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Effects of log storage on water quality and microbiology in experimental enclosures in Babine Lake, British… Wentzell, Paula Lanette 1987

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EFFECTS OF LOG STORAGE ON WATER QUALITY AND MICROBIOLOGY EXPERIMENTAL ENCLOSURES IN BABINE LAKE, BRITISH COLUMBIA. by PAULA LANETTE WENTZELL •a • A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DECREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Civil Engineering W e accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A April 30, 1987 © Paula Lanette Wentzel l , 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the THE UNIVERSITY OF BRITISH COLUMBIA, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Civil Engineering THE UNIVERSITY OF BRITISH COLUMBIA 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: April 30, 1987 ABSTRACT The environmental impacts of log storage on water quality and microbiology in Babine Lake, B.C. were studied in experimental enclosures. The enclosure work was a two phase study, with data collected during the summers of 1984 and 1985. The experiments performed in the field season of 1984 involved the addition of mixed lodgepole pine (Pinus contorta) and white spruce (Picea glauca) bark debris, in different amounts, to the enclosures to examine effects on water quality (dissolved oxygen, lignins-tannins, total organic carbon, total inorganic carbon, pH, and alkalinity), bacterial activity (heterotrophy), and phytoplanktonic primary production. The 1985 study attempted to simulate a shallow water log storage facility by adding logs to the enclosures. A comparison was made of differences in water quality (including the above measurements plus chemical oxygen demand, nutrients, volatile fatty acids, and carbohydrates), and sestonic bacterial and phytoplanktonic algal populations (enumeration and biomass calculations) between (i) lodgepole pine and white spruce, and (ii) the number of logs per enclosure. The bark enclosure study resulted in organic enrichment of the enclosure ecosystem. More than 50% of the total organic carbon (TOC) was attributed to leached lignins and tannins. The leachate was capable of modifying microorganism production. Phytoplanktonic algal biomass, measured by chlorophyll _a, was completely eliminated at high concentrations of bark. Short term laboratory studies with bark leachate inhibited microbial activity of natural aquatic bacteria, however, from the enclosure experiments, it was apparent that with time a population of microbes would develop to utilize the chemically complex leachate. The presence of utilizable organic material (i.e. wood sugars) and an active microbial community resulted in a significant decrease in the dissolved oxygen levels. ii The results of the log study found significant decreases in the amount of organic extractives leached from logs compared to the TOC concentration in the bark experiment. For example, the TOC concentrations in the 5 log enclosures were approximately 20 mg/l by the end of the 25 day test period (s 10% was lignin and tannin carbon) ; this value was significantly smaller compared to the bark experiments, where the TOC levels in the heavy bark treatment (20 kg) reached — 400 mg/l after 25 days. A comparison between the bark and log experiments (on an equivalent bark dry weight basis) found water quality degradation by bark leachates more severe than log leachates. The log leachate stimulated bacterial production and did not adversely affect phytoplankton biomass. The increase in bacterial production, a direct result of (i) an available carbon source, determined by TOC measurements and the very low concentrations of volatile fatty acids and reduced carbohydrates (rapidly utilized), and (ii) an increase in water temperature caused a decrease in dissolved oxygen levels. The spruce log enclosures for both June and July had higher heterotrophic uptake rates than the pine log enclosures. This indicated a difference in the chemical composition of the spruce and pine log leachates. This chemical difference was detected in the TOC and COD measurements and the microbial uptake kinetics experiment. These measurements found that the pine log leachate was more readily degraded (high affinity, V m a x / K t ) by a heterotrophic population than the spruce leachate, and that this difference could account for the higher dissolved oxygen demand of the pine log enclosures compared to the spruce logs. Detailed chemical analyses of the leachates in the presence of microbial inhibitors may help to delineate this discrepancy between the spruce heterotrophic uptake rates and the chemical and bacterial measurements of the pine log leachates. The enclosure studies indicated that log storage in a shallow, poorly flushed, littoral area of a lake would possibly result in an accumulation of organic iii components leached from the log and bark debris. The organic enrichment of the aquatic ecosystem would contribute to a potential decrease in dissolved oxygen, thus, negatively affecting fish habitat, but could increase microbial production. iv Table of Contents ABSTRACT ii LIST OF TABLES ix LIST OF FIGURES xi ACKNOWLEDGEMENTS xiii 1. INTRODUCTION 1 2. LITERATURE REVIEW 3 2.1 Location, morphology, and limnology of Babine Lake 3 2.2 History of bark and log leaching experiments 5 2.3 Chemical composition of wood and bark extractives 9 3. EXPERIMENTAL DESIGN, METHODS, AND MATERIALS 16 3.1 Experimental design of 1984 bark study 16 3.1.1 Experimental treatments 16 3.1.2 Enclosure preparation 18 3.2 Experimental design of 1985 log study 18 3.2.1 Enclosure preparation 19 4. METHODS OF ANALYSIS 22 4.1 Water quality 22 4.1.1 General water quality measurements 22 4.1.1.1 Dissolved oxygen and temperature 22 4.1.1.2 pH and alkalinity 22 4.1.2 Organic pollution indicators 23 4.1.2.1 Total organic and inorganic carbon 23 4.1.2.2 Chemical oxygen demand 24 4.1.2.3 Lignins and tannins 25 4.1.3 Nutrients and organic substrates 25 v 4.1.3.1 Total Kjeldhal nitrogen 25 4.1.3.2 Total phosphorus 25 4.1.3.3 Total reducing carbohydrates 26 4.1.3.4 Volatile fatty acids 26 4.2 Bacterial population 27 4.2.1 Biomass and numbers 27 4.2.2 Heterotrophy 28 4.2.3 Heterotrophic uptake kinetics 29 4.2.3.1 Bark leachate inhibition experiments 29 4.2.3.2 Bacterial uptake studies with log leachates 31 4.3 Phytoplanktonic algal population 31 4.3.1 Biomass and enumeration 31 4.3.2 Phytoplanktonic primary production 34 4.4 Comparison between the bark and log studies 34 4.5 Statistical analysis 35 5. RESULTS 36 5.1 Enclosure experiments with bark - 1984 36 5.1.1 Comparison of the lake and control data 36 5.1.2 Water quality 37 5.1.2.1 General water quality measurements 37 5.1.2.2 Organic pollution indicators 41 5.1.3 Bacterial population 44 5.1.3.1 Heterotrophy 44 5.1.3.2 Bark leachate inhibition experiments 47 5.1.4 Phytoplanktonic algal population 47 5.1.4.1 Phytoplanktonic biomass 4 7 5.1.4.2 Phytoplanktonic primary production 47 vi 5.2 Enclosure experiments with logs - 1985 48 5.2.1 Comparison of the lake and control data 48 5.2.2 Water quality 51 5.2.2.1 General water quality measurements 51 5.2.2.2 Organic pollution indicators 55 5.2.3 Bacterial population 62 5.2.3.1 Bacterial numbers, biomass, and heterotrophy 62 5.2.3.2 Heterotrophic uptake kinetics 68 5.2.4 Phytoplanktonic algal population 71 6. DISCUSSION 73 6.1 Enclosure experiments with bark - 1984 75 6.1.1 Comparison of control and lake results for bark and log studies 75 6.1.2 Water quality 76 6.1.3 Heterotrophy 83 6.1.4 Phytoplanktonic algal population 85 6.2 Enclosure experiments with logs - 1985 86 6.2.1 Water quality 87 6.2.1.1 Dissolved oxygen (DO) 87 6.2.1.2 Total organic carbon (TOC) and chemical oxygen demand (COD) 88 6.2.2 Bacterial population 91 6.2.2.1 Numbers, biomass and heterotrophy 91 6.2.2.2 Kinetic uptake study 95 6.2.3 Phytoplankton biomass 98 6.3 Comparison between the enclosure studies and the 3 year field study by Westwater Research 98 7. CONCLUSION 102 vii 8. APPENDIX A 1 0 4 9. APPENDIX B 1 0 9 10. ABBREVIATIONS • 1 1 2 11. REFERENCES 1 1 3 viii List of Tables 1 Main groups of wood extraneous compounds (Buchanan, 1975 ; Fengel and Wegener, 1985). 2 Chemical composition of bark extractives (Jensen et a!. 1975 ; Fengel and Wegener, 1985). 1 Summary of the 1984 enclosure experiments. 2 Experimental design of the 1985 enclosure study. 1 Process of preservation for analytical procedures for water quality, bacterial, and phytoplankton studies. 2 Volumes of solutions added to the bark leachate reaction flasks in microbial inhibition studies. 3 Volumes of solutions added to the log leachate reaction flasks in microbial activity studies. 1 Changes in dissolved oxygen, pH, and alkalinity over time in the 1984 bark enclosure studies done at Babine Lake. 2 Temporal changes in the concentration of organic and inorganic carbon compounds in the 1984 bark enclosure experiments at Babine Lake. 3 Temporal changes in heterotrophy and phytoplanktonic biomass and productivity in the 1984 bark enclosure experiments at Babine Lake. 4 A comparison between the pine log control enclosure and Babine Lake of significant changes in several water quality and bacterial measurements. 5 A comparison of significant changes in heterotrophy and bacterial numbers and biomass between the spruce log control enclosure and Babine Lake, 1985. 6 A summary of means and standard deviations of general water quality measurements for the 1985 pine and spruce log study at Babine Lake. ix 5.7 Effects of pine and spruce logs on dissolved oxygen in the 1985 June log enclosure experiments. 5.8 Temporal changes in total organic carbon in the pine and spruce log enclosure studies at Babine Lake, 1985. 5.9 Temporal changes in bacterial numbers in the pine and spruce log enclosure studies of 1985, Babine Lake. 5.10 Heterotrophic uptake kinetic values obtained on Days 0 and 10 from the 5 spruce and pine log enclosures. 5.11 Temporal changes in chlorophyll _a_ concentration in the pine and spruce log enclosure studies done at Babine Lake, 1985. 6.1 A calculation of the dry weight of bark added to the 1984 bark and 1985 log enclosure studies for water quality comparisons. 6.2 A comparison of water quality conditions in enclosures with 1 kg bark (July, 1984) and 1 log (July, 1985) over an 11 day period. 6.3 A comparison of water quality measurements between several researchers bark leaching experiments. 6.4 Calculations to determine the % of total organic carbon (TOC) comprised of lignins and tannins (L-T) and resultant chemical oxygen demand (COD). 6.5 A comparison of absolute inorganic carbon concentrations between alkalinity and TIC for the 20 kg bark enclosure of 1984. 6.6 Dissolved oxygen saturation for the 1985 log enclosure studies at Babine Lake. 6.7 A comparison of the uptake of 1 4 carbon per ug of bacterial biomass for the 1985 log experiments. List of Figures 2.1 Location of log transportation study areas in Babine Lake. 3.1 Diagram of an experimental enclosure. 5.1 Temporal changes in dissolved oxygen in the 20 kg bark enclosure at Babine Lake ; control = no bark. 5.2 Temporal changes in pH in the 20 kg bark enclosure experiment at Babine Lake, 1984 ; control = no bark. 5.3 Temporal changes in alkalinity in the 20 kg bark enclosure experiment at Babine Lake, 1984 ; control = no bark. 5.4 Temporal changes in total organic carbon in the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. 5.5 Temporal changes in the total inorganic carbon levels in the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. 5.6 Temporal changes in lignins and tannins concentration for the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. 5.7 Temporal changes in heterotrophic uptake of labelled glucose in the 20 kg bark experiment at Babine Lake, 1984 ; control = no bark. 5.8 Temporal changes in chlorophyll _a_ concentration in the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. 5.9 Effects of spruce and pine logs on dissolved oxygen in enclosures for the 1985 July log study period ; number of logs in treatment are given in parentheses. 5.10 Temporal changes in COD concentration for the pine and spruce log enclosure studies done at Babine Lake, 1985 ; number of logs in treatment are given in parentheses. 5.11 Linear regression relationship between changes in COD and dissolved oxygen concentrations for the Babine Lake pine log experiments of 1985. xi 5.12 Linear regression relationship between changes in COD and dissolved oxygen concentrations for the spruce log experiments at Babine Lake, 1985. 61 5.13 Changes in L-T concentrations in the 1985 spruce and pine log experiments at Babine Lake ; number of logs in treatment are given in parentheses. 62 5.14 Changes in bacterial activity and biomass in the 1985 pine log enclosure experiments done at Babine Lake. 64 5.15 Changes in bacterial activity and biomass in the spruce log enclosure experiments of 1985 at Babine Lake. 65 5.16(A) Bacterial biomass on initiation of the 5 pine logs enclosure at Babine Lake, 1985 ; X1562. 5 urn 67 5.16(B) Bacterial biomass on day 25 of the 5 pine logs enclosure experiment at Babine Lake, 1985 ; X1562. 5 urn 67 5.17 Lineweaver-Burk plot of S/V vs S on Day 10 for the 5 log pine and spruce enclosures an8 the control. 70 6.1 Aerial photograph of the Morrison dump site in mid-winter, 1984. 100 6.2 Aerial photograph of the Morrison dump site on May 14, 1985. 101 xii ACKNOWLEDGEMENTS This project was funded under Section 88.2 of the B.C. Forests Act. The students were funded by a GREAT Scholarship to E. Power from the B.C. Science Council and both a YEP university student summer grant and NSERC operating grant for P. Wentzell. I would like to thank Doug Reynolds, Brett Hall, and Jim Mclver for helping obtain logs for the experimental enclosure studies and Pat Ogawa of Houston Forest Products for donating lumber for construction of the experimental enclosures. Norm Aason, Ham Brown, and Jim Barker of the Granisle Power Boat Club are acknowledged for providing housing and marina facilities. I also thank Stewart Barnetson and Colin Harrison, managers of the Fulton River spawning channels, for providing laboratory facilities to conduct chemical analysis and store our equipment from 1984 to 1985. The assistance given by Susan Liptak and Paula Parkinson of the Environmental Engineering Laboratory, U.B.C., and Ralph Daley previously of the National Water Research Institute, West Vancouver, in water quality measurements and bacterial analysis is greatly appreciated. I thank Craig Peddie for his help in computer word processing, Tom Northcote for his valued opinions and comments editing this manuscript, and Ken Hall for his patience. And finally, I wish to express sincere gratitude to Beth Power for her helping hand in gathering samples, running analysis, constructive criticism, patience, and putting up with a stubborn and sometimes cranky associate. xiii 1. INTRODUCTION In 1982 the British Columbia Ministry of Forests, in response to a pine beetle infestation in the forests of the Morrison Arm region of Babine Lake, decided to accelerate logging plans in an attempt to salvage the infested timber. A transportation system was proposed that would involve the dumping, storage, towing, and dewatering of logs. The Department of Fisheries and Oceans reviewed the proposal and gave its conditional approval provided that a three year study be undertaken to determine the effects of log storage and transportation on fish populations and habitat of Babine Lake, with emphasis on sockeye salmon. The Westwater Research Center received funding under Section 88.2 of the B.C. Forests Act provided to the University of British Columbia as a grant from Houston Forest Products Ltd. to study the environmental impacts of log handling and storage activities on Babine Lake, British Columbia. The main objectives of the student thesis projects were to experimentally test hypotheses about the chemical and biotic effects of log storage in a shallow limnetic environment. This research was done in the summers of 1984 and 1985 and conducted in conjunction with Westwater Research Centre's three year study- of log transporation impacts in Babine Lake. The hypothesis that littoral water quality and microbiology populations are affected by log storage activities was tested in experimental enclosures. Experimental enclosures were used because conditions inside these large bags can be manipulated and easily monitored, as opposed to in situ work where experimentation is difficult. Wood and bark debris, and log rafts were placed in the enclosures to simulate conditions that could occur when logs are handled and stored in shallow 1 2 water areas. As one of the students in the project, 1 studied changes in (i) water quality conditions (dissolved oxygen, nutrients, lignins and tannins, organic carbon, chemical oxygen demand, pH, and alkalinity), (ii) microbial activity and biomass, and (iii) phytoplanktonic primary production and biomass. Beth Power, a graduate student in zoology, (i) studied the effects of wood and bark debris on the zooplankton population, (ii) performed short term bioassay tests to examine the toxicity of log and bark leachate on Daphnia pulex, sockeye fry (Oncorhynchus nerka), and rainbow trout (Salmo gairdneri), and (iii) examined the effect of bark and log leachate on the ability of sockeye fry to avoid predators. The results of this project should provide some of the first direct and manipulative experimental information regarding effects of log storage on lake water quality and related effects on bacterial and algal processes important to salmonid survival and reproduction. This research should help greatly in predicting log handling impacts on poorly circulated littoral areas of lakes. The following section is a literature review on the limnology and associated lake dynamics of Babine Lake, previous research concerned with assessing the environmental impacts from bark, wood debris, and logs, and the chemical composition of wood and bark. 2. LITERATURE REVIEW 2.1 LOCATION, MORPHOLOGY, AND LIMNOLOGY OF BABINE LAKE Babine Lake is the largest natural lake in the province of British Columbia. The lake is one of the major sockeye salmon producers in the province, and presently 90% of the Skeena River sockeye originate in tributaries surrounding the lake (Levy and Hall, 1985). Babine Lake is a long (150 km), narrow lake with an elevation of 708 m, located on the Central Interior Plateau of British Columbia, 580 km North of Vancouver at 55° N latitude and 123° W longitude (Figure 2.1). It forms one of the major drainage basins in the Skeena River system (Levy and Hall, 1985). It has an area of 490 km 2 , mean depth of 55 m, and a maximum depth of 186 m (Johnson, 1965). Based on a literature review of the limnology of Babine Lake (Levy and Hall, 1985), and considering trophic state indicators, i.e. primary production, chlorophyll _a, and nutrients, the trophic status of the lake best fits the oligotrophic classification. There are several morphometric factors to consider when evaluating the possible effects of log storage and transportation activities on Babine Lake. The small littoral zone combined with a deep mean depth, large volume of water (approximately 27 km 3 ; Narver and Andersen, 1968), and persistant strong winds (long fetches) do not allow for the formation of a stable thermocline. Therefore, I would postulate that the concentration of materials leached from the logs and the bark debris dislodged from the transportation of booms, would not build up to toxic levels in the epilimnetic zone of the main basin of Babine Lake, but would be dispersed and diluted without significant effect on the aquatic system. 