Effects of Water Quality on Photoautotrophic Periphyton

CONTRIBUTED PAPERS

SESSION II: AQUATIC BIOLOGY AND WATER QUALITY

Saturday, March 1, 2001

Moderator:

Susan Hendricks
Murray State University

Editor:

A. Floyd Scott
Austin Peay State University

91

EFFECTS OF WATER QUALITY ON PHOTOAUTOTROPHIC PERIPHYTON PRODUCTION AND PHOTOCHEMICAL EFFICIENCY OF A POLLUTION-INTOLERANT ALGA WITHIN MILLER CREEK,
ROBERTSON COUNTY, TENNESSEE

Alex S. Flynt, Jefferson G. Lebkuecher, and M. C. Bone

Department of Biology, Austin Peay State University, Clarksville, Tennessee 37044

ABSTRACT. Effects of water quality on growth and photochemical efficiency of a pollution-intolerant alga, photoautotrophic periphyton production, and periphyton photoautotrophic-heterotrophic biomass ratios were evaluated during the summer and fall of 2000 in the lower, middle and upper reaches of Miller Creek of Robertson County, Tennessee. The results were compared with effects of water quality in the lower reach of Buzzard Creek of Robertson County, Tennessee, known to have good water quality. Periphyton production was significantly greater in all three reaches of Miller Creek relative to Buzzard Creek during October of 2000. Photoautotrophic periphyton production, periphyton production, and rate of sediment accumulation were significantly greater in the upper reach of Miller Creek relative to the lower reach of Buzzard Creek during July and October of 2000. Photochemical efficiency of photosystem II and reproduction rate of the pollution-intolerant alga Selenastrum capricornutum following in situ growth during October of 2000 were significantly lower in Selenastrum grown in upper Miller Creek relative to Buzzard Creek. The results support conclusions from previous evaluations which indicate poor water quality in Miller Creek, especially in the upper reach of Miller Creek.

INTRODUCTION

Miller Creek is within the Sulphur Fork Creek watershed of Robertson County, Tennessee, and is targeted by the U.S. Natural Resource Conservation Service for implementation of best-management practices to improve water quality. Assessments of aquatic primary production were determined at selected sites within Miller Creek to provide data to evaluate the impact of planned-best management practices to improve water quality. This research is a supplement to other bio-monitoring studies being conducted in the watershed by The Center for Field Biology at Austin Peay State University.

The Sulphur Fork Creek watershed is located in the Western Pennyroyal Plain subsection of the Pennyroyal Karst ecoregion (Baskin et al. 1997). This subsection is characterized geologically by limestone, chert, shale, siltstone, sandstone, and dolomite. Soils are of the Pembroke, Crider, and Baxter series, forming a thin, loess mantle over limestone (Miller 1974). Vegetation is characterized as Western Mesophytic Forest consisting largely of Quercus and Carya species (Braun 1950). No single climax forest type occurs. Instead, a mosaic of types are present which are largely determined by edaphic factors and the diverse topography (Chester and Ellis 1989). The Sulphur Fork Creek watershed drains approximately 120,000 acres of the southern half of Robertson County and consists largely of highly erodible farmland supporting tobacco (planted using conventional tillage), numerous livestock operations, and suburban developments.

Metabolism and diversity in small streams are affected by changes in water quality that alter photoautotrophic components of the trophic base (Lebkuecher et al. 1998a). Autochthonous primary production is the major contributor to the trophic base of small streams, especially in those with decreased detritus input due to removal of riparian flora (Vannote et al. 1980, Lamberti and Steinman 1997). Because photoautotrophic periphyton are the most important photoautotrophic components of small lotic systems (Dodds 1991, Lambert and Steinman 1997), changes in water quality that affect photoautotrophic periphyton production in streams with little canopy cover may severely affect whole-stream ecological relationships.

Studying photoautotrophic production yields valuable information on the effects of water quality on the trophic relations of aquatic ecosystems (McIntire and Colby 1978, Naiman 1983). Nonpoint-source pollution (i.e., pollutants entering water bodies from surface runoff) is the most significant pollution problem in the watershed (M. T. Finley, APSU, personal communication). Different pollutants affect photoautotrophic production and physiological status of different genera in different ways (Lebkuecher et al. 2000). Thus, production of naturally occurring photoautotrophic periphyton as well as the status of the pollution-sensitive algae, Selenastrum capricornutum, grown in situ were evaluated during July and October of 2000.

