bacterial respiration and growth in vitro and in situ experiments on lake mud. It is suggested that antibiotics cannot be used to partition bacteria from total community respiration. 64 ACKNOWLEDGEMENTS My thanks to Mr. Rob Tyhurst and Mrs. Pamela Fraker for their contribution of supporting data. A special thanks to Dr. Ian E. Efford for his guidance and criticism and to the National Research Council for their support of this re-search. INTRODUCTION The use of antibiotics to selectively eliminate bacteria from algal cultures is a well established practice (Hunter and McVeigh, 1961). More recently, i t has become a feature of certain methods of partitioning community respiration into component parts (Hargrave, 1969; Smith et a l . , 1972). In these methods bacterial respiration is defined by the dif-ference in oxygen consumption of natural sediments and those to which antibiotics have been added. It must be assumed that this difference is due singularly to the elimination of the bacterial component of total community respiration. An attempt to use this method in the course of benthic com-munity studies suggested that the response of the community is not as simple as this assumption indicates. The results of these and further laboratory experiments are presented to support the contention that the antibiotic effect is a complex one, which cannot be directly related to bacterial metabolism. 66 METHODS I n i t i a l observations were made with the same experimental procedure and mud from the same lake used by Hargrave (1969). Samples of an intact sediment interface with approximately 100 ml of overlying water were removed from the lake bottom at a depth of 1 m in 12.5 cm x 5.0 cm glass cylinders. A combina-tion of neomycin (SO^) and streptomycin (SO^) was added to half of the cores to produce a total concentration of 50 mg/1. After measuring oxygen concentrations in the supernatant water the cores were sealed and incubated at constant temperature in darkness. In three hours the water was gently stirred and oxygen again measured in each cylinder. A comparison was then made of the oxygen consumption of treated and untreated sediments: this entire process was carried out at the lake site and was initiated within 20 min of taking the samples. As a result of inconsistent and contradictory observa-tions the method was modified for further experiments in June, 1970. To give additional time for antibiotic action injected samples were placed in the dark for 2 hr prior to the experi-ment. The total time of exposure to antibiotics was thus in-creased to 5 hr. Both respiration and primary production measurements were made successively in the same core. The effect of antibiotics on algal production could then be evaluated by comparing treated and natural cores incubated in the light. 67 A direct measurement was made of the respiration of bacteria isolated from lake sediment and cultured both sin-gularly and as a combination. Suspensions of bacteria were injected with various concentrations of antibiotics to a maximum of 150 mg/1. The effect of antibiotics on the res-piration of ten individual species was measured in this way as was the effect upon a combined culture of the dominant bacteria types found in Marion Lake. Finally the observed changes in community respiration were compared with the growth of bacteria populations. This was done on the assumption that actively growing bacteria would be u t i l i z i n g greater amounts of oxygen than static bac-t e r i a l populations and such a measure would thus provide a means of checking the observations of respiration. Samples removed from both treated and untreated sediments were plated and counted to determine whether the growth of bacteria pop-ulations had been inhibited. Changes in species composition were not examined as the object of the experiment was to examine the net effect of antibiotic treatment. RESULTS I n i t i a l observations with intact sediment demonstrated no consistent reduction in community respiration as a response to antibiotic treatment. Cores which had been treated with antibiotics showed either similar or higher respiration than 68 equivalent untreated cores (Fig. 1). This latter result was quite opposite to what would have been expected from a selec-tive elimination of the bacterial component. Further observations in June, 1970 produced similar re-sults despite a 2 hr increase in the time of exposure to antibiotics (Table 1). As both respiration and primary pro-duction were measured in the same sample core a further effect of antibiotic treatment became evident: cores to which anti-biotics had been added for respiration estimates showed a drastic reduction of net oxygen production in subsequent primary production measurement (Table 1). An increase in the concentration of streptomycin and neomycin to 200 mg/1 pro-duced no significant change in either of these observations. This was true also for changes in the mode of antibiotic addition, the degree of sample agitation and other modifica-tions of the method. Antibiotic treatment of bacteria cultures produced a wide variety of effects on respiration. While the respira-tion of combined culture of several common bacteria was re-duced only 20 % by antibiotics, individual cultures varied in response from an 80 % to a 30 % reduction in their oxygen uptake. A most important feature of these results is that the reduction in respiration was a gradual one whatever its fin a l extent. This means that regardless of the degree to which the bacteria are eventually affected the assumption 6 9 F i g . 1 Oxygen consumption of sediment core samples in the f a l l of 1969 curve a : cores t reated with ant ibiot ics at 50 mg/1 curve b : con t ro l cores l o I sept. i oct. nov. 71 Table 1 : Oxygen consumption and algal oxygen production in sediment core samples (June 1970) Community respiration (m.l. O^ hr. core) Control (untreated sediment) Antibiotic (50 mg. / 1.) area 1 .067 .098 area 2 .087 .101 2 -1 Primary production (ml. O hr. / core) Control (untreated sediment) Antibiotic (50 mg . 