3 4 Historical Dump Site • 1984 1s Historical Dewatering Site 1984 4 F O R T B A B I N E Morrison Arm Dump Site 1983 1984 1985 / BABINE LAKE./ / / / >. VANCOUVER 200 miles Existing Northwood Dewatering Site 1984 T O P L E Y 3 LANDING Future Houston Dewatering Site 1983 1984 1985 (from West, 1978. p.2) Existing Northwood Dump Site 984 1983 - pre-impact data 1984 - "extensive post treatment analysis" 1985 - post-impact data 10 15 miles Figure 2.1 Location of log transportation study areas in Babine Lake. 5 However, because the Morrison Arm area of Babine Lake has a sheltered littoral zone and a shallow mean depth (compared with the main basin of Babine Lake) there exists the possibility of aquatic habitat degradation. Therefore, the limnology of Babine Lake is an important factor to consider when assessing the enclosure results and predicting logging impacts in the lake. 2.2 HISTORY OF BARK AND LOG LEACHING EXPERIMENTS There are two areas of research concerned with the leaching of pollutants from wood and bark. Most of the literature is concerned with industrial timber processing ; this includes (i) woodwaste from debarking operations with the bark and wood debris dumped in landfills or left in piles at the mill site (Asano ef al. 1974 ; Atwater, 1980 ; Benedict et al. 1974a and 1974b ; Evans, 1973 ; Harger et al. 1973 ; Henricksen and Samdal, 1966 ; Phipps, 1974 ; Raabe, 1968 ; Schermer and Phipps, 1976 ; Slagle, 1976 ; Sproul and Sharpe, 1970 ; Thomas, 1977), and (ii) the discharge of pulp and paper mill effluent on receiving water quality (Kroner and Moore, 1953 ; Pearson, 1980 ; Poole ef al. 1978). The remaining literature includes work pertaining to the quantity and quality of leachates derived from various hardwood and softwood species, and their effect upon limnetic and benthic communities (Buchanan ef al. 1976 ; Duval, 1980 ; Graham and Schaumburg, 1969 ; Hansen et al. 1971 ; Pease, 1974 ; Peters, 1974 ; Peters ef al. 1976 ; Ponce, 1974 ; Schaumburg, 1973 ; Servici ef al. 1971 ; Gellman, 1971). Most of the woodwaste landfill and pulp and paper effluent studies are similar in experimental design and parameters measured. The leachate (pollution) , generated by laboratory and field studies is characterized using chemical oxygen demand (COD), biochemical oxygen demand (BOD), total organic carbon (TOC), pH, 6 odour, alkalinity, colour, and toxicity tests. Total Kjeldahl nitrogen (TKN), total phosphorus (TP), and metals are measured in municipal landfills where hogfuel is used as a cover. Most of the laboratory experiments are concerned with analyzing water soluble leachates (extractives) generated by fresh and aged softwood bark in static or constant (continuous) flow lysimeters or both (Benedict et al. 1974a and 1974b ; Henricksen and Samdal, 1966). The softwood species commonly studied includes Douglas fir (Pseudotsuga menziesii), western hemlock (Tsuga heterophylla), lodgepole pine (Pinus contorta), and western red cedar (Thuja plicata). The duration of the experiments changed between the researchers, for example, 1 0 - 4 0 hours (Asano ef al. 1974) and long term experiments of 22 days (Schermer and Phipps, 1976) to 4 years (Atwater, 1980). The high concentrations of wood and woodwaste used in the studies resulted in high concentrations of organic pollutants leached during the experiment. For example, Schermer and Phipps (1976) have a COD value of 8282 mg/l at the terminaton of their 23 day static leaching study with Douglas fir. Their results were similar to the data from the short term continuous flow experiments with softwood by Benedict ef al. (1974b), i.e. COD > 4500 mg/l after 23 days. Several authors initiated long term studies to determine both the mechanism responsible for leaching and the biodegradability of water soluble extractives (Benedict ef al. 1974a and 19746 ; Henricksen and Samdal, 1966 ; Phipps, 1974 ; Schermer and Phipps, 1976 ; Sproul and Sharpe, 1970 ; Thomas, 1977). The long term static and dynamic (constant flow) studies (minimum of 90 days), although involving different species of woodwaste and bark, give the following leaching characteristics : 1. biodegradability of the leachate is a function of the chemical nature of the water soluble compounds (extractives) found in each different species of tree, 2. initial high concentrations of COD, BOD, total solids, TOC, and colour 7 decrease with time (after = 4 to 10 days) because of (i) a decrease in readily available near surface water soluble compounds and (ii) a decrease in the concentration gradient or driving force in static experiments, 3. biphasic BOD where the initial stage is characterized by a high BOD (simple wood sugars) mediated, by heterotrophic bacteria and the second stage is represented by a lower BOD (compared to the rapid first stage) due to the slow decomposition by bacteria and fungi of lignin and other chemically complex structures (Benner ef al. 1986). For example, Raabe (1968) and Kroner and Moore (1953) found high concentrations (> 60%) of lignin present in receiving water 20 weeks after initial discharge, 4. the leaching mechanism is a function of the volume of contact water and contact time, i.e. potential BOD and COD are depleted more rapidly by a large volume of contact water than by a small volume of water. Existing literature on the effects of log storage on the aquatic environment can be classified into three categories : physical, effects, chemical effects, and biotic effects (Power and Wentzell, 1985). The most noticeable impacts of log handling on the aquatic environment are the physical changes that occur in the habitat. Chemical changes to water quality induced by log storage have been quantified, but data are generally obtained from laboratory studies. There is information on the effects of log storage on aquatic vegetation, benthic invertebrates, zooplankton, and fish, however, with the exception of the fry feeding experiments conducted by Levy ef al. (1982), most of the research is descriptive. Duval (1980) and Hansen ef al. (1971) provide good literature reviews on log handling and dumping in coastal public waters. Graham and Schaumburg (1969), Pease (1974), Schaumburg (1973), Peters (1974), and Gellman (1971) conducted laboratory and field studies to, firstly, characterize log leachates and, secondly, 8 determine the rate and mechanism of leaching soluble organics from logs and bark. Schaumburg (1973) investigates the magnitude of the pollution problem associated with log handling practices. The results indicate that the open ends and logs without bark are a major source of organic matter (i.e. wood sugars) and a possible pollution problem. For example, after a 7 day leaching period, a 150 year old Douglas fir log section, with bark, had a COD of 53 mg/l, compared to 189 mg/l from a similar log section without bark. The results of field studies by Schaumburg (1973) and Gellman (1971) indicates leachates from logs held in storage exert BOD and COD on the holding water, but not in sufficient concentrations to adversely affect water quality. However, the distribution and accumulation of logging debris on the sediment can affect epibenthic and benthic communities. Benthal deposits of bark can result in a decrease in abundance of infauna, low dissolved oxygen concentrations within the bark deposits exerted by the BOD of the bark, and, possibly, bark leachates toxic to the infauna (Pease, 1974) Pease (1974) conducted log leachate studies with unaltered sections of western hemlock, Sitka spruce {Picea sitchensis), red cedar, and yellow cedar (Chamaecyparis nootkatensis). The results of Pease's (1974) unaltered log sections are similar to Schaumburg (1973) and Gellman (1971), i.e. significant increases in COD, BOD, and TOC, and a decrease in pH. Buchanan ef al. (1976) did toxicity bioassays on larval and adult pink shrimp (Pandalus borealis), larval Dungeness crab (Cancer magister) and pink salmon fry (Oncorhynchus gorbuscha) with Sitka spruce and hemlock bark extractives. The bioassay tests have a dissolved oxygen concentration above 6 mg/l. Ponce (1974) also conducted toxicity bioassays with leachate from Douglas fir using guppies (Poecilia reticulata) and steelhead trout (Salmo gairdneri) as the test animals. The study results from Buchanan ef al. (1976) and Ponce (1974) indicates that leachate 9 toxicity is a function of the chemical composition of the water soluble fraction. For example, laboratory bioassays by Peters (1974) with the water soluble fraction of western red cedar heartwood leachate found the extraneous components responsible for respiratory distress leading to death in coho salmon (Oncorhynchus kisutch) fry. Increases in opercular movements in the salmon fry without an increase in oxygen consumption, may be the result of charged compounds (i.e. tropolone) complexing with the iron component of hemoglobin molecules and interfering with respiration (Peters, 1974). Servizi ef al. (1971) studied the effects of Douglas fir, lodgepole pine, Engelmann spruce (.Picea engelmannii), and Alpine fir (Abies lasiocarpa) bark deposits on the survival of sockeye salmon eggs (Oncorhynchus nerka) and alevins in flow through gravel boxes (dissolved oxygen s 4 mg/l). The results indicate that mortality is a function of Sphaerotilus (filamentous bacteria) growth (promoted by leachates from fresh bark) on the gills of the alevins, therefore, suggesting that the chemical nature of the leachates was not directly toxic to the test fish at that concentration of bark. The literature to date reveals limited studies on (i) the microorganisms responsible for degrading wood and bark extractives, (ii) microbial activity and growth kinetics, and (iii) poor laboratory simulations of the natural log storage environment. This thesis attempts to simulate an actual log storage facility while maintaining experimental control of the ecosystem. 2.3 CHEMICAL COMPOSITION OF WOOD AND BARK EXTRACTIVES The biotic water environment of the enclosures is chemically governed by the materials leached from the wood and bark debris, and the logs. The extent of water quality degradation (i.e. organic enrichment and dissolved oxygen demand) is 10 a function of the quality and quantity of the leachates extracted from the bark and logs. Therefore, knowledge of what is being leached from the logs and bark will help in understanding and interpreting the experimental results. There is a variety of water soluble chemicals contained in wood and bark. It is important to know the chemical nature of the extraneous components leached from the spruce and pine logs because the quantity and quality of aqueous extracts from the wood and bark would determine the extent of water quality degradation. Wise (1959) defines the term extraneous components as " ...a wide variety of organic (and some inorganic) substances that can be extracted with cold water and that are soluble in neutral solvents such as ether, chloroform, the alcohols, benzene, ligroine, and methylene chloride." The cell wall components consist of the structural compounds cellulose, lignin, and polyose (hemicellulose), which form interpenetrating systems insoluble in cold water (20 °C) and neutral solvents (Wise, 1959). The structural compounds of coniferous wood, mainly lignin and cellulose, are chemically similar irrespective of tree species ; however, the wood can vary in appearance, specific gravity, and strength (Barton, 1975). All woods contain a wide variety of organic and some inorganic substances that can be extracted with cold water and neutral solvents (Wise, 1959 ; Buchanan, 1975 ; Jensen ef al. 1975). These extraneous compounds are known as extractives and comprise 2% to 15% of coniferous wood (Barton, 1975). More recently, Fengel and Wegener (1984) have classed the organic matter as extractives and the inorganic matter as ash. Extractives are low molecular weight compounds compared to the structural components of mature wood. Extractives impart a distinct set of properties to each tree species and are known as personality chemicals (Fengel and Wegener, 1984 ; Barton, 1975). Wise (1959) states that extraneous compounds serve to fingerprint the wood. Some factors that affect the types and amounts of extractives present in 11 a tree are species, season, type of wood (heartwood vs. sapwood) and the distance from the butt of the tree. Some properties affected by extractives are odour, resistance to decay and insect attack, colour, and pulpability (Barton, 1975). Available literature illustrates an obvious difference between chemical components of bark and wood. Fengel and Wegener (1984) state that bark possesses polyphenols and suberin, a lower percentage of polysaccharides, and a higher percentage of extractives than wood. Chemical differences also exist between the inner bark, phloem, and the outer bark, rhytidome. Generally the content of extractives and polysaccharides decreases, and the content of lignin and polyphenolic substances increases from the inner bark to the outer bark (Fengel and Wegener, 1984). The function of the inner bark is to transport assimilates and to serve as a storage organ for food reserves, whereas the outer bark consists of dead cells and forms a protective layer against mechanical and chemical injury (Kurth, 1944). Tables 2.1 and 2.2 lists the major extraneous components found in bark and wood extractives. The carbohydrates, vitamins, and proteins would provide a utilizable source of carbon which would contribute to a dissolved oxygen demand. Several of the aromatic compounds are chemically complex (i.e. tannins and lignins) and would not cause an immediate dissolved oxygen demand, but would increase water turbidity and colour ; this would cause light attenuation in the water column. The terpenes are volatile, oily compounds which leave an oily residue on the surface of water. Resin acids and unsaturated fatty acids in hardboard plant effluent have been shown to be toxic to sockeye salmon (Oncorhynchus nerka) fingerlings if present in the water in concentrations S1 mg/l (Rogers ef al. 1979) Therefore, it is apparent that the water soluble compounds of wood and bark could directly or indirectly affect water quality and aquatic microorganisms in the enclosures. T a b l e 2.1 M a i n g r o u p s o f w o o d e x t r a n e o u s c o m p o u n d s ( B u c h a n a n , 1 9 7 5 ; F e n g e l a n d W e g e n e r , 1985 ) . E x t r a c t i v e s D e s c r i p t i o n L o c a t i o n f o u n d E x t r a c t i o n in t r ee S o l v e n t 1. A r o m a t i c ( p h e n o l i c ) c o m p o u n d s a. lignans b. s t i lbenes c . f l avono ids and derivatives d . tannins i) hydrolzyable tannins ii) c o n d e n s e d tannins (phlobaphenes) 2 . T e r p e n e s a. m o n o t e r p e n e s b. o x y g e n a t e d m o n o t e r p e n e s c . d i te rpene (resin ac ids) d . t r i terpene e. s e s q u i - and po ly terprenes f. t r o p o l o n e s and thujic ac id several d imer ic phenylpropane c o m p o u n d s l inked b e t w e e n B-C a toms pinosylvin predominant in pine s p e c i e s po lyhydroxy l ic phenols hydrolysis p r o d u c e s gallic and ellagic a c i d s , and sugars natural substances chemical ly der ived f r o m isoprene units, 2 - m e t h y l -butadiene ; acyc l ic and c y c l i c a - p i n e n e , gum turpentine o f mos t p ines o x y g e n containing m o n o t e r p e n e s s e c r e t e d by epithelical ce l l s and o c c u r as f ree acids not abundant 15 plus c a r b o n atoms c o m p o u n d s f o u n d in w e s t e r n r e d cedar and s p e c i e s o f the Cupressaceae ; exhibit ac id ic and aromatic proper t ies r o o t s , h e a r t w o o d , fo l iage, e x u d a t e s h e a r t w o o d , inner bark h e a r t w o o d leaves bark inner bark, s a p w o o d h e a r t w o o d h e a r t w o o d , s a p w o o d leaves, bark, exudates h e a r t w o o d h e a r t w o o d a c e t o n e , methanol ether a lcohol partially so lub le in water a lcoho l a l c o h o l , b e n z e n e , ether a l c o h o l , ether b e n z e n e , ether b e n z e n e , ether s team volatile t Table 2.1 cont'd 3. Aliphatic acids a. saturated acids b. unsaturated acids c. fats and oils d. waxes 4. Carbohydrates a. mono-, di-, and polysaccharids i) simple sugars- sucrose - fructose - glucose - arabinose b. glycosides c. starch d. pectic material e. cellulose f. polyoses (hemicellulose) 5. Alcohols a. sterols b. aliphatic alcohols palmitic acid fatty acids are found as triglycerides in the sapwood t ether t benzene, ether ether major components are not extractable (i.e. cell wall compounds) reserve food supply cell wall component high molecular weight polymer of p-D-glucose polymers of sugars sapwood, inner bark sapwood, inner bark sapwood, inner bark, heartwood heartwood t t t t water water water water t t t aliphatic rings generally found free and as esters and glycosides occur as ester components - hot water = no available information Table 2.2 Chemical composition of bark extractives (Jensen et al. 1975 ; Fengel and Wegener, 1985). Extractives Description Extraction Solvent 1. Aromatic (phenolic) compounds benzene, ether a. stilbenes b. polyphenols i) flavane derivatives ii) hydrolzable tannins iii) condensed tannins (phlobaphenes) iv) polyphenols acids classed according to molecular weight and solubility colouring materials ; occur in free state and as glycerides outer bark contains the low molecular weight tannins high molecular weight tannins ether, hot water alcohol alcohol, water ethanol, hot water 1% NaOH/100 *C 2. Terpenes and resin acids 3. Aliphatic acids a. saturated acids b. unsaturated acids c. fats d. waxes triterpenes are the predominant bark terpenes acids are secreted in response to damage benzene, ether 4. Carbohydrates a. cellulose b. hemicellulose i) pectins ii) gums c. mono-, di- and polysaccharides - not extractable - easily hydrolyzed, however not extracted with indifferent solvents t t - 1.87% spruce bark and 1.35% pine bark mild acid t t some are water soluble Table 2.2 cont'd 5. Alcohols a. sterols 6. Vitamins 7. Proteins - predominant sterol in pine is a-sitosterol - vitamins are stored in the fall and depleted by spring vitamins are present in significant amounts - bark acts as storage place for proteins and other assimilates ; amounts vary seasonally benzene some are water soluble some are water soluble, but most are not = no available information 3. EXPERIMENTAL DESIGN, METHODS AND MATERIALS The enclosures consisted of two parts ; a plastic bag and the float from which the bag was suspended. The bags were cylindrical in shape, with an enclosed bottom, and made of woven polyolefin fabric (1.5 m diameter and 2.1 m deep). The enclosures contained a volume of 3700 I and were not in contact with the lake sediment, thus there was no exchange of water (Figure 3.1). The collar was constructed of black plastic to slow fabric degradation by ultraviolet light ; the bag was made of clear, white plastic to allow maximum natural radiation inside the bag. The float was of plywood construction and flotation was provided by styrofoam blocks. The bag was suspended from the inside of the float and laced into place (Figure 3.1). Eight identical enclosures were built and moored inside Granisle Power Boat Club marina between June and August of 1984, and May and August of 1985. The marina site was approximately 3 m deep and provided protection from storms and vandalism. 