MATERIALS AND METHODS

Sampling Site Locations

The lower Buzzard Creek sampling site is located approximately 6.4 km from Highway 41/11, off Buzzard Creek Road, 100 m upstream of its confluence with Little Buzzard Creek. The lower Miller Creek site is located 9.6 km northeast of I-24, approximately 0.4 km downstream of the Carr Road bridge. The middle Miller Creek site is located at the Turnersville bridge on Turnersville road. The upper Miller Creek site is on the west fork of upper Miller Creek along Henry Gower Road, 0.8 km from the intersection with Sandy Springs Road.

Periphyton Sampling

Periphyton was sampled between July 16 and 22, 2000, and between October 1 and 7, 2000. Periphyton sampling methods followed the recommendations of Morin and Cattaneo (1992). The sampling procedures were designed to provide the most consistent environmental conditions at all sites, a prerequisite to comparing aquatic primary productivity among different sites (Naiman 1983). Commercial periphytometers holding acid-washed slides, placed parallel to flow, were submerged 5 cm below the stream surface. The periphytometers were placed in areas of similar flow (Lebkuecher and Houtman, 1999) and sunlight exposure (full exposure during the photoperiod, maximum 2000 Fmol photons·m^-2·s^-1). For all assays described below, periphyton was removed with a razor blade from both sides of the slides from each site.

Periphyton Ash-Free Dry Weight

Ash-free dry weight was determined following the methods of Clesceri et al. (1989). Periphyton from both sides of 12 slides from each site were scraped into preweighed, prefired, crucibles. Periphyton from three slides were combined to give four replicates. The periphyton was dried at 105EC for 24 h, cooled at 2% relative humidity, and weighed to the nearest 0.1 mg. The dried periphyton was ashed at 550EC for 2 h. Ash-free dry weight was determined following re-wetting the ash with deionized water to reintroduce water of hydration, drying at 105EC for 24 h, and cooling at 2% relative humidity.

Chlorophyll a Concentration

Chlorophyll a concentration was determined following the methods of Arnon (1949). Chlorophyll (chl) was extracted by grinding periphyton from four slides (1 slide per replicate) from each site with a mortar and pestle for 3 minutes in 80% acetone buffered with 2.5 mM NaPhosphate buffer, pH 7.8 at 25EC. The homogenate was filtered through Whatman no. 1 filter-paper circles. Optical density was determined at 663 nm and chl a concentrations calculated (Arnon 1949). Photoautotrophic periphyton biomass was calculated from the chl a concentrations using the equations of Clesceri et al. (1989).

Autotrophic Index

The heterotrophic nature of the periphyton community was evaluated by determining the autotrophic index (AI) and was calculated using the following equation (Crossey and La Point 1988):

AI = Ash-free wt of organic matter (mg/m²)

chlorophyll a (mg/m²)

In Situ Sensitive Algal Assay

Selenastrum capricornutum, a pollution-sensitive green alga and a standard assay organism (Bartsch 1971, Shubert 1984), was used to perform in situ growth assays (Koltz et al. 1975, Shubert 1984). Axenic Selenastrum capricornutum (University of Texas Culture Collection, Austin Texas) was cultured in 25 ml of Bold’s nutrient media with 0.15 g^.L^-1 penicillin for 3 d at 200 µmol photons^.m^-2.s^-1 and 25^OC to obtain cells in the exponential growth phase. Selenastrum capricornutum (2 X 10⁵ cells) were suspended in 6 ml of one-twentieth strength nutrient solution in 16 clear, 1.5 nm-porous membrane bags which allow rapid passage of stream nutrients (Spectrum, Laguna Hills, CA., Spectra/Por Biotech membranes, molecular weight cut off = 3,500 daltons, 10-mm diameter, 10-cm length). The algae-containing bags were suspended 5 cm below the surface parallel to flow between bars of a metal frame attached to the periphytometers at each site. Cells were harvested after 6 d of in situ exposure. Cells were counted using a hemocytometer. Three replicate counts were used to obtain the mean number of cells in each bag to calculate the number of population doublings per day.

