U. S. A. ; 'present address : Idaho National Engineering Laboratory, Biotechnology, P. 0. Box 1625, ...... Lock, M. A., R. R. Wallace, J. W. Costerton, R. M. Ventullo.
Hydrobiologia 171: 79-90 (1989) @ Kluwer Academic Publishers
Evidence of structural and functional adaptation in epilithon exposed to zinc Frederick S. Colwell', Sally G. Hornor & Donald S. Cherry Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A. ;'present address :Idaho National Engineering Laboratory, Biotechnology, P. 0.Box 1625, Idaho Falls, ID 83415-2203, U.S.A. Received 1 December 1986; in revised form 14 September 1987; accepted 20 November 1987
Key words: epilithon, periphyton, zinc, glucose respiration, protein, carbohydrate
Abstract Artificial streams were used to study the effect of chronic zinc dosing (0.00,0.05 or 1.00 mg Zn 1- ') on epilithic communities during summer and early fall, 1984 and 1985. Following zinc addition, epilithon was periodically measured for '4C-glucose and 14C-glutamaterespiration, ash-free dry weight (AFDW), protein, carbohydrate, chlorophyll a (Chl a), pheophytin a (pheo a), and colony forming units (CFU) on zinc amended and unamended media. Transient peaks in glucose respiration rates mg- ' protein and AFDW occurred within 5 to 10 days in streams dosed with 1.0 mg Zn 1- ' and coincided with decreases in AFDW, protein, carbohydrate, Chl a, and pheo a. Respiration of glutamate did not show a significant response to dosing (a = 0.05). Epilithic plate counts demonstrated more total and zinc-tolerant CFU in dosed than in control epilithon after 20 days. After 30 days, greater biomass and lower protein: carbohydrate ratios were evident in epilithon dosed with 1.0 mg Zn 1- ' compared to unamended treatments. Peaks in glucose respiration suggested the presence of zinc-tolerant heterotrophs transiently capable of increased respiration of glucose but not glutamate. Concurrently, minimal but measurable biomass inferred the persistence of zinc-tolerant microorganisms in zinc-dosed epilithon. By the end of the studies, results indicated that metal-tolerant populations replaced metal-sensitive organisms under conditions of elevated zinc concentration. These zinc adapted communities had lower potential value as a food source to grazers (low protein: carbohydrate ratio) and were no longer distinctive with respect to glucose respiration.
Introduction Zinc is an important model toxicant because it is abundantly distributed in aquatic systems due to both natural and anthropogenic sources (Spear, 1981). In addition to its role as a metal pollutant, zinc is a required trace element for many organisms (Lehninger, 1982). Although levels of dis-
solved zinc are estimated to be relatively low (< M or 0.07 mg 1- ') even in industrialized rivers (Shiller & Boyle, 1985), in some cases concentrations of this metal may exceed the U.S.EPA limit of 0.180 mg 1-' total recoverable zinc at a hardness of 50 mg CaCO, 1- ' (U. S.EPA, 1980; Weatherley et al., 1980). The epilithon or periphyton in streams consists
of a diverse assemblage of microorganisms including algae, bacteria, fungi, and protozoans which contribute to nutrient cycling, energy flow, and biodegradation in streams (Hynes, 1970). The quality of the epilithon (e.g., carbon: nitrogen or protein: carbohydrate ratio) can determine the physiological health of higher organisms dependent on this community as a source of food (McMahon et al., 1974). Additionally, heavy metals chelated by the anionic polysaccharide matrix produced by the epilithon may enter the base of the food chain (Foster, 1982; Lock et al., 1984; Filip et al., 1979; Sterritt & Lester, 1979; Patrick & Loutit, 1976). Studies dealing with the effects of zinc on epilithon are limited in the literature. Williams & Mount (1965) determined that 9.0 mg Zn 1- ' causes a shift from predominantly autotrophic to heterotrophic communities. Albright & Wilson (1974) noted an increase in glucose turnover time in planktonic microbial communities treated with 0.05 mg Zn 1- ' during short (3-day) studies suggesting that low zinc concentrations may effect the rate of processing of organic compounds by aquatic microogranisms. The influence of extended zinc dosing on nontaxonomic structure and