3.1 EXPERIMENTAL DESIGN OF 1984 BARK STUDY The experimental design of 1984 attempted to define a range of lethal and sublethal leachate concentrations, i.e. 20 kg and 1 kg bark experiments, respectively. 3.1.1 EXPERIMENTAL TREATMENTS The 1984 field season was concerned primarily with determining the effects of bark on the enclosure ecosystem. Table 3.1 lists the treatments (amounts of bark 16 17 plywood Figure 3.1 Diagram of an experimental enclosure. 18 added to the enclosures) and their duration for the 1984 study period. None of the treatments were replicated. 3.1.2 ENCLOSURE PREPARATION The enclosures were initially filled with a Par electric bilge pump. This was later replaced with a 3 horse power Honda water pump. The enclosures were always filled the day before the start of an experiment to allow the system to equilibrate with the surrounding waters. The bark consisted of a mixture of white spruce and lodgepole pine ; the ratio was 40% spruce to 60% pine. The mixture was representative of the ratio of trees being logged and stored at the Morrison Arm storage facility. The bark was fresh and obtained from nearby construction sites where spruce and pine logs cut in April were being peeled (debarked) for log homes. The bark included the rhizome and a thin strip of inner bark. Enclosures were sampled initially (before treatment) and then every 5 to 7 days for the duration of the experiment (2 to 4 weeks). The adjacent lake was also sampled to allow a comparison between the dynamics of the enclosures and the lake. All samples and in situ measurements were taken at a depth of 1 m. 3.2 EXPERIMENTAL DESIGN OF 1985 LOG STUDY The experiments of the 1985 field season closely examined the effect of log booms, simulated by log rafts (logs approximately 1.3 m in length and 14 cm in diameter) on enclosure water quality, bacterial activity and biomass, and phytoplanktonic algal biomass. The original eight enclosures of 1984 were used in the log study. The log enclosure studies of 1985 attempted to qualify the Table 3.1 Summary of the 1984 enclosure experiments. 19 Experiment Duration 20 kg bark 7 June to 8 July 5 kg bark 12 July to 26 July 1 kg bark 29 July to 9 August log raft* 17 July to 7 August = raft contained 5 pine logs and 3 spruce logs, freshly cut 1 week prior to the start of the experiment ; each log was approximately 1.3 m in length and 14 cm in diameter. environmental effects of pine and spruce leachates generated from a simulated log storage facility. This was done with a more realistic loading of logs to define a graduated range of effects from severe water quality degradation to no significant change in the enclosure environment. The effects of pine logs and spruce logs on the enclosure ecosystem were studied independent of one another, i.e. four enclosures for spruce logs and four enclosures for pine logs. The experimental design of 1985 is outlined in Table 3.2. 3.2.1 ENCLOSURE PREPARATION The enclosures were filled with filtered lake water (100 Mm) by a 3 horse power Honda water pump. The logs used for the study were harvested one week prior to the commencement of the experimental period. It is important to emphasize that the bark and logs used in the 1984 and 1985 experiments were fresh, i.e. had not been exposed to time and weather conditions. This enabled us 20 to simulate as closely as possible the actual logging and storage operations practised by Houston Forest Products at Morrison Arm where the felled logs are immediately bundled and dumped into the holding bay. All enclosures and the adjacent lake were sampled at 0, 2, 5, 10, 15, 20, and 25 day intervals at a depth of 1 m. The dissolved oxygen measurements were performed approximately every other day. Initial sampling, day 0, was conducted before logs were added to the enclosures. The first set of spruce and pine experiments was in June, where the average enclosure temperature was 9 °C. The experiments were repeated in July to examine the effect of water temperature (average enclosure water temperature was 15.6 °C) on enclosure dynamics. Table 3.2 Experimental design of the 1985 enclosure study. 21 Species of tree Experimental interval Number of logs/enclosure Pine June 26/05/85 to 20/06/85 C 1 3 5 July 02/07/85 to 27/07/85 C 1 3 5 Spruce June 02/06/85 to 27/06/85 C 1 3 5 July 09/07/85 to 03/08/85 C 1 3 5 C = control (no logs added) 4. METHODS OF ANALYSIS 4.1 WATER QUALITY The method of preservation, storage container used, and quantity of water sample preserved are outlined in Table 4.1. 4.1.1 GENERAL WATER QUALITY MEASUREMENTS 4.1.1.1 Dissolved oxygen and temperature The DO and temperature were determined in situ with a YSI Model 54 dissolved oxygen/temperature meter. The oxygen probe was calibrated at the beginning of each sampling day by wrapping the probe in a wet towel (simulates air saturation), taking the air temperature, and then correcting for both temperature and elevation. 4.1.1.2 pH and alkalinity The pH was determined with a portable Fisher pH Meter 119. The alkalinity was determined by titrating a 100 ml water sample with 0.02 N ^ S O ^ to a pH of 4.5 (Standard Methods, 1985). The alkalinity was calculated with Equation 4.1 : alkalinity as C a C 0 3 (mg/l) = A x N x 50,000 (4.1) ml of sample 22 23 Table 4.1 Process of preservation for analytical procedures for water quality, bacterial, and phytoplankton studies. Analysis Volume of sample Storage container Method of preservation TOC, TIC, and VFA 5 - 7 ml glass vials frozen (-10 °C) L-T 250 ml high density polyethylene sample bottles frozen (-10 °C) COD, TKN, TP, and TRC 500 ml high density polyethylene sample bottles H 2 S 0 4 to pH<2, cold room where : A = ml of standard acid N = normality of the standard acid. 4.1.2 ORGANIC POLLUTION INDICATORS 4.1.2.1 Total organic and inorganic carbon Total carbon (TC) and total inorganic carbon (TIC) were analyzed with a Beckman Model 915A Total Carbon Analyzer (detection limit = 0.1 mg/l) by the combustion infrared method (Standard Methods, 1985). Two standard curves were made, one for Total Carbon (10, 20, 30, 50, and 100 mg carbon/I from 1.0625 g of crushed and dried KHCgH 4O 4 /500 ml distilled water) and the other for Total Inorganic Carbon (5, 10, 20, 30, and 50 mg carbon/I from 0.4404 g N a 2 C 0 3 and 24 0.3497 g NaHCOg/100 ml distilled water). The water samples were thawed and brought to room temperature. To determine Total Carbon, 50 n\ of sample was injected into a heated (950 °C), packed ceramic combustion tube in a stream of ' oxygen (Total Carbon Channel). The water sample was vapourized and the carbon oxidized to CC>2 and measured by a nondispersive type of infrared analyzer. The TIC was measured on the TIC Channel where the furnace temperature was 150 °C. The volume of water sample injected was 50 n\ and the carrier gas was oxygen. TOC was found by subtracting the TIC results from the TC data. 4.1.2.2 Chemical oxygen demand The chemical oxygen demand (COD) was performed according to the dichromate reflux method (detection limit = 0.01 mg/l) detailed in Standard Methods (1985), except the sulfuric acid reagent was added directly to the flasks and not through the top of the condenser. Equation 4.2 was used to calculate COD: COD (mg/l) = (blank - sample) x N FAS x 8000 (4.2) ml of sample where N FAS ml dichromate x 0.25 ml ferrous ammonium sulfate standard blank blank titre of distilled water, dichromate, sulfuric acid reagent sample = sample titre FAS standard = consists of distilled water and dichromate 25 4.1.2.3 Lignins and tannins Lignins and tannins (L-T) were determined by a spectrophotometric technique where the aromatic hydroxy! groups of these plant constitutents were reacted with tungstophosphoric and molybdophosphoric acids to form a coloured (blue) complex (Standard Methods, 1985). The water samples were thawed, brought to room temperature, the reagents added to the samples and standards (0.5, 1, 2, 3, 5, and 10 mg/l as tannic acid), and left for 1/2 hour to allow for colour development. The absorption of the coloured complex was determined with a 1 cm cell on a Pye Unicam SP8-100 Ultraviolet Spectrophotometer (detection limit = 0.1 mg/l) at 700 T?m and the concentration determined from the standard curve. 4.1.3 NUTRIENTS AND ORGANIC SUBSTRATES 4.1.3.1 Total Kjeldhal nitrogen Total Kjeldhal nitrogen (TKN) was prepared by rigorous acid digestion and determined spectrophotometrically by formation of the ammonium salicylate complex method as outlined in Technicons' Industrial Methods 329-74W and 376-75W. The samples were analyzed with a Technicon Autoanalyzer II (detection limit = 0.2 mg/l) at a wavelength of 660 7 7 m . The final TKN concentration was determined from a standard curve developed for the automated analysis. 4.1.3.2 Total phosphorus Total phosphorus (TP) was measured spectrophotometrically by the stannous 26 chloride reduction method where ammonium molybdate and stannous chloride reacted with the water sample to form a phosphomolybdenum complex (Standard Methods, 1985). The coloured complex was read within 10 minutes of colour development with a 1 cm cell on a Bausch-Lomb Spectronic 88 (detection limit = 0.1 mg/l) at 690 p . A standard curve was generated from 0.5, 1, 2, 3, and 5 mg/l of Fisher Phosphate Standard Solution (1 mg phosphorus/1 ml). 4.1.3.3 Total reducing carbohydrates A colourimetric method was used for quantitative estimation of carbohydrates (Keleti and Lederer, 1974). The standards consisted of 0.4, 1.2, 2.4, and 3.4 mg/l of dextrose. Absorption of the coloured complex was made on a Bausch-Lomb Spectronic 88 at 690 rjm using a 1 cm cell (detection limit = 0.1 mg/l). 4.1.3.4 Volatile fatty acids Preliminary measurements of the volatile fatty acids (VFA) acetic, propionic, and butryic were measured on a Hewlett Packard 5750 Gas Chromatograph. However, the concentration of VFA's was below the detection limit of the gas chromatographic technique, ^ 2 mg/l. Therefore, an alternate method for determining low VFA concentrations (Barcelona et al. 1980) was used (detection limit = 0.1 Mg/l). The water samples were derivatized and measured with a Hewlett Packard 5880A Series Gas Chromatograph. 27 4.2 BACTERIAL POPULATION 4.2.1 BIOMASS AND NUMBERS The total number of bacteria were counted by the acridine orange direct count (AODC) epifluorescent technique as modified by Daley and Hobbie (1975). Nuclepore filters were stained with an irgalan black solution (2 g/l in 2% acetic acid). On sampling days, the prestained filters were rinsed with wash water (lake water filtered with Type HA 0.2 um Millipore filters). The stained filters were placed on a small filter assembly, 1 ml of sample water was added and 4 ml of wash water. The final volume was filtered and washed down with an additional 5 ml of wash water. The filter was placed in a gas-tight petri dish and allowed to air dry for 8 hours. The petri dishes were then labelled with the volume of water sample filtered, and taken to the National Water Research Institute in North Vancouver for counting. Counts were made with a Leitz Ortholux microscope (with an internal magnification of 1.25X) equipped with 12.5X oculars, an NPL 100X oil immersion objective, and a lamp and filter system detailed in Daley and Hobbie (1975). The air dried filters were placed on a filter assembly, stained for 5 minutes with 0.2 um filtered acridine orange, filtered, and washed with 5 ml of 0.2 um (Type HA Millipore) wash water. The filter was placed on a glass slide with a drop of Cargille A Immersion Oil, a cover slide added, and the bacteria counted at 1562X magnification. Random fields were counted until over 400 bacteria were enumerated. These counts were converted to counts/ml (Appendix A). Counts were performed on the volume of wash water and acridine orange used for sample preparation, and subtracted from the sample counts. 28 Bacterial biomass was determined by using a micrometer and measuring the length and width of rods and the diameter of the spheres. The measurements were converted to cell volumes and ultimately to grams of carbon/ml. The calculations for this procedure are detailed in Appendix A. The biovolume for the filamentous bacteria present during the latter stages of the study were found by counting the number of times a filament crossed the counting grid (Brock, 1978). The determination of cell biovolume is listed in Appendix A. 4.2.2 HETEROTROPHY Heterotrophy, a measure of microbial activity, or the uptake of radioactive isotope, i.e. 1 4C-glucose for the bark study and 1 4C-acetate for the pine and spruce log enclosure experiments, by microorganisms, was an in situ experiment performed in each enclosure and the lake. Isotope preparation involved the dilution (with 0.2 um filtered lake water) of 4 microCurrie's (MQ) of ^4C-gIucose (specific activity = 346 uG/Mmole) to a volume of 30 ml in 1984 and 4 uCi of 1 4C-acetate (specific activity = 57.6 uCi/umole) to a final volume of 30 ml in 1985. Four 10 ml syringes were rinsed with the sample water and 9 ml of sample drawn into each syringe. The control was injected with 0.2 ml of concentrated formaldehyde. After a 5 minute period to kill the control, 1 ml of isotope solution (1 ml = 0.0537 Mg glucose or 1 ml = 0.06985 Mg acetate) was injected into each syringe. The 4 syringes were then suspended in an enclosure at a depth of 1 m and incubated for 3 hours. This procedure was repeated for each enclosure and the lake. At the end of the incubation period the samples were filtered (0.2 um, 25 mm, cellulose nitrate membrane filters), and the filters transferred to scintillation vials, where they were inactivated with 10 ml of PCS scintillation solution 29 (Amersham Searie). The scintillation vials were transported to the University of British Columbia (U.B.C.) and counted on a Nuclear Chicago Isocap 300 scintillation counter operated in an external standard mode to correct for quenching. The raw count data was corrected to yield microbial uptake rates in ug of glucose or acetate/l/h. An example calculation for determining the uptake rates is presented in Appendix A. 4.2.3 HETEROTROPHIC UPTAKE KINETICS 4.2.3.1 Bark leachate inhibition experiments The inhibition of white spruce bark leachate to microbial activity was determined in a laboratory experiment with a bark concentration equivalent to 10 kg bark/3700 I water. After a contact period of 12 days, the bark leachate was filtered through CFC filters and then refiltered with 0.2 jum, cellulose nitrate membrane filters. The bacterial population used for the inhibition test consisted of 50 ml of settled primary sewage (ensured a population of bacteria) and 400 ml of pond water (dilution of sewage while maintaining an isotonic solution). The radiolabelled glucose (specific activity = 333 uCi/umole) was prepared to a final volume of 30 ml with sterile, distilled water. Table 4.2 lists the volumes of isotope, sample (sewage + pond water), formalin, sterile distilled water, and leachate added to each 25 ml reaction flask. There were 2 leachate concentrations used for the experiments ; 2 ml and 5 ml additions. 30 Table 4.2 Volumes of solutions adde microbial d to the bark leachate reaction inhibition studies. flasks in Solutions Volumes added (ml) to flasks Blank-C Control Blank-L Leachate 1. Sample 4.0 4.0 4.0 4.0 2. Formalin 0.2 0 0.2 0 3. Make-up water^ 5.0 5.0 0 (3.0)+ 0 (3.0) 4. Leachate 0 0 5.0 (2.0) 5.0 (2.0) 5. Isotope 1.0 1.0 1.0 1.0 (1) = sterile distilled water * = numbers in brackets represent volume differences for the 2 ml addition of bark leachate After adding the isotope, the incubation flasks were fitted with tight sealing rubber caps with small plastic cups containing folded GFC 2.4 cm glass fibre filters saturated with 0.1 ml of 1 M hyamine hydroxide to absorb 14CC>2. At the end of the 2 hour incubation period, the samples were killed with 0.2 ml 5 N F^SO^. The flasks were swirled and allowed to sit for 20 minutes to collect the ^^CO^ To determine particulate uptake, the water samples were filtered through 0.2 /im cellulose nitrate filters, the filters transferred to scintillation vials, and dissolved in 10 ml PCS scintillation solution. The GFC filters were removed from the plastic cups, placed in scintillation vials and 10 ml of PCS scintillation fluid added ; this provided information on the amount of CC>2 respired by the microorganisms. Samples were counted as outlined in Section 4.2.2. 31 4.2.3.2 Bacterial uptake studies with log leachates A comparison of bacterial dynamics involved in the utilization of organic solutes was done on samples from the lake, control, spruce (5 logs), and pine (5 logs) enclosures taken at the beginning of the experiment (day 0) and at day 10. The kinetic uptake procedure used was adopted from Hall ef al. (1972) and the organic solute used was 1 4C-acetate (specific activity = 57.6 uCi/umole). The isotope was diluted to 0.1333 uCi/ml with 0.2 um filtered lake water. Uptake was measured at 5 duplicate solute concentrations, 20, 40, 60, 80, and 100 ug/l of unlabelled solute. Table 4.3 lists the volumes of isotope, water sample, and unlabelled acetate added to the 25 ml reaction flasks. A blank for each solute concentration was fixed with 5 N H2SO4 prior to isotope addition. After adding the isotope, 0.1365 ug 1 4 C-Ac. /ml , the incubation flasks were prepared in the same method outline above in Section 4.2.3.1. The final solute concentration (labelled + unlabelled acetate) of the flasks was 30.6, 48.8, 67.0, 85.1, and 103.3 ug Ac./I. At the end of the 3 hour incubation period, the samples were killed with 0.2 ml 5 N H 2 S 0 4 , prepared and preserved as outlined in the previous section. 