Physiological Status Following the In Situ Assay

The physiological status of Selenastrum capricornutum grown in situ was evaluated using chl a fluorescence transients measured with a Plant Efficiency Analyzer (PEA; Hansatech Limited, Kings Lynn, Norfolk, England). Cells suspended in stream water (0.4 ml) were placed inside the fluorescence chamber for 3 min to oxidize primary electron acceptors prior to fluorescence induction (Lebkuecher et al. 1999). Fluorescence transients were measured during a 2-s flash of red light (2000 µmol photons^.m^-2.s^-1) provided by an array of six light-emitting diodes (peak at 650 nm). The fluorescence signals were detected using a PIN-photodiode after passing through a long filter (50% transmission at 720 nm). Origin fluorescence yield (F_O) was calculated by determining the line of best fit through the data points from the first 4-12 µs of fluorescence induction (Lebkuecher 1997). Relative concentration of photosystem II was determined as the variable fluorescence yield (F_V), obtained by subtracting F_O from the maximum fluorescence yield (F_M)(Lebkuecher et al. 1999). The photochemical efficiency of photosystem II was determined as F_V/F_M (Lebkuecher and Eickmeier 1991).

Statistical Methods

Statistical analyses followed the recommendations of Sokal and Rohlf (1981) and Day and Quinn (1989). The experimental design employed a model I analysis of variance with equal replication (Zar 1984). Means are determined to be significantly different if they were dissimilar at the experiment-wise error rate of alpha = 0.05 probability level using the Tukey-Kramer Honestly Significant Difference Test (Sokal and Rohlf 1981).

RESULTS AND DISCUSSION

Primary production is the rate at which inorganic carbon is assimilated into organic form and can be measured as the rate of photoautotrophic biomass accumulation (Lamberti and Steinman 1997, Lebkuecher et al. 1998b). Approximately 1.5% of the dry weight of algae is chl a, and the rate of chl a accumulation per unit area is widely used to determine aquatic primary production (Keithan and

Lowe 1985, Lamberti and Steinman 1997). Aquatic primary production is largely dependent on water quality and is most often increased by decreased water quality resulting from nutrient loading (Baxter 1977, Lebkuecher et al. 1996). Streams pick up dissolved and particulate pollutants from agricultural, silvicultural, and urban sources. This nonpoint source pollution is the major contributor to poor water quality in West Tennessee (Finley et al. 1992, Hupp 1992). The negative effect of nutrient loading from nonpoint-source pollution in upper Miller Creek is indicated by the high values of primary production, as determined by mg chl a/m^2.d (Baxter 1977, Keithan and Lowe 1985), in upper Miller Creek relative to less disturbed Buzzard Creek during July and October (Table 1). Significantly increased ash-free dry weight of periphyton and sediment-accumulation rate in upper Miller Creek in July relative to Buzzard Creek also reveal that upper Miller Creek was more polluted relative to Lower Buzzard during the July sampling period.

Organic pollution of the waterway is indicated by high concentrations of heterotrophic biomass (Lowe and Pan 1996). The middle Miller Creek site is heavily impacted by animal access and this organic pollution is reflected by the high AI value during October relative to lower Buzzard Creek. Autotrophic index values typically increase downstream due to higher concentrations of organic matter in lower stream reaches which, in turn, support a larger biomass of heterotrophs (Vannote et al. 1980). The similar AI values of the upper and middle reaches of Miller Creek relative to the lower reaches of Buzzard Creek during July and October (Table 1) indicate poor water quality at the upper and middle Miller sites.

Table 1. Periphyton characteristics, sediment weights (plus periphyton ash), and autotrophic indexes determined from artificial substrates in Miller and Buzzard creeks. Means ± SE represent four replicate determinations. Means followed by the same superscript letter are not significantly different at the experiment-wise error rate of alpha = 0.05 probability level.