function in the epilithon has not been studied. To determine the effects of zinc on epilithon, we used outdoor artificial streams continuously dosed with zinc and measured a range of functional and structural characteristics of epilithon during summer and fall of two field seasons. Epilithic respiration of glucose was used as an indication of community response to a highly labile carbon source. Measurement of glutamate respiration was added during the second field season to calculate the glucose: glutamate (gluc: glut) respiration ratio which is a useful measure of health in algal communities (Gillespie et al., 1976; Griffiths et al., 1982). Epilithic biomass was estimated by ash-free dry weight (AFDW), protein, and carbohydrate, accepted methods of quantifying epilithon (American Public Health Association, 1985; Brock & Brock, 1967; Bott & Brock, 1970; Clark et al., 1982). The protein: carbohydrate (P :C) ratio offers information regarding the nutritional
value of epilithon for consumers of this material (McMahon et al., 1974). Analysis of epilithic chlorophyll a (Chl a) and pheophytin a (pheo a) pigment concentrations during the second field season provided an estimate of biomass that is specific for algae. The Chl a:pheo a ratio is also used as an algal health indicator (American Public Health Association, 1985). Heterotrophic plate counts on solid media variously amended with zinc (Hornor & Hilt, 1985)were used to determine whether zinc-tolerant bacteria become established in epilithon continuously dosed with the metal.
Materials and methods Study site and stream design. Glass rods (5.4 cm2 surface area) separated by 2.2 cm were incubated in an attached, vertical position in river-fed cial streams along the New River at Glen Lyn, Virginia, U. S.A. Streams had approximate volumes of 27 1, current velocities of 14 cm sec- ', and, with a 1.2 1min-' influent flow rate, the water in each stream was renewed every 23 min (see Belanger et al., 1985). The estimated submerged surface area (including glass rods) was 0.64 m2. In 1984 studies, nine artificial streams were used, representing three replicates of each of three zinc treatments: 0.00, 0.05, and 1.00 mg Zn 1- ' (0.77 and 15.30 pM Zn). Six streams were used during 1985, with three replicates of each of 0.00 and 1.00 mg Zn 1-' treatments. Rods were colonized with epilithon in streams 10 days before starting metal dosing. One day before dosing with zinc, snails from the New River were placed in all streams (approximately 80-110 snails m W 2 ; mean snail diameter was 1 cm) as part of an additional experiment (Farris, 1986). In 1984, two 30-day dosing periods were started; one on June 23, (J84), and another on September 7, (S84). Glucose respiration, AFDW, protein, and carbohydrate in epilithon were measured 0,2,5, 10,20, and 30 days after zinc introduction. In 1985, two 20-day dosing periods were started on August 22, (AM), and on September 29, (S85). Shorter (20-day) dosing periods were used during 1985
since the critical effectof zinc on the epilithon was noted soon after its introduction. During A85, epilithic glucose and glutamate respiration, heterotrophic adaptation to zinc (plate counts), AFDW, protein, and photosynthetic pigments were measured 0,5, 10, and 20 days after starting zinc dosing. During S85, the same sampling schedule was followed, but only epilithic AFDW, protein, and pigments were measured. Streams were cleaned, painted, and recolonized between studies. Glucose and glutamate respiration. Three rods with intact epilithon were removed randomly from each of three zones in each stream and placed into 250-ml jars containing 60 ml water from the corresponding stream. Respiratory rates for glucose and glutamate were determined using a modification of the method of Williams & Askew (1968). A trace amount of [U-14C] glucose (specific activity: 345 mCi mmole- ' ; 0.44 pg 1- ' final concentration) or [U-14C] glutamate (specific activity: 290 mCi mmole- ' ; 0.21 pg 1- ' final concentration) were added to separate jars containing the glass rods. Contents were mixed and jars sealed and incubated in the dark at ambient water temperature with gentle agitation. Killed controls were used to correct for abiotic production of 14C02.Five to fifteen min incubations were used so that < 10% of the labeled substrate would be respired. Biological activity was stopped by the injection of 2 ml of 6N H2S04 (final pH < 2.0). A dry filter paper in each jar was saturated with 0.15 ml of phenethylamine allowing absorption of 14C02. 