4.3 PHYTOPLANKTONIC ALGAL POPULATION 4.3.1 BIOMASS AND ENUMERATION The algal biomass indicator used was chlorophyll _a_ (chl _a). Chi _a_ was determined by filtering known volumes of water samples through 47 mm diameter GFC filters. Approximately 1 ml of MgCO.. (slurry) was added to the remaining few 32 Table 4.3 Volumes of solutions added to the log leachate reaction flasks in microbial activity studies. Solutions Volumes added to flasks (ml) 1. Sample 9.0 9.0 9.0 9.0 9.0 2. Unlabelled acetate 0.2 0.4 0.6 0.8 1.0 3. Make-up water 0.8 0.6 0.4 0.2 0 4. Isotope 1.0 1.0 1.0 1.0 1.0 ml of each filtrate. The filters (replicates) were then placed in petri dishes and frozen. Upon return to the Environmental Engineering Laboratory at U.B.C., the chl _a samples from the bark studies were extracted with 11 ml of 90% acetone in graduated centrifuge tubes over a 24 hour period in a dark, cold room (4 °C ; Strickland and Parsons, 1972). The samples (including replicates) were centrifuged (1500 rpm), the supernatant drawn off, the volume recorded, and the fluorescence measured on a Turner Designs Fluorometer. Equation 4.3 describes the chl _a_ standard curve ; Equation 4.4 was used for volume corrections : * = Fo (4.3) (0.968 - 0.00035Fo) 33 chl _a_ (ug/I) = $' x O.Q1(litres)+ (4.4) volume of water filtered where : Fo = fluorometer reading $ = uncorrected chl _a_ concentration = averaged uncorrected chl _a_ cone. * = volume of extraction liquid To ensure the complete extraction of chl _a_ from the samples, extraction solvents and technique were changed for the log study samples. The frozen filters were placed in graduated, screw top centrifuge tubes and 10 ml of 2:1 (volume : volume) chloroform : methanol extractant added (Wood, 1985). The extraction process was allowed to proceed for 5 hours in a dark, cold room (4 °C), the tubes were centrifuged (1500 rpm), the supernatant removed, volume recorded, and fluorescence promptly measured on the Turner Designs Fluorometer. A new standard curve was generated for the chloroform : methanol extraction solvent, and the final chl _a_ concentrations were determined from the values obtained from the curve and Equation 4.4. For phytoplanktonic algal identification and enumeration a 150 ml sample was collected from the lake and the enclosures, preserved with 1 ml of Lugol solution, and returned to U.B.C. A volume of the sample water was placed in a 25 ml settling chamber, covered with a microscope slide, and left undisturbed for 24 hours (Standard Methods, 1985). The preserved phytoplankton were enumerated with a Nikon Phase/Contrast Inverted Microscope. 34 4.3.2 PHYTOPLANKTONIC PRIMARY PRODUCTION Primary production was determind by the in situ uptake of radioactive 1 4 C-HCO- j" isotope (Strickland and Parsons, 1972). Preparation involved the dilution of 40 MCI" of 1 4 C - H C 0 3 " with filtered 0.2 /zm lake water to a volume of 22 ml. Four 300 ml BOD bottles were rinsed and filled with sample water. One BOD bottle was injected with 1 ml of concentrated formaldehyde (control) and a second was injected with 5 ml of DCMU, a photosynthetic inhibitor (Legendre et al. 1983). After a 5 minute period to kill the control and inactivate the DCMU sample, 1 ml of isotope solution (1 ml = 1.818 MCI) was injected into each bottle. Four bottles were suspended in each enclosure and the lake for a 6 hour incubation period. At the end of the incubation period, the BOD bottles were filtered (0.45 Mm membrane filters), transferred to scintillation vials, acidified to remove any unincorporated 1 4 C - H C 0 3 " , and then inactivated with 10 ml of PCS scintillation solution. The scintillation vials were counted by the same method outlined in Section 4.2.2. The data were corrected and the uptake rates calculated in rjg C/l/hr (Strickland and Parsons, 1972). 4.4 COMPARSION BETWEEN THE BARK AND LOG STUDIES Experiments with similar bark weights were used to quantitatively compare the bark and log study results. The bark for the 1984 study was dried in an oven at 110 °C for 24 hours to determine the dry weight of bark added to the enclosures (Table 6.1). For the 1985 study, the logs were peeled, the bark oven dried for 24 hours at 110 °C, and the results reported in Table 6.1. 35 4.5 STATISTICAL ANALYSIS The test results of the bark and log studies were statistically analyzed with U.B.C. ANOVAR, a statistics package providing analysis of variance and covariance adapted by M. Creig and D. Osterlin from B.Y.U. Documentation (1977). A two-way ANOVA (a = 0.05) was performed on the 1984 test results to determine the significance between the 20 kg bark treatment and control. The two-way ANOVA was used on the 1985 log study results to test significance between treatments (number of logs) and control, between species (pine and spruce), over time (25 days), and at different water temperatures (i.e. month to month variations). The statistical analysis for 1985 included the significance of the above listed interactions as well as relationships between any combination of the above, i.e. species x time x number of logs, species x time x month, etc. 5. RESULTS 5.1 ENCLOSURE EXPERIMENTS WITH BARK - 1984 The 20 kg bark experiment meet the criteria for statistical analysis, i.e. homogeneity of variance. However, the 5 kg, 1 kg, and 8 logs treatments violated Bartlett's homogeneity of variance assumption, therefore, statistical interpretation of the data was limited. For example, there was a significant decrease in dissolved oxygen in the 20 kg bark treatment (Figure 5.1). Although a similar decrease in oxygen in the 5 kg and 1 kg bark experiments could not be statistically validated, there was a trend similar to that observed in the heavy bark enclosure. Considering that the 20 kg bark enclosure results could be statistically tested and the difficultly in graphing all the bark treatments because of differences in (i) sampling intervals and experimental duration, and (ii) temporal changes, the results of the heavy bark treatment and its control were graphed, and the data from the remaining treatments were tabulated. 5.1.1 COMPARISON OF THE LAKE AND CONTROL DATA A statistical comparison (ANOVA a = 0.05) was made between the lake and control enclosures to investigate impoundment effects on water quality and biological measurements. There was no significant difference between control and lake for alkalinity, dissolved oxygen, lignins and tannins, primary production, and chlorophyll ja_ (Tables 5.1, 5.2, and 5.3). However, pH and heterotrophy values were statistically different between the control and the lake (Tables 5.1 and 5.3) ; this may be 36 37 attributed to the contained or static environment of the sealed enclosures. Since the changes in pH and heterotrophy were small, the control and lake values were considered similar. 5.1.2 WATER QUALITY 5.1.2.1 General water quality measurements (i) Dissolved oxygen (DO) Figure 5.1 demonstrates the severe and significant DO demand created by 20 kg of bark. The sharp decrease in DO concentration observed in the 20 kg bark enclosure was also observed in the lower bark treatments, however, the severity of the trend was proportional to the bark concentration. For example, after 5 days, the DO in the 20 kg bark treatment had dropped to less than 1 mg/l while in the 5 kg bark enclosure it took 14 days to reach a similar DO concentration (Table 5.1). For the 1 kg bark enclosure, the DO was still at 6 mg/l after 11-days (Table 5.1). The enclosure experiment which contained 8 logs showed an oxygen consumption approximately equal to that of the 5 kg enclosure (Table 5.1). (ii) pH and alkalinity « There was a steady and significant decrease in pH in the 20 kg bark enclosure, compared to the control, where the pH values dropped from 7.30 to 5.55 in 31 days (Figure 5.2). There was a pH decrease in the 5 kg, 1 kg, and 8 logs experiments, however, the changes were small (< 1 pH unit, see Table 5.1). Table 5.1 Changes in dissolved oxygen, pH, and a l k a l i n i t y over time in the 1984 bark enclosure studies done at Babine Lake. DO (mg/l) pH A l k a l i n i t y (mg/l) Treatment Days T C L T C L T C L 5 kg bark 0 10. .6 10. .4 10, .2 7.13 7 . 17 7 .02 38 .0 37 .5 38 .5 7 3. .3 9 .9 10. 1 6.55 7 .46 6 .91 35 .0 38 .0 37. .5 14 0. .4 9 .4 10. .9 6.76 7 .74 7 .61 36 .0 39 .5 38. .5 1 kg bark 0 9 . 4 9. . 8 9. 8 7 . 30 7 .01 7 . 13 37 . 5 38 .0 39. O 4 7. .9 8 .6 9. .0 6.75 7 . 13 7 .26 37 .0 39 .0 39. ,5 11 6. 0 8, .9 8. 6 6.93 7. .29 7, . 12 37 .5 38 . 5 39. ,5 8 logs 0 9. .4 9. .7 9. 5 7 . 10 7 .31 7 .00 37 .0 39 .0 39. .0 7 4 . 7 9 .8 10. 1 6.43 7 . 20 6 .96 36 .9 37 .5 38. .0 14 0. 8 9 .6 9. 2 6.26 7 . 29 7. . 14 35 .0 38 .0 37. .5 21 1 . 1 8 . 6 8 . 7 6 . 27 7 . 13 7 . 0 4 . 35 .O 38 .O 38. ,5 T C L = t r e a t m e n t = c o n t r o l e n c l o s u r e ( n o b a r k ) = 1 a k e 39 12 v I I 1 1 1 1 1 1 ' 0 5 10 15 2 0 2 5 3 0 3 5 TIME (days) Figure 5.1 Temporal changes in dissolved oxygen in the 20 kg bark enclosure at Babine Lake ; control = no bark. 8 Figure 5.2 Temporal changes in pH in the 20 kg bark enclosure experiment at Babine Lake, 1984 ; control = no bark. 40 — 60 10 -Control O O 2 0 k g B a r k I 0 5 10 15 2 0 2 5 3 0 3 5 TIME (days) Figure 5.3 Temporal changes in alkalinity in the 20 kg bark enclosure experiment at Babine Lake, 1984 ; control = no bark. ^ IOOOJ 2 o 0 5 10 15 2 0 2 5 3 0 3 5 TIME (days) Figure 5.4 Temporal changes in total organic carbon in the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. 41 In the 20 kg bark enclosure experiment, the alkalinity dropped from 44 mg CaCCy i to 20 mg CaCCyi by the 10th day of the experiment (Figure 5.3). However, the alkalinity gradually increased to 31 mg CaCOyi by the end of the experiment. There were no significant decreases in alkalinity values exhibited by the other treatment enclosures (Table 5.1). 5.1.2.2 Organic pollution indicators (i) Total organic and inorganic carbon (TOC and TIC) The TOC concentration of the 20 kg bark enclosure increased from 7.8 mg/l to 390 mg/l during the 31 day experimental period (Figure 5.4). The increase in organic enrichment with the addition of 20 kg bark, was exhibited in the other bark treatments in lesser degrees of severity (Table 5.2). The high levels of TOC indicated that a substantial amount of organic material was being leached from the bark. For the 1985 log study, the organic matter was characterized to determine the general chemical composition of the leachate. The increase in TIC concentration in the 20 kg bark enclosure was significant compared to the control enclosure (Figure 5.5). However, there was no significant change in inorganic carbon levels in the 5 kg, 1 kg, and 8 logs enclosure (Table 5.2). The increase in inorganic carbon in the 20 kg enclosure from 6.7 mg/l to 28.6 mg/l in 5 days should have been accompanied by an increase in alkalinity since the alkalinity in Babine Lake is mainly attributed to the bicarbonate and carbonate anions. However, the alkalinity decreased within the experimental time span. The discrepancy between inorganic carbon measurements is discussed in Section 6.1.2. Table 5.2 Temporal changes In the concentration of organic and Inorganic carbon compounds in the 1984 bark enclosure experiments at Babine Lake. TOC (mg/l) TIC (mg/1) L-T (mg/l) Treatment Days T C T C T C L 5 kg bark 0 9.0 7 8 2 . 0 14 114.0 1 kg bark 0 9.0 4 10.5 11 10.9 8 logs 0 8.0 7 16.0 14 21 8.1 5.3 6.7 11.5 5.5 4.7 8.5 6.1 6.0 7.7 4.2 6.5 8.5 4.0 6.5 8.8 6.5 6.0 6.1 4.0 9.2 9.8 4.2 4.2 0.4 0.4 0.5 22.5 0.6 0.4 3 5 . 0 0.5 0.5 0.5 0.4 0.5 1.3 0.5 0.4 11.2 0.5 0.4 0.4 0.5 0.4 2.9 0.5 0.4 3.8 0.4 0.4 4.8 0.4 0.9 T = t r e a t m e n t C = c o n t r o l e n c l o s u r e ( n o b a r k ) L = l a k e 43 E 4 0 3 0 -o CD CC < o y 2 0 z < o rr 5 « H < I-o / 0 20 kg Bark Cont ro l 10 15 2 0 2 5 3 0 3 5 TIME (days) Figure 5.5 Temporal changes in the total inorganic carbon levels in the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. CD E CO 1 0 0 0 -1 0 0 - 2 0 kg BarkT 10-i CO Cont ro l — O — -0.1-0 10 15 2 0 2 5 3 0 3 5 TIME (days) Figure 5.6 Temporal changes in lignins and tannins concentration for the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. 44 (ii) Lignins and tannins (L-T) L-T were leached rapidly from the 20 kg bark enclosure i.e. 338 mg/l in 10 days (Figure 5.6). This increase in the concentration of L-T was very significant (a = 0.05) compared to the control enclosure and paralleled the increase in the TOC levels. In the 5 kg bark enclosure, the L-T concentration after a two week leaching period was 10 times lower compared to the 20 kg experiment, while the 1 kg enclosure only contained 11.2 mg/l after 11 days (Table 5.2). The 8 log experiment had a L-T concentration of 4.8 mg/l (Table 5.2) after three weeks, significantly less than L-T levels in the 20 kg and 5 kg bark experiments. 5.1.3 BACTERIAL POPULATION 5.1.3.1 Heterotrophy The addition of 20 kg bark and 8 logs to the experimental enclosures caused an apparent enhancement of heterotrophy. There was a significant increase (a = 0.05) in glucose uptake rates in the 20 kg bark' study after materials had leached into the water for 7 days or longer (Figure 5.7) ; for example, the uptake of glucose increased from 0.1 jug Glu./l/h at the beginning of the experiment to 7.3 Mg Glu./l/h in 24 days. There was an increase in microbial activity in the 8 log enclosure over a two week period, i.e. 0.2 Mg Glu./l/h to 16.4 Mg Glu./l/h (Table 5.3). However, the uptake of glucose dropped to 1.0 Mg Glu./l/h during the third week. The observed trends in microbial activity are discussed in Section 6.1.3. Table 5.3 Temporal changes In heterotrophy and phytoplanktonic biomass and p r o d u c t i v i t y in the 1984 bark enclosure experiments at Babine Lake. Heterotrophy (,,g G l u . / 1 / h ) Primary production ( n g c/l/h) Chlorophyl l a (j>g/1) Treatment Days 5 kg bark 0 7 14 0.5 1 .5 1 .6 0.7 0.5 0.7 0.2 0.6 0.6 0.9 0.2 0.3 1 .2 1 .2 1 .4 0.3 0.7 1 . 1 0. 1 0 0 0.2 0.2 0.3 0. 1 0. 1 0. 1 1 kg bark 0 4 11 0.4 2.5 0.3 1 .0 0.2 0.7 0.8 0.6 0.9 0.2 0 0.9 0.2 0.4 0. 1 0.2 0 0. 1 0. 1 0. 1 0. 1 0. 1 0. 1 8 logs 0 7 14 21 0.2 8.5 16.4 1 .0 0.2 0.2 1 . 1 1.5 0. 1 0.3 1 . 1 1 .4 0.7 0.2 0. 1 0.3 0.7 0.2 0.4 0.4 0.6 0.6 1 .0 0.7 0. 1 0 0 0. 1 0. 1 O. 1 O. 1 0. 1 0. 1 T = t r e a t m e n t C = c o n t r o l e n c l o s u r e (no l o g s ) L = l a k e 46 ^ 10 UJ < z> LU V> O o —I o 8-O 0-0 20 kg Bark Control 10 15 2 0 2 5 TIME (days) 3 0 3 5 Figure 5.7 Temporal changes in heterotrophic uptake of labelled glucose in the 20 kg bark experiment at Babine Lake, 1984 ; control = no bark. ^ 0 . 2 0 -=3. CD > 0.15 0 . 1 0 -X o § 0 0 5 0 . 0 0 -\ \ I 20 kg Bark 0 5 10 15 2 0 2 5 3 0 3 5 TIME (days) Figure 5.8 Temporal changes in chlorophyll _a_ concentration in the 20 kg bark enclosure at Babine Lake, 1984 ; control = no bark. 47 5.1.3.2 Bark leachate inhibition experiments There was a significant decrease in the net uptake of labelled glucose in the white spruce bark inhibition experiments. The control (no leachate) had an average uptake rate of 14.1 Mg Glu./l/h (n = 3), while the uptake rates of both the 2 ml and 5 ml leachate additions had reduced the heterotrophic activity to zero. Thus, there was complete inhibition of natural sewage microbial populations after a 2 hour incubation period with bark leachates. 5.1.4 PHYTOPLANKTONIC ALGAL POPULATION 5.1.4.1 Phytoplanktonic biomass There was a rapid and significant decrease in chlorophyll _a_ in the 20 kg bark enclosure from 0.2 Mg/l to 0 in 10 days (Figure 5.8). This decreasing trend was also apparent in the 5 kg and 1 kg bark and 8 logs enclosures where chlorophyll _a_ was below detection level by the end of the experimental period (Table 5.3). 5.1.4.2 Phytoplanktonic primary production The changes in phytoplanktonic primary production in the 5 kg bark, 1 kg bark, and 8 logs enclosure experiments are presented in Table 5.3. In the 20 kg bark enclosure, an initial productivity rate of 0.8 Tjg C/l/h was reduced to zero by the fifth day of the experiment ; this decrease in productivity paralleled the drop in the phytoplanktonic biomass. The 8 log and 5 kg bark experiments also showed some inhibition of phytoplanktonic algal production with reductions of 50% - 70% 48 after 7 days. No changes in primary production were observed in the 1 kg treatment. 5.2 ENCLOSURE EXPERIMENTS WITH LOGS - 1985 5.2.1 COMPARISON OF THE LAKE AND CONTROL DATA A statistical comparison (ANOVA a — 0.05) was made between the control enclosures and Babine Lake for reasons outlined in Section 5.1.1. There was a significant increase in organic carbon and chemical oxygen demand in the lake during the first 5 days of the experiments with pine logs in June when compared to the control enclosure. These increases were possibly caused by the resuspension of organic material from the lake sediment, the result of a severe wind storm that occurred during the first two days of the experiment. Generally, there was a significant increase over the June and July 25 day test period in heterotrophy, bacterial numbers and biomass, and chl _a_ in the control enclosures (no logs) during studies with both tree species compared to the lake values (Tables 5.4 and 5.5) ; this was probably caused by the sealed (static) enclosure design. The bacterial biomass in the June control enclosures and lake fluctuated over the test interval, however, by the end of the experiments (both spruce and pine) biomass in the control was greater (3 to 4 times) than the lake values. Table 5.4 A comparison between the pine log control enclosure and Babine Lake of s i g n i f i c a n t changes In several water qua l i t y and bac te r ia l measurements. T1me (days) 0 5 10 15 20 25 Test Measurement C L C L C L C L C L C L i n t . June COD (mg/l) 18.15 15.72 19.00 36.00 19.OO 30.00 15.72 11.79 13.76 17.10 24.70 27.50 TOC (mg/l) 6.5 4.8 6.0 13.3 6.5 6.5 10.5 9.5 7.5 7.0 10.0 6.5 Bact . no. 4.1 3.7 5.7 4.4 - 3.9 7.2 4.3 10.3 4.0 18.2 9.5 (c ts /ml x 10 s) Bact . b i o . 22.2 10.1 - - - - - - - - 43.0 11.2 ( „ g C / l ) C h l . a ( „ g / l ) 1.4 1.2 1.7 1.1 1.4 1.4 1.8 1.4 1.3 1.1 1.4 1.5 duly Bact . no. 9.9 10.9 8.0 6.0 9.5 6.9 11.0 4.8 6.5 4.8 7.2 6.5 (c ts /ml x 10 5) Bact . b iO. 72.8 . 72.0 - - 105.0 32.0 - 24.2 18.3 ( „ g C / l ) Heter. 