Assay	Lower Buzzard	Lower Miller	Middle Miller	Upper Miller
July 2000
Chlorophyll (mg/m^2.d)	0.31 ± 0.05^a	0.52 ± 0.06^ab	0.35 ± 0.02^a	0.64 ± 0.10^b
Photoautotrophic biomass (mg/m^2.d)	20.8 ± 3.5^a	35.2 ± 4.4^ab	23.7 ± 1.4^a	42.6 ± 6.4^b
Periphyton dry weight (ash-free) (mg/m^2.d)	102.2 ± 5.2^a	188.5 ± 16.9^b	107.4 ± 11.3^a	272.6 ± 19.3^c
Sediment weight (mg/m²d)	1019 + 66^a	1685 + 147^a	1135 + 377^a	3019 + 233^b
Autotrophic index	361 ± 65^a	380 ± 78^a	310 ± 44^a	449 ± 48^a

October 2000
Chlorophyll (mg/m^2.d)	0.05 ± 0.00^a	0.08 ± 0.01^ab	0.10 ± 0.01^b	0.16 ± 0.02^c
Photoautotrophic biomass (mg/m^2.d)	3.05 ± 0.16^a	5.67 ± 0.53^ab	6.77 ± 0.53^b	10.4 ± 1.1^c
Periphyton dry weight (ash-free) (mg/m^2.d)	15.6 ± 5.4^a	61.5 ± 3.6^b	43.0 ± 3.9^b	61.5 ± 5.8^b
Sediment weight (mg/m^2.d)	131 ± 12^a	664 ± 53^b	221 ± 24^a	470 ± 46^c
Autotrophic Index	356 ± 143^a	750 ± 94^b	438 ± 60^ab	403 ± 47^ab

Determination of the effects of water quality on the physiological status of primary producers is a prerequisite to understanding and monitoring changes in watershed ecosystems (Shubert 1984, Clesceri et al. 1989, Naiman 1983, de Madariaga and Joint 1992). The photochemical efficiency of photosystem II (PS II) is an indicator of the efficiency in which absorbed light energy is convertedinto initial electron flow (Lebkuecher and Eickmeier 1993) which, in turn, is used to convert inorganic carbon into organic molecules. This measurement is very accurate and is widely used as an indicator of photoautotroph physiological status (Powles 1984, Bjorkman and Demmig 1987). Significantly lower growth rate (cell doublings/d) and lower photosystem-II photochemical efficiency of the pollution-intolerant alga, Selenastrum capricornutum, grown in upper Miller Creek during October suggest poor water quality relative to the other sites (Table 2).

CONCLUSIONS

Our study provides information on the effects of water quality on periphyton primary production, periphyton autotrophic-heterotrophic relationships, and the growth and physiological status of a pollution-intolerant algae at different sites within Miller Creek. As a whole, the data indicate that Miller Creek has poor water quality relative to Buzzard Creek, especially the west fork of upper Miller Creek, and are consistent with earlier evaluations of water quality within Miller Creek (Lebkuecher and Houtman 1999). In conclusion, the rate of endogenous photoautotrophic-periphyton production was greatest in the west fork of upper Miller Creek during July and October which reveals the negative impact of nonpoint-source pollution. The low growth rate and low PS-II photochemical efficiency of pollution-intolerant Selenastrum capricornutum grown in situ during October support the conclusions from the periphyton characteristics which indicate poor water quality in the west fork of upper Miller Creek relative to the other sites.

Table 2. Growth and photochemical efficiency of photosystem II of Selenastrum capricornutum grown in situ in Miller and Buzzard creeks. Means ± SE represent four replicate determinations following 6 d of growth and if followed by the same superscript letter are not significantly different at the experiment-wise error rate of alpha = 0.05 level of probability.

Assay	Lower Buzzard	Lower Miller	Middle Miller	Upper Miller
July 2000
Cell doublings/d	2.6 ± 0.1^a	5.5 ± 0.8^b	4.1 ± 0.3^ab	2.4 ± 0.5^a
Photochemical efficiency of PS II (F_V/F_M)	0.76 ± 0.01^a	0.73 ± 0.00^b	0.71 ± 0.01^b	0.72 ± 0.00^b
October 2000
Cell doublings/d	2.7 ± 0.1^a	2.0 ± 0.1^bc	2.4 ± 0.2^ab	1.4 ± 0.5^c
Photochemical efficiency of PS II (F_V/F_M)	0.71 ± 0.01^a	0.72 ± 0.00^a	0.72 ± 0.01^a	0.53 ± 0.00^b

ACKNOWLEDGMENTS

The research was funded by the Tennessee Department of Agriculture (Environmental Protection Agency 319 Fund, contract ID nos. 98-06718-00 and Z-00-097944-00) and The Center for Field Biology of Austin Peay State University. We thank Drs. E. W. Chester, Mack Finley, and Floyd Scott for reviewing the manuscript and offering suggestions for improvement.

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