14C02 absorption occurred during 1.5 h of gentle shaking after which filter papers were placed into 10-ml cocktail (4 g PPO and 0.1 g POPOP 1- ' toluene [Sigma Chem. Co.]) and counted by liquid scintillation. The sample channels ratio method was used to correct for quench. Ash-free dry weight, protein, carbohydrate, and pigment determinations. Epilithon from each stream was scraped from 3 to 10 glass rods and homogenized in a Waring blender and stored at - 20 " C until assayed for AFDW, protein, carbo-
hydrate, Chl a, and pheo a. AFDW was determined by subtracting combusted weight (1 h at 500 " C) from dry weight (dried to constant weight at 105 "C) for samples of replicated zinc treatments (American Public Health Association, 1985). Protein was extracted from homogenate by treating three times with 0.5 N NaOH at 80 " C for 10 min (Rausch, 1981) and analyzed using the micro-biuret method with bovine serum albumin as a standard (Itzhaki & Gill, 1964). Recovery of bovine serum albumin was 90% after extraction. Carbohydrate was determined by the phenolsulfuric acid method (Herbert et al., 1971). Chl a was estimated by measuring the optical density (O.D.) of an acetone extracted epilithon sample at 663 nm (American Public Health Association, 1985). Subsequently, the sample was acidified and measured at O.D. 665 nm to derive pheo a. The ratio of O.D. 663 nm before acidification-to0.D.665 nm after acidification provided an estimate of the Chl a:pheo a ratio. Heterotrophic counts. Total heterotrophic plate counts, expressed as colony forming units (CFU), were determined for the initial and 20th days during the A85 study using the method of Hornor & Hilt (1985). Appropriate dilutions of epilithon homogenate from each stream were spread onto triplicate plates containing 114 strength Nutrient Agar (Difco) amended with 0.0, 1.0, or 10.0 mg Zn 1- '. Colonies were counted after 5-day dark incubations at 20 " C. Water chemistry. Temperature, pH, alkalinity, hardness, conductivity, and ammonia were determined by standard methods (American Public Health Association, 1985). Total zinc was measured by flame atomic absorption spectrophotometry. Periodically, nitrate, sulfate, and phosphate were measured by ion chromatography (Dionex) (Tabatabai & Dick, 1983). Dissolved organic carbon (DOC) was measured in filtered water samples (Gelman glass fiber filters, type A/E, 0.3 pm pore size) on each sampling day during the A85 study using a Model 800 Total Organic Carbon Analyzer-0.1. Corporation (Menzel & Vaccaro, 1964).
Statistical methods. A repeated measures or split plot design (without replication or blocking) was used to analyze experiments (Milliken & Johnson, 1984). Zinc was the between subject (whole plot) factor and time was the within subject (subplot) factor. For 1984 studies, all epilithon structural and functional characteristics and water chemistry measurements were compared by treatment on a given study using Analysis of Variance (ANOVA) (Sokal & Rohlf, 1981). Duncan's Multiple Range Tests were performed after noting significance using ANOVA to determine differences among treatments on a given study day. During the 1985 studies, water chemistry measurements from each sample day and heterotrophic plate counts were compared using ANOVA. The nonparametric Mann-Whitney test which utilizes a rank-sum protocol (Sokal & Rohlf, 1981) was used to distinguish sample distributions on each study day for mean glucose and glutamate respiration rates, as well as AFDW, protein, Chl a, and pheo a measurements. A nonparametric analysis was used for these variables since samples exhibited a non-normal distribution. The a = 0.05 level was used for all statistical tests.
Results
Among water chemistry measurements, only total zinc concentration and pH were significantly different between treatments on a given study day (Table 1). Total zinc levels in control streams ranged from below detectable limits High (