0.3 0.1 0.2 0 1.0 0.4 0.1 0.1 0.3 0.3 0.7 0.2 ( „ g A c . / l / h ) C h l . a ( v g / l ) 1.4 1.2 1.6 1.0 1.2 0.9 1.0 1.0 1.5 1.1 1.4 1.1 T e s t 1nt. = t e s t I n t e r v a l C = c o n t r o l e n c l o s u r e (no l o g s ) L = l a k e Bact. no. = b a c t e r i a l numbers Bact. b i o . = b a c t e r i a l biomass Heter. = h e t e r o t r o p h i c a c t i v i t y Chl a = c h l o r o p h y l l a Table 5.5 A comparison of s i g n i f i c a n t changes In heterotrophy and bac te r i a l numbers and biomass between the spruce log control enclosure and Babine Lake, 1985. Time (days) 0 5 10 15 20 25 Test Measurement C L C L C L C L C L C L in t . June Bact . no. 7.5 6.4 6.6 6.6 8.3 6.0 47.8 5.1 9.6 6.2 6.8 4.3 (c ts /ml x 10 5) Bact . b i o . 51.4 42.0 - - 121.4 89.6 - 88.7 27.8 ( „ g c / i ) Heter. 1.0 0.1 0.1 0.2 0.3 0 0.4 0 0.7 0.3 ( „ g A c . / l / h ) Ju ly Bact . no. 12.0 7.9 13.6 9.0 11.7 9.1 11.7 10.7 10.4 11.9 10.5 12.5 (c ts /ml x 10 5) Heter . 0.1 0 0.3 0.2 0.3 0.2 0.4 0.3 0.3 0.3 0.3 0.1 ( „ g A c . / l / h ) T e s t I n t . = t e s t I n t e r v a l C = c o n t r o l e n c l o s u r e (no l o g s ) L = l a k e B a c t . no. = b a c t e r i a l numbers B a c t . b i o . = b a c t e r i a l biomass H e t e r . = h e t e r o t r o p h i c a c t i v i t y 51 5.2.2 WATER QUALITY There was no significant change (a = 0.05) in any of the spruce or pine experimental enclosures in alkalinity, pH, total Kjeldhal nitrogen, total inorganic carbon, or phytoplanktonic algal numbers compared to the control enclosures. Total phosphorus, volatile fatty acids, and carbohydrate values were below the detection limits of the analytical methods. A summary of the means and standard deviations for dissolved oxygen, temperature, chemical oxygen demand, total organic carbon, lignins and tannins, and chlorophyll _a_ are listed in Table 5.6. 5.2.2.1 General water quality measurements (i) Dissolved oxygen (DO) There was a significant decrease in June DO concentration after 20 days for the 5 and 3 log treatments for both tree species (Table 5.7). From Table 5.6, the average June DO concentrations of spruce 5 and 3 log treatments were higher compared to pine (surface areas of the pine and spruce logs were the same, i.e. uniform length and diameter). For example, in the 5 log treatment, the average oxygen concentrations were 9.7 mg/l and 9.0 mg/l for the spruce and pine logs, respectively. Therefore, pine may exert a slightly higher oxygen demand than spruce. In July, a significant decrease in the DO levels was apparent after 5 days for both the 5 and 3 log experiments for both tree species (Figure 5.9). For example, the DO in pine 5 and 3 log treatments decreased from 10.3 to 2.5 mg'/l and 10.3 to 3.0 mg/l, respectively, over the 25 day experimental period. The oxygen demand decreased with a reduction in the number of logs. Although a similar DO deficit trend was exhibited by the spruce study in July, pine exerted a significantly greater oxygen demand at all 3 log loadings. The DO concentrations for Table 5.6 A summary of means and standard deviat ions of general water q u a l i t y measurements f o r the 1985 pine and spruce log study at Babine Lake. Parameter DO (mg/l) Temp. C O COD (mg/l) TOC (mg/l) L-T (mg/l) Chl a ( „ g / l ) Tree Test No. Av. S.D. Av. S.D. Av. S.D. Av. S.D. Av. S.D. Av. S.D. sp. Int. of logs Pine June C 10. .9 0. . 6 9. .9 1 .7 16 . 58 4 .85 7 . 9 1 . 8 0. 3 0. 1 1 . .5 0. 2 1 10. .5 1 . .0 9, ,9 1 .6 . 27. .07 7 .72 11 . 0 1 . ,9 0. ,4 0. 1 1 , 8 0. 1 3 9 . 5 0. .8 9. .5 1 .6 34. .54 1 1 .35 13. . 1 3. .4 0. ,9 0.4 1 , .5 0. 2 5 9. ,0 1 . . 1 9. .5 1 .5 40. ,63 17 . 18 16. . 1 5 . 7 1 , ,0 0.4 1 . .5 0. 3 Ju ly C 9. .4 0, ,4 14 , .9 1 .9 21 . 97 7 .00 10. . 1 2 . 6 0. ,4 0 1 . 4 0. .2 1 8 . 9 0. .9 14. 9 1 .9 27 , 60 10 .79 12. .2 4 . 7 0, ,6 0. 1 1 , .4 0. .3 3 6 , 7 2 .0 14. .9 1 .9 39. .30 25 .50 18 . 1 4 . 7 1 , ,0 0.4 2. . 3 1 . 0 5 6 , . 1 2. .3 14. .9 1 .9 41 . 17 19 .87 13 .8 5 .  1 0, .9 0.3 1 .8 0. .6 Spruce June C 1 1 . .0 0 .4 9 .0 2 .2 23. .32 2 . 28 10 .5 1 , .9 0, . 5 0. 1 1 .4 0, 2 1 10. .5 0. . 7 9. .0 2 .3 22 . 27 4 . 24 9 .4 0 .5 0. .6 0. 1 1 .4 0. .2 3 9 .9 0 .7 8 .6 2 .2 35 .86 12 .99 12 . 2 0 .6 0, .9 0.2 1 . 5 0. .2 5 9 .7 0. .9 8 .6 2 .2 35. .01 1 1 .76 12 .9 2 .8 1 , 2 0.5 1 .7 0. .4 Ju ly C 9 .4 0 .4 15 .7 1 . 1 19. .88 0 7. . 1 1 , .8 0, .4 0 0 .8 0. .2 1 9. .0 0. .6 15 .7 1 . 1 28 .86 8 .54 4 .0 1 , . 1 O, . 5 0 0 .9 0. 2 3 7 . 8 1 .3 15 .7 1 . 1 31 . 69 16 .01 7 .6 2, .8 0, .9 0.3 1 .0 0. 3 5 6 .8 2 .2 25 .7 1 . 1 33 . 33 18 .80 9 . 3 2, .7 1, .2 0.5 1 .0 0. .2 Tree sp. = t r e e s p e c i e s T e s t i n t . = t e s t i n t e r v a l C = c o n t r o l e n c l o s u r e (no l o g s ) Av. = average S.D. = s t a n d a r d d e v i a t i o n Table 5.7 E f f e c t s of pine and spruce logs on d isso lved oxygen in the 1985 June log enclosure experiments. Time (days) Tree sp. No. of 0 2 5 10 15 18 20 22 25 logs Dissolved Oxygen (mg/l) Pine C 11.6 11.5 11.1 11.4 10.7 10.9 10.4 10.8 9.8 1 12.4 11.4 10.8 10.2 10.1 10.0 9.5 9.8 9.6 3 11.1 10.0 10.0 9.3 8.8 8.7 8.6 9.2 9.5 5 11.5 9.7 9.7 8.4 8.2 8.1 8.0 8.6 9.1 Spruce C 10.8 11.3 11.4 11.3 11.4 10.4 11.4 11.0 10.6 1 10.6 11.6 11.2 10.8 11.2 10.0 10.5 10.0 9.6 3 11.1 10.9 10.7 9.9 10.1 9.6 9.8 9.6 9.2 5 11.2 10.8 10.6 9.4 9.8 9.2 9.6 9.4 9.0 Tree sp. = tree species C = control enclosure (no logs) 5 4 0 5 10 15 2 0 2 5 3 0 TIME (days) Figure 5.9 Effects of spruce and pine logs on dissolved oxygen in enclosures for the 1985 July log study period ; number of logs in treatment are given in parentheses. 55 the pine studies were consistently lower than the spruce values for the same time span and study period. 5.2.2.2 Organic pollution indicators (i) Total organic carbon (TOC) The general trend exhibited by pine and spruce was an increase in TOC values with an increase in the number of logs placed in the enclosures (Table 5.8). There was a significant increase in TOC concentrations leached from the 3 and 5 log enclosures for pine and spruce compared to the control enclosure. There was no difference between the pine June and July TOC values which indicated that water temperature did not influence the amount of organic matter leached from pine logs. However, there was a significant decrease in the amount of TOC leached from all spruce log experiments in July than June. For example, the mean TOC concentration for 5 spruce logs over the June 25 day experimental period was 12.9 mg/l compared to 9.3 mg/l for the same time span in July (Table 5.6). (ii) Chemical oxygen demand (COD) The general trends exhibited by both tree species was a significant increase in COD values, over the 25 day test interval, for all 3 log loadings compared to the control results (Figure 5.10). For example, the COD values for the June 5 pine logs experiment increased from 19.6 mg/l to 70.7 mg/l between Days 0 and 25. The COD concentrations for July displayed a similar trend. There was no significant difference in COD values between the months, therefore, water temperature did not influence the amount of oxidizable organic Table 5.8 Temporal changes 1n to ta l organic carbon in the pine and spruce log enclosure s tudies at Babine Lake, 1985. Time (days) Tree Study No. of 0 2 5 10 15 20 25 species per iod logs Total Organic Carbon (mg/l) Pine June C 1 3 5 6.5 8.0 7 . 3 6.0 8.5 9.5 10. 3 11.0 6.0 11.5 13.0 20.5 6.5 11.5 14.0 16.8 10.5 13.5 14.8 17.5 7 . 5 10.0 15.0 19.0 10.0 12.7 17.5 22.0 Ju ly C 1 3 5 10.0 11.5 11.2 6 . 1 14.5 19.0 14.5 9.0 12.5 17.0 12.8 11.0 13.3 20.0 12.3 7.7 15 .O 23 .8 13.5 11.5 12.5 16.8 16.8 6.8 11.5 24.5 21.5 Spruce June C 1 3 5 8.0 9.0 11.5 8.7 10.5 10.0 1 1.5 14.0 10.0 10.0 12.5 12.5 9.5 9. 5 12.0 11.0 11.5 9.3 13.0 15.8 13.5 8.8 12.8 15.5 Ju ly C 1 3 5 9.3 2.3 8.0 6.5 5.2 3.0 7.2 6.8 5.8 4.9 13.5 8.0 6.2 4 . 7 5.8 7.8 9.0 5.5 4.5 12.5 5 . 5 3.5 7 . 3 10.5 9.0 4 . 2 7 . 2 12.9 C = c o n t r o l e n c l o s u r e (no l o g s ) 8 0 0 8 0 0 PINE | JULY ~ i i i i i i 0 5 10 15 2 0 2 5 3 0 TIME (days) SPRUCE JUNE I . (5) : A . / / . (3) i i i i i lULY] (5) 0 5 10 15 2 0 2 5 3 0 TIME (days) Figure 5.10 Temporal changes in COD concentration for the pine and spruce log enclosure studies done at Babine Lake, 1985 ; number of logs in treatment are given in parentheses. on 58 matter leached from the spruce and pine logs. The DO data was plotted against the COD results using a computer program for the best fit linear regression to determine if a temporal relationship existed between DO and COD (Figures 5.11 and 5.12). The linear regressions were tested by covariance analysis to determine differences between the slopes of the lines and, if the slopes were the same, between levels. The June experiments for both species of trees (except for the spruce control enclosure) found no significant difference existed between the slopes of the control, and 3 and 5 log treatments ; the slope of the 1 log experiment was slightly higher compared to the other enclosures. Therefore, the number of logs in the enclosures did not significantly affect COD or DO compared to the control enclosure. There was no significant difference between levels for (i) the June 1 spruce log and control enclosures, and (ii) the June and July pine control and .1 log experiments. This indicated that the spruce and pine 1 log treatments of June and July had no significant affect on COD and DO compared with the control enclosures, i.e. the decrease in DO and the increase in COD in the 1 log experiments were the result of the static, non-flushing environment of the enclosures. In July, there were significant changes in the slopes of the spruce and pine 3 and 5 log enclosures compared to the June slopes, which indicated a temporal relationship between COD and DO measurements. (iii) Lignins and tannins (L-T) The L-T results for 1985 are shown in Figure 5.13. There was a significant increase in L-T concentration over the test duration in the 5 and 3 pine and 6S 09 62 spruce log enclosures, although there was no significant difference in the amounts of L-T leached between June and July for both tree species. 5.2.3 BACTERIAL POPULATION 5.2.3.1 Bacterial numbers, biomass, and heterotrophy The bacterial numbers and biomass for all the June spruce and pine log treatments increased significantly (a = 0.05) over the duration of the experiments. Bacterial numbers (Table 5.9) indicated a change in the physical size of a population (counts/ml), however, enumeration did not depict an accurate account of microbial growth, i.e. change in morphological forms from coccoids to filaments. Therefore, biomass measurements (Mg C/l) were made to best represent changes in the microbial communities of the enclosure experiments (Figures 5.14 and 5.15). Calculations for the conversion of bacterial numbers to biomass are in Appendix A. Because of the extensive amount of time required to perform biomass measurements, calculations were done for only three sampling intervals (0, 10, 25 days). The bacterial biomass for the June 5 pine and spruce log enclosures increased several orders of magnitude from day 0 to the end of the test period. For example, the initial microbial biomass concentration in the 5 pine and spruce log enclosures was 30.1 Mg C/l and 59.8 Mg CJ, respectively ; this increased to 1370 Mg C/I and 1890 Mg C/l (respectively) after 25 days (Figures 5.14 and 5.15). However, in July, a significantly smaller bacterial biomass had developed in the pine and spruce enclosures, compared to the June concentrations. In the 5 spruce logs enclosure, the June biomass increased from 59.8 Mg C/l to 1890 Mg C/l over the 25 day test period (Figure 5.15). This increase in biomass was four Table 5.9 Temporal changes in bac te r ia l numbers in the pine and spruce log enclosure s tudies of 1985, Babine Lake. Time (days) Tree Study No. of 0 2 5 10 15 20 25 spec i es per i od 1ogs Bacter ia l Numbers (counts/ml x 10 5) Pine dune C 4.1 7.1 5.7 - 7.2 10.3 18.2 1 4.2 8.7 7.4 8.6 13.2 11.8 13.9 3 2.0 - 9.3 21.0 18.8 14.9 22.4 5 3.8 - - 27.7 26.2 15.4 18.6 duly C 9.9 6.4 8.0 9.5 11.0 6.5 7.2 1 9.3 8.3 10.6 - 3.5 15.6 19.1 3 12.3 5.9 11.5 14.8 5.5 18.9 12.6 5 8.2 7.7 7.7 7.4 3.4 21.6 22.2 Spruce dune C 7.6 4.5 6.6 8.3 47.8 9.6 6.8 1 8.6 2.8 7.0 12.6 17.0 9.8 8.1 3 8.2 5.3 10.8 12.0 11.8 8.8 24.8 5 7.8 6.2 9.9 13.9 11.5 3.9 33.1 duly C 12.0 16.4 13.6 11.7 11.7 10.4 10.5 1 6.7 15.6 10.2 14.5 15.7 9.9 12.5 3 9.8 15.6 13.1 8.6 13.4 116.7 1.9 5 11.6 19.1 13.4 8.5 12.6 201.0 21.3 C = c o n t r o l e n c l o s u r e (no l o g s ) 64 — 3 EJ 1000 Yl 800 CO < o CD 6 0 0 -4 0 0 CC £ 2 0 0 -U < CO 0 L e g e n d • Control S 1 Log [Z2 3 Logs • 5 Logs / / mLii 0 10 25 TIME (days) 0 10 25 TIME (days) Figure 5.14 Changes in bacterial activity and biomass in the 1985 pine log enclosure experiments done at Babine Lake. 65 — 6 ™ 1600-1400-CO 1200-GO < 1000-i CJ < CD 200-JUNE JULY Legend I Control 1 Log YZ2 3 Logs | • 5 Logs ^7 0 10 25 TIME (days) 0 10 25 TIME (days) Figure 5.15 Changes in bacterial activity and biomass in the spruce log enclosure experiments of 1985 at Babine Lake. times larger than the July 5 spruce logs experiment where the initial biomass of 68.2 Mg C/l increased to 430 Mg C/l by the end of the experiment (Figure 5.15). The increase in bacterial biomass reflected a succession of bacterial species in the log enclosures, i.e. initially small cocci ( 0.5 Mm diameter) and rods ( 0.75 - 1 Mm length) to filamentous microorganisms ( 100 + Mm length) and larger cocci and rods by the conclusion of the June experiments (Figures 5.16(A) and 5.16(B). The predominant bacterial morphological forms in the July log experiments were coccoids of 0.5 - 0.75 Mm diameter and small rods of 1.0 - 1.5 Mm (length). It was assumed that the presence of the aforementioned bacterial forms were introduced into the enclosures from a resident population found on the logs. The rapid growth of the various microbial species was accompanied by an increase in microbial activity (biomass was able to assimilate the simple radiolabelled solute). The June heterotrophic uptake rates increased with time for all spruce and pine log treatments, however, acetate uptake values for the spruce enclosures were significantly higher compared to the enclosures containing pine logs. For example, the June acetate uptake rates by heterotrophic organisms in the 5 spruce logs enclosure increased from 0.1 Mg Ac./l/h to 3.2 Mg Ac./l/h over 25 days ; for the 5 pine logs experiment, microbial activity increased from 0.2 to 2.1 Mg Ac./l/h for the same study interval (Figures 5.14 and 5.15). Microbial activity for all spruce log treatments was similar between June and July, i.e. for the 5 spruce log study acetate uptake increased from 0.1 Mg Ac./l/h for both June and July to 3.2 and 5.2 Mg Ac./l/h after 25 days, respectively. The uptake for the pine logs, especially the 1 and 3 log enclosures, were significantly higher in July compared to the June values. ure 5.16(A) Bacterial b iomass on in i t iat ion of the 5 pine logs enclosure at Babine Lake, 1985 ; X1562. 5 um Figure 5.16(B) Bacterial b iomass on day 25 of the 5 p ine logs enc losure exper iment at Babine Lake, 1985 ; X1562. 5 um 68 5.2.3.2 Hetero t roph ic uptake kinetics Data for the kinetic uptake study done in July, was plotted using a modified Lineweaver-Burk plot of S/V vs S, where S is the solute concentration and V is r o ' o the solute uptake rate. A computer program plotted the best fit linear regression to the data (Figure 5.17) ; the kinetic values of maximum uptake velocity or V m a x (ug/i/h), turnover time or (h), and active transport constant or affinity constant K t (h) were obtained from the plot and are listed in Table 5.10. It was assumed that the microbial activity in the control enclosure was statistically similar to that in the spruce and pine enclosures at the beginning of the experiment, thus, initial day kinetics for spruce and pine enclosures is represented by the control data. A detailed discussion on assumptions of the kinetic study and definition of the kinetic values is presented in Appendix B. (i) Net uptake for log exper imental enc losures a. Cont ro l The increased V m a x arid decreased T^  values after 10 days in the control enclosure indicated that the heterotrophic population had increased in biomass (Table 5.10) This observation was supported by an increase in the biomass and 1 4 C uptake data measured over time at a single substrate concentration. The increased V m a x values from 6.8 to 10.9 ug/I/h in 10 days, indicated a larger biomass to be saturated. The T^  decreased from 3.8 hours to 3.1 hours, which implied that the species composition of the bacterial population present after 10 days was more capable of utilizing acetate as an organic substrate than the population present at the beginning of the experiment. This statement was 69 Table 5.10 Heterotrophic uptake kinetic values obtained on Days 0 and 10 from the 5 spruce and pine log enclosures. Enclosure Time (day) Kinetic Results max (Mg/l/h) t (h) K t (Mg/D V IK. max t (h) Control 0 Control 10 Spruce (5 logs) 10 Pine (5 logs) 10 6.8 10.9 142.9 142.9 3.8 3.1 1.5 0.8 25.9 33.6 219.7 109.7 0.3 0.3 0.6 1.3 supported by an increase in the ratio of V /K.. ' 1 ' max t It is important to realize that the changes in the kinetic values in the control after 10 days were small and not highly significant (a = 0.05). Therefore, changes in growth and organic solute uptake kinetics were probably the result of the static (i.e. no water-renewal) environment of the enclosure. b. Pine There was a significant difference in kinetic measurements after 10 days in the 5 pine logs enclosure. The increased V m a x values indicated an increase in the heterotrophic population and possible eutrophication (Table 5.10). The decrease in T^  from 3.8 h to 0.8 h and the increase in the affinity ratio V m a x/ K t, 0.3 h to 1.3 h (Table 5.10) implied that the water soluble extracts from the logs (i.e. low molecular weight organic acids) were rapidly utilized. m a x 240 220 200 180 160 140 120 100 80 60 40 20 0 20 40 60 80 100 120 S (jjg/l) Figure 5.17 Lineweaver-Burk plot of S/V vs S on Day 10 for the 5 log pine and spruce enclosures and the control. o o 71 c. Spruce The V m for the 5 spruce logs enclosure was the same as for the 5 pine max r ° 1 log enclosure (Table 5.10). However, this did not imply that the heterotrophic population and uptake kinetics were identical. The T^  and values for spruce were significantly different compared to both the pine and control data, i.e. the spruce values for K t and T^  were double the pine values of 109.7 ug/l and 0.8 h, respectively (Table 5.10). The affinity ratio of V m /Kj for spruce was 0.6 h, half the affinity value of pine, therefore suggesting a chemical difference between the two tree species. 