11.) area 1 .126 .002 area 2 .162 .001 72 of an immediate effect is not a valid one. Results of bacteria plate counts showed a similarly variable effect of antibiotic addition. A comparison of untreated bacteria with bacteria exposed for 5 hr to 50 mg/1 of the combined antibiotics demonstrated a variety of res-ponse in both (Table 2). In one instance an actual increase resulted from a 5 hr exposure. Of twelve individual species tested (Table 3) nine grew well on a medium including strep-tomycin at 50 mg/1 and three grew well on a medium containing 50 mg/1 each of streptomycin and neomycin. DISCUSSION Two assumptions must be made in relating the observed effect of antibiotic addition to the respiration of bacteria. The f i r s t of these is that bacterial respiration w i l l be totally or at least largely eliminated by the action of the antibiotics. The second assumption is that the response of a community to antibiotics i s a singular one due simply to the selective elimination of the bacteria component. Evi-dence presented here suggest that neither of these assump-tions is valid. The v a r i a b i l i t y of antibiotic inhibition indicates that there is no response common to a l l bacteria species present in the sediment. This has two potential effects on the in-terpretation of results at the community level. The f i r s t is that i f laboratory studies are used to calibrate the impact 73 T a b l e 2: The effect of ant i b i o t i c s on the growth Q f b a c t e r i a in sediment samples taken f r o m M a r i o n L a k e Set T r e a t m e n t 1 W. A . W/o. A . 2 W. A . W/o. A . 3 W. A . W/o. A . 4. W. A . W/o A . P l a t e counts made at times: 0 (start) 2 -§• h r s . 5 h r s . 7 5 28 10 11 10 267 5 4 4 4 3 8 6 4 1 1 3 4 24 8 10 22 7 8 N.B. W. A . s i g n i f i e s treatment with 50 m g / l s t r e p t o m y c i n and n eomycin before incubation; W/o A . s i g n i f i e s no antibiotic t r e a t -ment. Data c o u r t e s y of P. F r a k e r 74 Table 3: The effect of antibiotics on the gr owth. of individually cultured bacterium species (96 hr. exposure) Species Code 3 Control h Streptomycin: 50 mg/1 h 150 mg/1 h Streptomycin Neomycin 50 7 h h h -8 m m m -21 h h h 1 42 h m h h 43 h h h -46 h m m -21 h h h h : no growth 1 : light growth m : medium growth h : heavy growth Data courtesy of R. Tyhurst 75 of antibiotics on bacterial populations as a whole the result w i l l be distorted according to which species are measured individually. If, for example, a more susceptible population is tested the observed results w i l l not be applicable to the bacteria component as a whole. Furthermore, the combined response of even a representative portion of the dominant species is not likely to be simply the sum of the individual responses. If some affected species are releasing their c e l l contents into a medium where other unaffected bacteria are s t i l l growing i t is most likely that the remaining bacteria w i l l be stimulated by the sudden avai l a b i l i t y of nutrients. The resulting total respiration would in such a case be a complex of declining and expanding populations and bear no direct relation to natural bacterial metabolism. The plate counts show a similar situation. While some populations de-clined with exposure to the antibiotics, others were either unaffected or, in at least one instance, showed an actual increase. It may be argued that experiments conducted under laboratory conditions with growing bacterial cultures may show responses not typical of the situation in the lake i t s e l f . However, observations made in this way were quite consistent with those made with intact sediment and do serve to indicate at least the potential response of the individual components. A further complication exists in the time required for the antibiotics to take effect as the action of even those 76 a n t i b i o t i c s which did eventually r e s u l t i n a r e s p i r a t i o n reduction was a gradual one. The assumption of a constant e f f e c t throughout the experiment demands, however, an im-mediate act i o n . There i s also l i t t l e doubt that the a n t i b i o t i c s used a f f e c t more than simply the b a c t e r i a l component of the ben-thi c community. The d i s r u p t i o n of a l g a l a c t i v i t y observed i n t h i s study i s well substantiated by observations of other workers; Foter et a l (1953) i n a study of the e f f e c t s on algae of a n t i b i o t i c s at concentrations (20 mg/l) less than those used in these experiments stated "of the f i v e a n t i -b i o t i c s tested against s i x representative a l g a l cultures, streptomycin and neomycin were the most e f f e c t i v e in preventing growth", and Vance (1966) reached the same conclusion in p a r t i c u l a r reference to blue-green algae. It i s thus l i k e l y that at least a portion of the observed a n t i b i o t i c e f f e c t i s one on the a l g a l rather than b a c t e r i a l component. This further confuses the already complex res-ponse of the bacteria themselves. It i s not my intention to draw conclusions as to the exact e f f e c t of a n t i b i o t i c s on the various components of the benthic community. The evidence presented here demonstrates simply that the net e f f e c t of a n t i b i o t i c s i s the r e s u l t of a complex of responses not only in the b a c t e r i a l population but i n other parts of the community as w e l l . As such i t i s most u n r e a l i s t i c to measure b a c t e r i a l r e s p i r a t i o n by examining the response of a::mud community to a n t i b i o t i c s . 77 REFERENCES Foter, M. J., C. M. Palmer and T. E. Maloney. Antialgal properties of various antibiotics. Antibiotics and Chemotherapy. 111(5). 1953. Hargrave, B. T. Epibenthic algal production and community respiration in the sediments of Marion Lake. J. Fish. Res. Bd. Canada, 26:2003-2026. 1969. Hunter, E. 0., Jr. and I. McVeigh. The effects of selected antibiotics on pur cultures of algae. Am. J. Bot. 48:179-85. 1961. Smith, K. L. Jr., K. A. Burns and J. M. Teal. 1972. In situ respiration of benthic communities in Castle Harbor, Bermuda. Mar. B i o l . 12:196-199. Vance, B. D. Sensitivity of Microcystis aeroginosa and other blue-green algae and associated bacteria to selected antibiotics. J. Phycology 1:125-128. 1966. 


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