5.2.4 PHYTOPLANKTONIC ALGAL POPULATION From the data in Table 5.11, it was apparent that neither the number of logs nor the tree species had any adverse effect (a = 0.05) on the phytoplanktonic biomass as measured by chlorophyll _a_ (chl _a). In the July pine log enclosures there may have been a stimulation of phytoplanktonic algal standing crop (biomass) because the chl _a_ concentrations increased compared to the control. There was an increase in chl _a_ values in both the 5 and 3 log treatments, i.e. the chl _a_ values for 3 pine logs, July experiment, increased from 1.4 ug/l to 4.0 Mg/l over the experimental period compared to fairly constant values (1.0 - 1.6 Mg/l) ' n the control enclosure over the same period. There was no significant increase with time in chl _a_ concentrations during June in any of the pine or spruce enclosures. However; the July chl _a_ spruce values were significantly lower than the June concentrations, i.e. for the enclosure that contained 5 spruce logs, the average June and July chl _a_ dropped from 1.7 to 1.0 Mg/l (Table 5.6). Table 5.11 Temporal changes In ch lorophyl l a concentrat ion In the pine and spruce log enclosure s tud ies done at Babine Lake, 1985. Time (days) Tree Study No. of 0 2 5 10 15 20 25 species Per iod logs Chlorophyl l a ^ t^^ /^ ) P i ne dune C 1.4 1.5 1.7 1.4 1.8 1.3 1.4 1 1.6 2.0 2.0 1.9 1.8 1.7 1.8 3 1.2 1.2 1.4 1.6 1.4 1.4 1.9 5 1.4 1.6 1.3 1.7 1.4 1.1 1.9 duly C 1.4 1.4 1.6 1.2 1.0 1.5 1.5 1 1.4 1.4 1.9 1.5 1.5 1.3 0.9 3 1.4 1.4 2.6 1.4 2.1 3.0 4.0 5 1.4 1.5 2.4 1.9 1.3 1.5 3.0 Spruce dune C 1.6 1.5 1-7 1.6 1.1 1.4 1.3 1 1.6 1.6 1.6 1.1 I.3 1.6 1.4 3 1.6 1.7 1.7 1.2 1.3 1.8 1.3 5 1.7 1.9 1.8 1.8 1.0 2.2 1.6 duly C 1.0 1.0 0.9 0.6 0.9 0.8 0.6 1 0.9 1.1 1.1 0.8 1.1 1.0 0.7 3 1.0 1.1 1.0 0.6 1 .4 0.8 0.7 5 1.0 1.0 1.1 0.7 1.3 0.8 0.9 C = c o n t r o l e n c l o s u r e (no l o g s ) 6. DISCUSSION Most research concerned with pollution from log dumping and storage operations is divided into two categories. Firstly, laboratory experiments where pollutants are leached from log sections, and secondly, organic extractives leached from bark in both laboratory and in situ studies. Schaumburg (1973) performed laboratory and field experiments to determine the water pollution potential associated with (i) water storage of logs and (ii) the loss of bark from logs during different phases of logging operations, specifically, the in situ distribution, extent, and rate of benthic deposits and decomposition. There was considerable difference between the results of the 1984 bark enclosure study and the 1985 log enclosure experiments. The qualitative and quantitative differences between the bark and log studies were : 1. chemical differences ; various materials leached from the bark (i.e. inner and outer bark) compared to the logs (i.e. cambium and heartwood exposed by freshly cut ends), and 2. physical differences ; surface area, quantity and size of the bark chips placed in the enclosure. The Babine Lake enclosure project attempted to make quantitative and qualitative comparisons between the bark and the log experiments to discern if either bark or logs were a major source of pollution to the aquatic environment. To compare the experimental results, it was necessary to consider experiments with similar bark weights. From Table 6.1, the 1 kg bark experiment had a bark dry weight of 0.9 kg, comparable to the enclosure which contained 1 log where 0.7 kg of bark (peeled from the log) was exposed to the water. 73 74 Table 6.1 A calculat ion of the dry weight of bark added to the 1984 bark and 1985 log enc losure studies for water quali ty compar isons. 1984 1985 Treatment Bark<1> Treatment Pine Spruce (Bark) (kg, dry wt) (logs) (kg, dry wt of bark)<2> 1 kg 0.9 1 0.7 0.7 5 kg 4.3 3 1.8 1.9 20 kg 17.0 5 2.6 2.4 (1) = contained a 60:40 mixture of pine and spruce bark (2) = average of June and July logs The quantity and quality of extractives leached from the bark and logs was a function of surface area and the chemical composition of the tree tissues. For example, the bark used in the 1984 experiments was peeled from fresh cut pine and spruce logs and placed in the enclosures where both the outer bark and a portion of the inner bark were in contact with the water. The inner bark (phloem) contains more extractives and polysacchrides than the outer bark (Section 2.3). Therefore, the contact water leached from a large surface area that included two anatomically and chemically different structures. The log study of 1985 involved the addition of logs to the enclosures, thus, the enclosure water contacted and leached extractives from the outer bark and the exposed ends of the logs. A comparison of water quality data from the 1 kg bark and 1 log studies (Table 6.2) over an 11 day period illustrated that on a comparable dry weight basis, the water quality conditions in the 1 kg bark enclosure degraded at a faster rate compared to the 1 log studies. Therefore, in active log dumping and storage areas, 75 Table 6.2 A compar ison of water qual i ty condi t ions in enclosures wi th 1 kg bark (July, 1984) and 1 log (July, 1985) over an 11 day per iod . Parameter 1 kg ba rk* 1) P ine* 2 ) S p r u c e ^ ' D O (mg/l) T O C (mg/I) L-T (mg/l) Ch l _a_ (Mg/l) 9.4 - 6.0 9.0 - 19.3 0.5 - 11.2 0.1 - 0.3 10.4 - 9.0 11.5 - 13.6 0.4 - 0.7 1.4 - 1.5 9.6 - 9.1 2.3 - 4.9 0.6 - 0.5 0.9 - 0.8 (1) = equivalent to 0.9 kg dry wt. (60 : 40 pine and spruce bark mixture) (2) = 1 log enclosure experiment in July, equivalent to 0.7 kg of dry wt. bark loose floating and sinking bark would have a greater negative effect on water column and benthic ecosystems, compared to floating logs. 6.1 ENCLOSURE EXPERIMENTS WITH BARK - 1984 6.1.1 COMPARISON OF CONTROL AND LAKE RESULTS FOR BARK AND LOG EXPERIMENTS The statistical (a = 0.05) increase in heterotrophy, bacterial biomass, phytoplanktonic algal biomass, and organic enrichment in the control enclosures compared to the lake values were probably the result of the contained (sealed) environment of the enclosure (no exchange of water with the lake) compared to the dynamic, wind mixed lake (i.e. resuspension of nutrients from the sediments). These results are consistent with Parson's et al. (1977) nutrient enrichment study in 76 controlled experimental ecosystems. In the control enclosure (seawater, no nutrients) phytoplanktonic algal biomass, measured by chlorophyll _a, increased slightly by the end of the 30 day experiment. There was no significant change in heterotrophic activity (determined by V ) in the control enclosure with time. Therefore, it was concluded that the data collected from the lake was not statistically different compared to the data generated by the control enclosure and provided a basis for comparisons between a perturbed system (bark treatments), i.e. Morrison Arm logging operation, and an unperturbed environment (control), i.e. Babine Lake. 6.1.2 WATER QUALITY The 20 kg bark experiment resulted in high concentrations of lignins and tannins (L-T), colour, and total organic carbon (TOC) and rapid decreases in dissolved oxygen (DO), pH, and alkalinity. Similar trends of increased organic pollution, determined by chemical oxygen demand (COD) and biochemical oxygen demand (BOD) measurements, resulted from bark leaching experiments by Sproul and Sharpe (1970), Benedict et al. (1974a), and Gellman (1971). However, there are significant differences in experimental design, bark concentrations, and analyses performed between the Babine Lake study and previous research. The 20 kg bark experiment was the highest density (volume of bark : volume of water) performed during the Babine Lake experiments. The bark was a 60 : 40 mixture of fresh (recently peeled) pine and spruce in 3700 litres of water or 5.4 g of bark/I. This bark loading density was much lighter (200 times) compared to experiments performed by Sproul and Sharpe (1970), Asano ef al. (1974), and Gellman (1971). Table 6.3 compares relative bark concentrations involved in the various leaching studies and several water quality measurements. Table 6.3 A comparison of water quality measurements between several researchers bark leaching experiments. Parameters Sproul (1970) Gellman (1971) Asano (1974) Benedict (1974a) Babine (1984) Bark studied Exp. desig nO) Extraction water Duration of study Temperature Bark density*6) - softwood, hardwood - leaching of barkpile hardwood and softwood - dist. water(4) - 0 - 6 9 days - 2 0 * C - 2 0 0 g bark/I softwood, hardwood leaching of fresh and aged softwood and hardwood mix water(5) 0 - 8 5 days room temp. 1 6 0 g fresh bark mix/I lodgepole pine fresh bark distilled water 1 0 - 4 0 hours 2 5 " C 2 5 % , 18%, 1 0 % wood chip;water volume - softwood, hardwood -- leaching of fresh and aged softwood and hardwood mix - distilled water - 0 - 8 5 days - room temp. - N/A lodgepole pine, white spruce static enclosure leaching of 6 0 : 4 0 pine and spruce lake water (4) 0 - 3 1 days 4 * - 12 * C 5.4 g fresh mixed bark /I 1. COD (mg/l)(2) Sample day 0 1 10 20 30 2. BOD (mg/l) Sample day 0 10 20 30 2 5 1 0 2 5 2 0 3 1 9 0 1 3 2 0 7 8 0 9 9 0 1 3 5 0 1 3 2 0 0 1 0 0 0 2 2 0 0 2 9 0 0 3 5 0 0 0 6 0 0 1 2 5 0 1 5 0 0 2 1 0 0 3 0 0 0 3 5 0 0 0 6 0 0 1 1 0 0 1 5 0 0 (3) 8 .7 1 3 8 . 7 1 2 1 . 6 1 16 .8 (1) (2) (3) zz brief description of study involved in static laboratory leaching of bark = data acquired via extrapolation of reseachers' studies = conversion of L - T carbon content of the T O C to equivalent C O D (Appendix A) (4) (5) (6) = no added poison (dist.=distilled) = no description of water = wet weight of bark 78 From Table 6.3, there are numerous differences between the Babine Lake bark study and the data collected by the other researchers. These differences include the following : 1. duration of the study, i.e. 40 hours to 85 days, 2. types and species of bark used, i.e. softwood and hardwood mixtures, and 3. concentration of bark leached and the leaching water, i.e. the bark concentrations of other researchers was 200 times greater than the bark concentration of the Babine Lake study. Although the 1984 experiment did not measure COD or BOD, the TOC measurements were converted into equivalent COD values to allow comparisons between my results and previous research (Table 6.4 ; Appendix A). The trends of increased COD and BOD with high concentrations of bark observed by Sproul and Sharpe (1970), Gellman (1971), Benedict et al. (1974a), and the Babine 20 kg bark experiment, indicated that bark in an aquatic environment can be a serious point source of organic pollution. The 1 kg bark experiment or 0.3 g of bark/I, was the lightest concentration of bark used in the Babine Lake leaching studies, and, even at this relatively low level of bark, water quality measurements of TOC and L-T increased and the DO decreased (Table 6.2). The L-T leached from the Babine Lake 20 kg bark experiment contributed to the red-clay colour of the water, however, because these complex compounds are relatively resistant to rapid microbial degradation, other extractives were probably responsible for the rapid depletion of oxygen. Duval (1980) describes a biphasic BOD where an increase in BOD in the first few days rapidly decreased, replaced by a long term or ultimate BOD. The initial high BOD is mostly exerted by sugars and other soluble, utilizable organics leached from the bark. The benthal bark decomposition experiment by Sproul and Sharpe (1970) gives a twofold explanation Table 6.4 Calculations to determine the % of total organic carbon (TOC) comprised of lignins and tannins (L-T) and resultant chemical oxygen demand (COD). 20 kg Treatment L-T L-T as carbonO) TOC % TOC as L-T COD ( 3) carbon (^ ) (day) (mg/i) (mg/l) (mg/l) % (mg/l) 0 0.6 0.3 7.8 3.8 0.7 5 155.0 83.1 148.5 55.8 190.9 10 330.0 176.9 293.5 60.3 406.9 17 327.5 175.5 - - -24 345.0 184.9 360.0 51.4 425.3 31 370.0 198.3 390.0 50.8 456.1 (1) = 53.6% of tannic acid standard is carbon (2) = portion of L-T that is carbon -r TOC (3) = calculated equivalent COD attributed to L-T (Appendix A) 80 to the rapid rise and decline in the initial B O D . Firstly, the initial BOD is exerted by sugars, rapidly utilized so that after the first 4 days of the experiment there is very little measurable sugar. The second explanation involves a component of the leaching mechanism, whereby, after surface organics have gone into solution, the leaching rate (a function of changes in the concentration gradient with time) would have decreased and this "...slower rate of leaching would then show up as lower BOD values." (Sproul and Sharpe, 1970). The long term BOD is exerted by the gradual decomposition of lignins and other chemically complex structures by lignoceliulolytic bacteria and fungi (Raabe, 1968 ; Benner ef al. 1986). Although, the exact composition of the TOC was not known in the Babine Lake bark studies, calculations determined that approximately 54% of the TOC was contributed by L-T organic carbon (Table 6.4). However, it was assumed that extractives leached from the inner bark (mono- and polysacchrides) contributed to the oxygen deficit created by the heterotrophic population (Section 6.1.3). The acidic nature of these L-T materials was most likely responsible for the initial lowering of the pH. However, after 10 days a slight increase in pH was discernible in all bark treatments. It was possible that acidic groups comprising a portion of the chemical composition of lignin and tannin molecules, effectively titrated the alkalinity as they were leached from the bark. Also, microbial modification of some of the macromolecules would slowly generate some CO2 which would come to equilibrium as H C O y (pH of 6.8 to 7.3) and help to generate some alkalinity. Sproul and Sharpe (1970) found during laboratory simulated benthic deposits (aerated columns) that the pH increased due to the production of CO^= and H C O y by benthic microorganisms. The initial decrease in alkalinity, observed in the 20 kg bark treatment and subsequent increase with time, contradicted the inorganic carbon (TIC) data which 81 exhibited a rapid increase from initial sampling to Day 10, followed by a gradual decline to the end of the experiment. To explain the discrepancy between the two methods of measuring inorganic carbon, alkalinity and TIC were compared on an absolute carbon basis (Table 6.5). It was possible that some of the TIC was locked up in cellular material not readily titrated by the alkalinity determination. From Table 6.5, the TIC measurement was greater than the alkalinity concentration ; it was possible that the TIC determination was removing labile carboxy groups from the L-T compounds that would not be measured by the alkalinity titration. A more probable explanation for the disparity between alkalinity and TIC values may be found in the definition of alkalinity. The alkalinity measurement is the sum of the total titratable bases in a water sample ; this includes the carbonate ( C 0 3 = ), bicarbonate (HCO^"), and hydroxide (OH") molecules. It does not include the dissolved carbon dioxide (C0 2 ) or carbonic acid ( H 2 C 0 3 ) components of the carbonic acid system. The TIC measurement includes dissolved C 0 2 plus the alkalinity values, therefore, the TIC minus that carbon as alkalinity equals the carbon present in the water as C 0 2 -At the beginning of the 20 kg bark experiment, the pH was 7.30 and the alkalinity was 43 mg/l as CaCO^. After 10 days the pH dropped to 5.55 and the alkalinity carbon effectively converted to TIC. The Henderson-Hasalbach equation (Equator. 6.1) can calculate the relative proportion of alkalinity to C 0 2 as the pH dropped 7.30 to 5.55. pH = pK n + log H C 0 3 " (6.1) H 2 C O s where: prC = 6.35 82 Table 6.5 A comparison of absolute inorganic carbon concentrations between alkalinity and TIC for the 20 kg bark enclosure of 1984. Days Alkalinity Total Inorganic Carbon (mg Inorg. C/l) (mg Inorg. C/l) 0 5.3 6.7 5 4.0 28.5 10 2.4 23.5 17 3.5 24 4.1 12.5 31 3.7 16.0 therefore, 7.30 = 6.35 + log H C C y H 2 C 0 3 8.91 = H C 0 3 " H 2 C 0 3 There are 9 molecules of H C O y for each molecule of H 2CC> 3 at pH 7.3, however, by day 10 of the experiment, the decreased pH resulted in a subsequent drop in the carbon ratio.' Therefore, 5.55 = 6.35 + log H C O y H 2 C 0 3 0.15 = H C C y H 2 C 0 3 83 At the lower pH of 5.55, there were 6.6 molecules of r ^ C O ^ for every molecule of HCOj", therefore, the measurable alkalinity decreased. The difference between TIC and alkalinity was possibly a function of the definition of alkalinity and not a discrepancy between either measurement. Therefore, the subtracted difference between TIC and the alkalinity data in Table 6.5 is an indicaton of the amount of dissolved C 0 2 in the water sample (heterotrophy will generate C 0 2 as assimilated organics are respired). 6.1.3 HETEROTROPHY The microbial activity for the 20 kg bark enclosure had peaked by 3 weeks and then started to decline. This increase in heterotrophy, especially when compared to the normal growth rates of bacteria, was relatively slow. The slow uptake rate of radiolabelied glucose may have been attributed to the inhibitory nature of the tannins, dilution of the radioisotope or both. Microbial activity was measured by the uptake rate of radioactive glucose ; if there was unlabelled glucose leached from the bark (hydrolyzed from carbohydrates) it could effectively dilute the labelled glucose and give a lower uptake rate. Once the natural unlabelled glucose was metabolized the uptake rate of the added labelled glucose would increase. This problem could only be resolved by monitoring actual glucose or sugar concentrations over the experimental period and determining an actual turnover time. The dilution of labelled glucose by unlabelled glucose leached from the bark would explain the rapid rate of oxygen depletion compared to the relatively slow uptake rates of the bacteria during the first 2 weeks of the experiment. Also, the rapid decrease in phytoplankton biomass, dieoff, could also have stimulated microbial activity with unlabelled nutrients. 84 The decrease in heterotrophy by the end of the 20 kg bark and 8 logs experiments may have been a result of nutrient limitation. By the end of the experiment, readily available glucose and other carbohydrates, would be sufficiently depleted to cause potential nutrient limitation resulting in a decrease in uptake rates. The initial increase in heterotrophy and subsquent decline was similar to the initial segment of the biphasic BOD described by Sproul and Sharpe (1970). The second stage BOD would be accomplished by microorganisms capable of utilizing the chemically complex structural components of bark. The degradation of lignified plant materials into digestible by-products is performed by a limited population of bacteria (procaryotes) and fungi (eucaryotes). Benner ef al. (1986) states that bacteria are the predominant degraders of the lignin and polysaccharide components of lignocellulosic detritus in aquatic environments and fungi are apparently the major decomposers of terrestrial lignocellulosic materials (Kirk ef al. 1977). Previous research on microbial degradation of plant matter in aquatic environments by Federle and Vestal (1982) and Lee ef al. (1980) indicate a biphasic utilization of plant detritus ; an initial high fungal biomass with an increasing bacterial biomass over time. The colonization and degradation of aquatic plant material' predominantly by either bacteria or fungi is apparently a function of the physical (i.e. temperate or tropical) and chemical (i.e. acidic or alkaline) environment. The main conclusion is that bacteria and fungi are capable of degrading lignocellulosic detritus with some of the degradative end products (lignin and polysaccharides) incorporated into microbial biomass (Benner ef al. 1986). Therefore, it was possible that a portion of the lignins leached from the bark experiments may have been assimilated into microbial biomass provided enough time had elasped for the establishment of a population of lignocellulolytic microorganisms. There was a higher glucose uptake rate in the 5 kg bark and 8 log experiments when compared to the 20 kg bark experiment. This suggested either inhibitory or dilution effects by the higher bark loading on microbial activity. However, the 1 kg bark treatment showed lower microbial activity which could have meant that lower concentrations of utilizable organic solutes from the bark were limiting the growth of microorganisms and their ability to take up glucose. Thus, there appeared to be a balance between solutes necessary to stimulate microbial activity and leachates which cause some inhibition at higher concentrations. The inhibition experiments conducted with laboratory leachate from 2.7 g white spruce bark/I (equivalent to 10 kg of bark/3700 I water) resulted in a very significant (a = 0.05) decrease in the microbial uptake of radiolabelled glucose. This indicated that bark leachates from white spruce where inhibitory to the metabolism of the sewage and pond water microorganisms. In the enclosures, this inhibition may not be as pronounced because the microbes present will have had time to adapt to the leachate properties. The assimilation process would result in a shift in the species composition of the microbial community. Such a change in bacterial composition was apparent in the log study (Section 6.2.2.1). 6.1.4 PHYTOPLANKTONIC ALGAL POPULATION There were several problems encountered while conducting the phytoplanktonic primary productivity tests. The major concern was the high radioactive counts in the formaldehyde bottle (blank) compared to the live samples. The high counts for the blanks may have been the result of improperly washed filters. The high blank counts resulted in low productivity found in the lake (average = 0.7 rjg C/l/h over 21 days for the log. raft study) compared to values obtained by other researchers, i.e. = 2.3 tig C/l/h (Stockner and Shortreed, 1975) at a 86 station north of Topley Landing (Figure 2.1). Therefore, the phytoplanktonic primary production measurement was not repeated for the 1985 log study due to inherent difficulties associated with the technique. The observed reduction in chlorophyll _a_ and phytoplanktonic primary productivity in the 20 kg bark enclosure was similar to results obtained by Howard et al. (1979). Howard ef al. (1979) did experiments to determine the effect of kraft softwood pulp mill effluent colour on phytoplanktonic primary production. Although the effluent colour concentration (pH maintained > 9) studied by Howard ef al. (1979) was not as high compared to the Babine Lake 20 kg bark enclosure experiment, there was a decrease in phytoplanktonic primary production, attributed to shading effects on the effluent colour. The decrease in phytoplanktonic biomass and productivity in the 20 kg bark enclosure may be the result of the inhibitory and possible toxic nature of the resin acids (pH = 5.55) and a decrease in light transmission, a result of the highly coloured waters (tannins). However, I was unable to partition the results to determine the major contributing factor in the annihilation of the phytoplanktonic primary producers. 6.2 ENCLOSURE EXPERIMENTS WITH LOGS - 1985 Discussion of the log enclosure study data was limited by the number of analysis that produced measurable data. For example, as stated in Section 5.2.2. there was no significant change in any of the enclosures in' alkalinity, pH, total Kjeldhal nitrogen, total inorganic carbon, or phytoplanktonic algal numbers compared to the control enclosures. Also, total phosphorus, volatile fatty acids, and carbohydrates were below the detection limits of the analytical techniques. However, the low concentrations of VFA and carbohydrates were the result of their rapid 87 utilization since the literature indicates these materials are present in wood extractions. Therefore, those data which were detected, measured, and deemed significantly different compared to any combination of interactions (Section 5.2) are discussed in the following sections. 6.2.1 WATER QUALITY 6.2.1.1 Dissolved oxygen (DO) There was a greater decrease in DO levels for the 3 and 5 pine and spruce log treatments in July compared to the 3 and 5 log enclosure experiments of June. The COD concentrations increased with time and number of logs in both months. However, there was very little difference in the leachate concentration between months (COD measurement) ; this indicated that the warmer water temperature of July did not influence the rate of extractive leaching. Therefore, the low DO levels in the July enclosures were probably the result of a combination of factors ; a higher biochemical oxygen demand (BOD) and a decrease in the oxygen solubility with an increase in water temperature. Firstly, a higher BOD would be caused by an increase in utilizable organic substrate or bacterial biomass/activity or both. Although the concentration of oxidizable materials was similar (no significant difference) in June and July, the concentration of TOC was significantly lower in all the July spruce log experiments compared to June ; this suggested that a larger fraction of the July TOC was oxidizable, indicating more organic substrate available for the heterotrophs. An increase in COD or TOC does not guarantee an increase in BOD because the organic matter being oxidized may have a complex chemical structure and, therefore, biologically difficult to degrade. 88 Unfortunately, the concentration of VFA's and carbohydrates present in the enclosures were below the detection limits, thus, we were unable to compare either the organic makeup of the June and July leachates or determine if the oxygen depletion was a direct function of an increased BOD. If BOD data were available, then the ratio of BOD : COD and the BOD k-rate would provide a generalization of the biodegradability of the leachate. Although the bacterial biomass at all log concentrations for both the spruce and pine enclosures had significantly decreased in July from the June concentrations, the heterotrophic uptake rates (activity) for acetate were similar for both tree species between June and July. Rapid turnover of the bacterial population would have caused an increase in oxygen consumption, while maintaining a relatively constant biomass. Bacterial activity is discussed in detail in Section 6.2.2. Secondly, by calculating the oxygen saturation percentage (DO values divided by the oxygen saturation concentration for that particular water temperture, Table 6.6), it was possible to determine that the sharp decrease in July pine and spruce DO levels could be attributed to a combination of lower oxygen solubility, an available organic carbon source, and bacterial activity. 6.2.1.2 Total organic carbon (TOC) and chemical oxygen demand (COD) There was a significant temporal decrease in the amount of TOC leached from all the spruce log enclosures in July compared to the June values. The reason for this decrease in spruce TOC concentrations in July may be a function of when the logs were cut (1 week prior to the commencement of the June and July experiments). Different harvest times may result in a difference in the availability of water soluble organic matter and the microorganisms ability to utilize various components of the TOC. Also, I was measuring a steady state concentration of Table 6.6 Dissolved oxygen saturation for the 1985 log enclosure studies at Babine Lake. Time (days) Tree Study No. of 0 2 5 10 15 20 25 species period logs Oxygen Saturation (percent) Pine June C 116.7 110.5 106.6 111.6 98.9 92.7 96.0 1 124.7 109.5 103.7 109.7 93.7 84.7 99.4 3 110.2 96.1 93.9 91.1 80.4 76.6 91.3 5 114.1 93.2 92.4 86.9 74.9 71.3 87.4 July C 105.6 107.6 96.4 95.4 104.3 102.3 95.9 1 106.7 110.9 94.3 87.2 96.7 88.6 85.3 3 105.6 102.0 84.1 69.7 63.4 57.9 32.0 5 105.6 100.9 80.0 63.6 59.8 47.7 26.6 Spruce June C 111.8 117.7 109.5 95.9 109.5 105.4 94.5 1 103.8 113.6 107.6 91.7 107.6 97.0 85.6 3 106.6 104.6 102.8 83.8 94.6 89.5 82.0 5 107.6 105.6 101.8 78.9 91.9 87.7 80.2 July C 104.3 101.5 104.5 109.9 100.0 103.3 98.9 1 104.3 101.3 103.4 105.4 94.6 92.4 89.1 3 105.4 98.5 97.0 89.7 72.8 79.3 65.2 5 105.4 99.7 93.8 85.2 60.9 57.6 36.4 C = control enclosure (no logs) 90 TOC and, therefore, not accounting for rapid removal of utilizable organics due to higher temperatures and microbial activity, thus, the leaching rate may not have changed. The lignins-tannins (L-T) composition of the TOC was different between the bark and log experiments. In the 20 kg bark experiment, S 50% of the TOC of the was attributed to L-T carbon (Table 6.4). However, by the end of the July 5 pine and spruce log experiments, approximately 3.2% and 8.3% of the TOC measured was L-T carbon (see Table 6.4 for calculations). This indicated a difference in the chemical composition of the TOC between the bark and log leachates. The difference in leachate composition would influence the microbiological dynamics of the enclosure ecosystem. Both Schaumburg (1973) and Pease (1974) had similar increases in COD concentrations with high log densities : water ratios compared to the Babine Lake study. Schaumburg (1973) performed 7 day static log leaching experiments with 2 log sections of Douglas fir, ponderosa pine, and hemlock measuring BOD, BOD k-rate, COD , BOD : COD ratio, and reducing sugars. The log sections were approximately 15.5 inch (in.) diameter x 20 in. length and were submerged in tanks containing approximately 100 I poisoned water. After all logs had their exposed ends sealed, the logs were then treated, i.e. for Douglas fir, 1 log with bark was submerged in one tank and the other fir log had its bark removed before being submerged in another tank. Schaumburg (1973) concluded the following from the softwood log experiments after removal of the poison : 1. an increase in BOD, BOD k-rate, COD, BOD : COD, and reducing sugars, with time, for all 3 species, and 2. that most logs without bark had higher concentrations of the above measurements, indicating that the exposed cambium layer leached more 91 organics and nutrients than the unaltered logs. The three significant differences between the Babine Lake experiments and Schaumburg's (1973) was (i) the volume of log : volume of water, i.e. 5 pine logs for Babine had a combined volume of 4.5 ft^/3700 litres or 0.035 m^/m^ water, much smaller than 0.6 m^/m^ (Schaumburg, 1973), (ii) the physical state of the log, i.e. peeled, and (iii) poisoned water which allowed the accumulation of readily utilizable organics, i.e. sugars. Although the Babine Lake data showed an increase in COD with time, the lack of sugar and BOD data limit the scope and accuracy of applying the findings to in situ situations. 6.2.2 BACTERIAL POPULATION 6.2.2.1 Numbers, biomass, and hetertrophy The uptake rate of 1 4 C acetate in the June experiments was greater in the spruce enclosures compared to pine enclosures, however, pine logs had a higher dissolved oxygen demand than spruce. It was possible that the bacterial population in the pine enclosures was capable of utilizing a wider and more available range of unlabelled organic matter for metabolism and growth than the spruce microbial population. Also, the spruce heterotrophs may have been more adept in taking up simple organic molecules, i.e. acetate, than the pine microorganisms. Therefore, discrepancies in the uptake rates between the tree species were possibly a function of the chemical composition of the extractives and bacterial species composition. The uptake of acetate in the 1 and 3 pine log experiments in July was (i) greater than occurred in June, and (ii) higher compared to the 5 pine logs 92 enclosure in July. However, the dissolved oxygen concentration in the 5 pine logs enclosure was 2.5 mg/l by day 25, thus, it would appear that the microbial population was creating a dissolved oxygen demand. The lower rate of acetate uptake in the 5 pine logs experiment was probably a function of isotope dilution due to possible leaching of higher levels of acetate from the logs. It was difficult to rationalize how the lower bacterial biomass of July, for all log concentrations of pine and spruce, was able to maintain uptake rates similar to or greater than the June heterotrophic values. There are several possible explanations for the observed discrepancies. The smaller bacterial biomass that developed in July had a better ability to assimilate the radioisotope demonstrated by their higher uptake ratios of Mg of carbon uptake : Mg of carbon biomass (Table 6.7). The assimilation ability of the smaller morphological forms can be attributed to a number of factors. Firstly, the smaller bacteria have a higher surface area : cell volume ratio compared to the filamentous forms, and secondly, higher water temperature. Assuming that the bacterial uptake of acetate follows Michaelis-Menten enzyme kinetics, the aforementioned anomaly may be caused by the effects of temperature on enzymatic reactions. The rate of enzyme-catalyzed reactions generally increases with temperature, within the temperature range in which the enzyme is stable and retains full activity. The rate of most enzymatic reactions approximately doubles for each 10 °C rise in temperature (Q-JQ — 2.0 ; Lehninger, 1982). The average water temperture for the June experiments was 9 °C. The temperature increased to 15.6 °C for the July studies and, although, the bacterial biomass decreased in July (compared to June), the higher water temperature probably increased the rate of enzyme reactions and acetate uptake. Thirdly, if a greater proportion of the acetate was respired by the microorganisms in July, it would have given a lower net uptake by the cellular material. Also, a faster turnover of the cellular material and recycling of a limited Table 6.7 A comparison of the uptake of 1 4carbon per ug of bacterial biomass for the 1985 log experiments. Sample days Tree species Month Number of logs 0 10 25 (Mg 14C/Mg C/h x 10"3) Pine June C 5.10 - 0.91 1 6.10 - 0.49 3 2.30 - 0.91 5 2.90 - 0.61 July c 0 - 12.00 1 0.01 12.20 12.0 3 0.64 2.20 1.62 5 1.40 0.43 2.30 Spruce June C 1 3 5 0.74 0 0.51 0.65 0.89 1.40 4.10 1.60 3.30 3.70 1.70 0.68 July C 1 3 5 0.76 0.34 0.26 0.48 3.70 6.40 6.90 0.63 2.20 9.90 11.00 4.80 C = control (no logs) nutrient pool in the enclosures could help to keep the bacterial biomass lower and more active. Lower oxygen concentrations in July support the observed overall higher metabolic activity. Fourthly, predation by heterotrophic protozoans on the bacterial biomass in July could also help explain the observed results. The predatory action of protozoa on the bacterial community is a widely recognized trophic link in aquatic systems. 94 There are abundant field observations and laboratory evidence that heterotrophic microflagellates and ciliates are active predators of bacteria in a variety of environments, i.e. marine and freshwater ecosystems, and activated sludge treatments plants (Sherr and Sherr, 1984 ; Azam ef al. 1983 ; Fenchel, 1977 and 1982 ; Cude, 1979). Sherr and Sherr (1984) state that in situ bacterioplankton production utilizes 10% - 50% of total phytoplanktonic productivity, and although bacterial numbers and biomass tend to be relatively low and constant, bacterial productivity indicates the biomass is turning over rapidly. Heterotrophic protozoan grazing is a major consumer of bacterioplankton. Laboratory evidence has found that microflagellates and ciliates consume 10 to 250 bacteria per protozoan cell per hour (Sherr and Sherr, 1984 ; Berk ef al. 1976 ; Fenchel, 1982). Cude (1979) studied the microflora composition of an activated sludge continuous culture system. In laboratory experiments with defined inocula, morphological structure and cell size of the bacteria were an important factor in selectivity of protozoan prey items. For example, filamentous, spiral, and floe forms of bacteria are resistant to protozoan grazing (compared to single-cell bacteria) by virtue of morphology. Therefore, defined bacterial cultures with a protozoan population are predominantly filamentous. These observations exemplify that species diversity can be increased by increasing the complexity of the system. Thus, certain bacterial populations wouid be overgrown by better adapted bacteria if competition for substrates were the only selection factor (Gude, 1979). For example, in protozoa free cultures, the single celled bacteria would have a competitive advantage over filamentous forms in substrate aquisition. However, the same population could compete successfully if an additional selection factor, i.e. predation, were introduced into the system (Cude, 1979). The results of Gude's (1979) experiments offer a possible explanation to the observed discrepancies between June and July log enclosure studies. It is possible 95 that protozoan predation upon singled-celled bacteria enabled the filamentous population to become the dominant bacterial species during the June time period. In July, the bacterial biomass was smaller (compared to June) and comprised predominantly of coccoids (0.5 um to 0.75 urn.) with only a few visible strands of filaments. This suggests possible intense protozoan grazing, low relatively constant bacterial numbers and biomass with high secondary production. Unfortunately, protozoan biomass for the 1985 enclosure studies was not measured, therefore, the above explanation is based on conjecture and theory. 6.2.2.2 K inet ic uptake study . (i) Limitat ions of the k inet ic uptake study There were several drawbacks with the kinetics study ; 1. the chemical composition of the leachate was not determined, 2. an unknown in situ acetate concentration. Although the in situ solute concentration was less than 0.1 Mg/', isotope dilution was a possible problem. If unlabelled acetate was leached from the logs in concentrations greater than 20 ug/l, it could have effectively dilute the labelled acetate and give a lower uptake rate, and 3. the values obtained from this study described the dynamics of the heterotrophic population for the July study period. For example, the kinetic values of the bacterial biomass were a function of water temperature, sealed bags (nutrient limitation), leachate composition, and initial stocking concentrations of bacteria, phytoplankton, and zooplankton. 96 The results of the experiment cannot be superimposed upon the June experimental results because of differences in the aforementioned physical, chemical, and biological conditions. For example, the July bacterial biomass for spruce and pine was significantly smaller compared to the June biomass, therefore, the July values for V m a x , T^ , and probably would not apply to the heterotrophic dynamics of the June experiments. (ii) Pine The increased V m a x value for the 5 pine logs enclosure suggested that the composition of the heterotrophic community had increased compared to the initial population. The increase in heterotrophic biomass was probably the result of a population of bacteria having been introduced into the enclosure by the pine logs which proliferated under the conditions of leachate generation (Figures 5.16(A) and 5.16(B)). The increased biomass and acetate data confirm an increase in the bacterial population. The drop in T^  coupled with the increase in V m a x- /Kj. indicated that the bacterial biomass in the 5 pine logs enclosure had a high affinity for the extractives (i.e. acetate) leached from the logs and that the extractives were utilizable. This was confirmed by the low levels of volatile fatty acids in the enclosure. (iii) Spruce Although the V m a x of the spruce kinetic experiment was the same for the pine experiment, the result of an increase in bacterial biomass (Figure 5.15 and Table 5.10), the Kt and T^  values for spruce were higher ; this indicated a difference in log leachate composition between the two species of trees. A chemical difference between log leachates was confirmed by comparing both the 97 COD values, where more oxidizable material was leached from pine vs spruce (i.e. 47 mg/l COD and 32 mg/l COD after 10 days, respectively) and the affinity ratio of V /K (5 spruce logs was half that of the pine value). This difference in ITlaX I extractive composition dictated the degree or intensity of heterotrophic utilization of the leachate. For example, the bacterial population in the spruce log enclosure was not able to utilize the log leachate as quickly as the population that developed in the pine enclosure (as indicated by lower dissolved oxygen). (ii) Impl icat ions The biomass, 1 4C-acetate uptake, and kinetic uptake data provided information on the microbial dynamics of log storage in a slow flushing environment. The bacterial study, coupled with the DO, COD, and TOC results, indicated : 1. temporal differences in bacterial biomass and chemical composition of log extractives, 2. a quantitative and qualitive difference in the organic matter leached from pine and spruce, that directly affected the ability of heterotrophs to utilize the leachate, 3. the leachate from pine had a faster turnover time and higher degree of biodegradability than spruce and the chemical composition of the pine extractives had more oxidizable matter than spruce ; this suggested that pine leachates were more susceptible to microbial degradation than spruce log leachates (supported by the lower dissolved oxygen values for pine compared to spruce), and 4. the low T and high V /K. ratio for pine and spruce indicated a potential eutrophication problem in a log storage facility. 98 6.2.3 PHYTOPLANKTON BIOMASS There was no significant increase or decrease in the phytoplanktonic algal biomass for the enclosure experiments. However, there was significantly lower phytoplankton biomass present in the July spruce enclosures compared to the June values. By comparing the lake and control chl _a_ data, the difference between June and July chl a values may be the initial stocking concentration of the phytoplankton. The 1985 studies indicated that there was no apparent toxic effects from the logs on phytoplanktonic biomass. 6.3 COMPARISON BETWEEN THE ENCLOSURE STUDIES AND THE 3 YEAR FIELD  STUDY BY WESTWATER RESEARCH In conjunction with the 1984 and 1985 enclosure studies, Westwater Research Center conducted a 3 year investigation commencing in June 1983 to compare pre-impact data (prior to the initiation of fall logging and log transportation at the Morrison Arm dump site and. Topley Landing dewatering site) to post-impact conditions studied from May to August of 1985 (Levy ef al. 1984, and Levy ef al. 1985a and 1985b). Intensive sampling stations were set up at both the dump site and the dewatering site with results from the affected areas compared to reference sites (Figure 2.1). Colour, turbidity, L-T, conductivity, DO, temperature, COD, and bacterial biomass measurements were conducted at depths of 0, 1, and 2 m on May 15, May 27, June 11, June 21, and August 8, 1985. The water quality results from both active sites were a function of location and lake morphology. Water quality measurements at the dewatering site did not show any degradation when compared to the reference stations or to the pre-impact data collected in 1983 (Levy ef al. 1984). It was concluded that the 99 unsheltered dewatering site, adjacent to the main basin of the lake, was subjected to strong winds that blow the long fetch of the basin which resulted in good water circulation and a well mixed water mass (characterized by a weak thermocline ; Levy et al. 19856). However, the water mass in the sheltered dump bay at Morrison Arm became stratified and inhibited water circulation, thus, creating a potential precursor for water quality degradation. Figures 6.1 and 6.2 depict the enclosed, sheltered log storage bay at Morrison Arm. For example, the post impact water quality at the dump and storage site deteriorated by May 27 when DO dropped to & 5 mg/l from > 8 mg/l on May 15 (Levy ef al. 1985b). The DO deficit coincided with an increase in COD in the surface waters (average of 40 mg/l for sampling depth range of 0 - 2 meters) compared to the reference site average of 24 mg/l (0 - 2 meters). The DO concentration at the 2 meter reference site remained constant at approximately 10 mg/I. The rapid warming of the surface waters in these protected areas of Morrison Arm dump site created ideal conditions which stimulated microbial activity to utilize organic materials leached from the logs resulting in deterioration of water quality. By July 16, all logs had been moved to the dewatering site at Topley Landing ; the DO in the dumping bay rebounded to ^ 8 mg/l by August 6, 1985. Assuming that a uniform bundle of logs is only 50% submerged in the Morrison Arm storage area and a thermocline at 2 m (leachate mixing in the top 2 m of water), the 166,000 m 3 of logs stored in Morrison Arm, 1985, would represent a loading level of approximately 0.4 m^/m3 water (Hall, unpubl.). This is an order of magnitude higher than the 5 log enclosures where the log loading level was 0.035 m 3 / m 3 water. Although, the log volume : water volume was greater at Morrison Arm than the enclosures, the decreased DO concentrations in the enclosures was paralleled by a similar decrease in DO at Morrison Arm ; this 100 Figure 6.1 Aerial photograph of the Morrison dump site in mid-winter, 1984. 101 Figure 6.2 Aerial photograph of the Mor r i son dump site on May 14, 1985. implied that similar leaching mechanisms and trophic level interactions were occurring under experimental conditions and in si tu. 7. C O N C L U S I O N The objective of the bark and log studies was to determine the effects of log dumping and storage on the Babine Lake aquatic environment, specifically, changes in water quality and microbiology. A major concern generated by the environmental impact studies was the dissolved oxygen deficit. The depletion of D O was a function of heterotrophic activity, chemistry of the wood and bark, the physical limitations of the enclosures, and oxygen solubility (function of water temperature). The sealed (no water-renewal) construction of the enclosures approximated the morphological scenairo of a shallow, sheltered, non-circulating aquatic ecosystem, and allowed the buildup of organic carbon leached from stored logs and log debris. The quality and quantitity of extractives leached from the logs and bark provided a readily available and utilizable organic source for an enterprizing population of bacteria. Microbial activity combined with the leached organic carbon produced a D O demand. From the enclosure experiments, I would hypothesize that the storage of logs in the enclosed, protected bay of Morrison Arm would result in organic enrichment, D O depletion, and degradation of water quality. The storage facilities and morphology of Morrison Arm provided ample conditions for the processes of organic enrichment and oxygen depletion. For example, the shallow and protected bay of the storage facility would restrict wind generated reaeration of the surface waters, and with the low volumes of water exchange (tracer studies revealed a flow of 3 m /^hr ; Power, 1987), would allow for an accumulation of organic material. These two conditions would contribute to a potentially severe D O deficit which could adversely affect habitat quality of the aquatic environment of Morrison Arm. 102 103 From the bark studies, it was found that bark and logs were significant contributors to the degradation of the enclosure environment. Therefore, in a log dumping and storage operation trees that have lost bark during the bundling and dumping phase of operations create a pollution potential via (i) concentration gradient of leached organic compounds above the 2 m thermocline, and (ii) loose bark and benthal accumulation. The 3 year study of Morrison Arm by Westwater Research Centre found that the benthic accumulation of bark had resulted in the annihilation of the benthic community. In conclusion, I would recommend for future log storage operations : 1. more stringent criteria for the siting of log dumping and storage facilities, i.e. locate the storage sites in areas where there is adequate water circulation to prevent the buildup of organics and the exertion of a BOD to such a degree that low oxygen conditions can develop, 2. rapid removal of logs from constricted circulation areas especially if warm epilimnetic conditions develop, and 3. possible aeration of the dump and storage areas to oxygenate the water and destratify the system, i.e. this is easily accomplished in winter logging areas where an aeration system is already in place to keep the storage bay ice free in winter. 8. APPENDIX A 1. Calcu la t ion of microbia l uptake of label led acetate. i) specific activity of acetate = 57.6 mCi/mmole ii) molecular weight of acetate = 59, therefore iii) specific activity = 0.976 uCi/ug of acetate Calcu la t ion example ; heterotrophy experiments i) 100 X = 15,000 dpm ii) 1 ml of isotope = 150,000 dpm iii) 1 uCi = 2.2 x 10 6 dpm iv) 0.976 MCi x 2.2 x 10 6 = 2,145,200 dpm/Mg acetate v) 150,000/2,145,200 = 0.06985 Mg acetate added to each syringe vi) acetate uptake expressed as Mg solute/l/hr was determined by ; uptake = dpm of syringe x 0.6985 Mg Ac. x _1_ hours x 10 ml T x 1000 ml 150,000 dpm 3 9 ml 10 ml where ; Ci = Currie dpm = disintegrations per minute = 10 ml is the sum of 9 ml sample water + 1 ml isotope. 104 105 2. Bacteria numbers (counts per ml) Calculation example ; June, Day 25 for 5 pine logs i) total filter count (within a grid of 50 units x 50 units) = 326 ii) total number of fields = 7 iii) mean field count (i ii) = 46.6 iv) volume of sample water filtered = 1 ml v) areal microscope conversion factor* = 40,000 vi) total bacterial count/ml (iii iv x v) = 1.863 x 10** * = (area of filter) -4- (area of counting grid) for that particular microscope power 3, Bacteria biomass i) measure (randomly) dimensions of rods, cocci, and filaments, keeping tally of the number of rods and cocci ; count the number of times a filament crosses (intercepts) the grid. ii) assume volume of cocci = sphere ; therefore, volume of sphere = r^ 7r4/3 iii) assume volume of rod = cylinder ; volume of cylinder = r^-irl where ; r = radius I = length iv) use the following equation to determine the volume of rods and cocci in sample volume of bacteria in sample = (Av. cell vol. rods) x (% of rods) + (Av. cell vol. coccoids) x (% of coccoids)) 106 where ; Av. = average cell = bacterium vol. = volume Calcu la t ion example ; June, Day 25 for 5 pine logs v) Total number (no.) of bacteria measured = 17 no. of rods measured = 13 or 76% no. of cocci measured = 4 or 24% vi) grams of carbon/ml = (0.75 Mm 3 x 0.76) + (0.524 um 3 x 0.24) = (0.696 Mm3/cell) x 5.6 x 10" 1 3 g of carbon/Mm3*1) = 3.9 x 1 0 - 1 3 g of carbon/cell x. counts/ml (1.863 x 10 6, bacteria counts/ml from previous Section (vi) = 7.26 x 10"^ g carbon/ml for rods and cocci where ; (1) = 5.6 x 10" 1 3 average carbon content/vol. of bacteria (Bratbak, 1985) Calcu la t ion example ; June, Day 25 for 5 pine logs vii) assume volume of filaments = volume of cylinder viii) tally no. of grids counted = 4 ix) tally no. of filaments per grid ; i.e. first grid had 1 filament x) count no. of times the filament(s) intercepts the grid ; i.e. 9 xi) length of filament (within grid) = 9 x 5 Mm (width of 1 grid unit) = 45 Mm xii) for grids with more than 1 filament, summate the lengths of all filaments xiii) total length of filaments from the 4 grids = 410 Mm 107 xiv) average length/grid (xiii viii) = 102.5 Mm xv) average length/ml = 102.5 Mm x 40,000 (areal conversion factor) = 4.1 x 10 6 Mm 3 xvi) average filament vol./ml = 1.16 x 10^ Mm3/ml xvii) average g carbon/ml = 1.16 x 10^ Mm3/ml x 5.6 x 10"^3 g carbon/Mm3 = 6.49 x 10"7 g carbon/ml Total bacterial biomass = (xvii) + (vi) = 6.49 x 10"7 + 7.26 x 10"7 = 1.37 x 10"6 g carbon/ml = 1370 x 10' 6 g carbon/I 4. Percentage of TOC as tannic acid (L-T measurement) i) tannic acid = ^y^^2^4d ii) molecular weight = 1701 iii) molecular weight C = 12 % tannic acid that is carbon = 76 X 12 = 912 g, therefore (912 1701) x 100 = 53.6% of tannic acid is carbon 108 5. Conversion of L-T carbon of TOC into equivalent COD (i) COD = C + 0 2 o C 0 2 where : C = TOC 0 2 = COD (ii) know 53.6% of L-T is carbon and that the oxidization of L-T is represented by, CvcH^ O - . + 660 0 o 76CO 0 + 2 6 H „ 0 /o 52 4b 2 2 2 therefore, 2112 g of 0 2 is required to oxidize 912 g of C. 912 mg/l C = 2112 mg/l 0 2 1 mg/l of C = 2.3 mg/l 0 2 (COD) By multiplying the L-T carbon by 2.3, the COD for the L-T portion of the TOC can be determined. 9. APPENDIX B The microbial kinetics of solute uptake was performed assuming the following assumptions : 1. Michaelis-Menten uptake kinetics, substrate-saturation curve (Wright and Hobbie, 1965 ; Hobbie and Wright, 1965 ; Allen, 1968), where the rate equation for a 1 substrate enzyme catalyzed reaction is described by Equation B.1. V = V S (B.1) o —max— K t + S where : V = maximum uptake rate max r V Q = substrate uptake rate S = substrate concentration K t = active transport constant, 2. steady-state conditions, where the rate of forming the enzyme-substrate complex is equal to the rate of breakdown of the enzyme-substrate complex (Button, 1986), 3. natural substrate concentration is equal to zero (Hall ef al. 1972) ; VFA concentrations < 0.1 ug/l from analytical measurements, 4. uniformly labelled substrate is added to a mixed, random population of heterotrophs, and 5. no significant removal of substrate by phytoplankton (Wright and Hobbie, 1965). 109 110 (i) Def in i t ion of the k inet ic values a. M a x i m u m uptake rate or V max (ug/I/h) V, max is an indicator or measure of the relative size of a population of heterotrophs able to utilize a given substrate, therefore, V is considered to be max proportional to biomass. V max enables one to compare the heterotrophic activity of different bodies of water and is a good indicator of pollution (high V m a x = eutrophication ; Gocke, 1977a ; Wright and Hobbie, 1966). The V m a x value is a function of (i) temperature, (ii) population size, composition, and physiological state of the heterotrophic population, and (iii) availability of substrate. b. Turnover t ime or T^ (hours) Turnover time is the time required by a natural population of heterotrophs for the complete uptake of a concentration of substrate equivalent to the natural concentration. Thus, T^  is an indicator of the intensity of heterotrophic utilization and a measure of the biological importance of a substrate (Gocke, 1977a and 1977b ; Allen, 1967). For example, a low T^  indicates that the substrate is rapidly utilized. High T t values suggests (i) that the substrate is not utilized to any large extent, (ii) only a small population of heterotrophs are present, (iii) zooplankton c. Act ive transport constant or (Mg/l) The Michaelis-Menten K{ is used as an indicator of the affinity of an organism for a substrate ; a lower K indicating higher affinity (Wright and Hobbie, grazing. 111 1966 ; Law and Button, 1977 ; Healey, 1980). The Michaelis-Menten rate equation (Equation B.1) defines K^ . as the substrate concentration at which the initial reaction velocity is half maximal (1/2 V x = K t ; Lehninger, 1982). is independent of the number of heterotrophs but is a function of the heterogeniety of a heterotrophic population (Hobbie and Wright, 1965). The term V ' /K. is used to best describe the affinity (attraction) between max t ' acetate (substrate) and bacteria (Healey, 1980 ; Button, 1986). The V m a x / K t value (slope of the rate equation of V q vs S ) evaluates the competitve ability of an organism in a nutrient limited system. An increased ratio of V m a x / K ^ indicates a strong affinity for the substrate present. 10. ABBREVIATIONS 1. i.e. = example 2. DO = dissolved oxygen 3. COD = chemical oxygen demand 4. BOD = biochemical oxygen demand 5. TOC = total organic carbon 6. TIC = total inorganic carbon 7. L-T = lignins and tannins 8. VFA = volatile fatty acids 9. TRC = total reducing carbohydrates 10. uCi = microCurries 11. ug carbon (C)/l/hr = micrograms of carbon per litre per hour 112 11. REFERENCES 1. A l i en , H.L. 1967. Acetate utilization by heterotrophic bacteria in a pond. Hidrologiai Kozlony 7 : 295-297. 2. A l l en , H.L. 1968. Acetate in fresh water: natural substrate concentrations determined by dilution bioassay. Ecology 49 : 346-349. 3. A P H A , A W W A , and W P C F . 1985. Standard methods for the examination of water . and wastewater. 15 t n ed. 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