Assessment of Epiphytic Algae (periphyton) as a Component of Kettle Pond Monitoring at cape cod national seashore (2004)
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Assessment of Epiphytic Algae (periphyton) as a Component of Kettle Pond Monitoring at CAPE COD NATIONAL SEASHOre (2004) Stephen M. Smith 99 Marconi Site Rioad Wellfleet, MA 02667 Introduction Periphyton refers to communities of algae in aquatic systems that are attached to the sediment surface or to aquatic, macrophyte vegetation. In the Kettle Ponds of Cape Cod National Seashore (CACO), periphyton can be abundant in the littoral zone, where it thrives in clear shallow waters primarily on the underwater surfaces of emergent vegetation (Figure 1). The biomass and taxonomy of periphyton communities are used as indicators of water quality in aquatic systems worldwide.  
Figure 1. Periphyton (indicated by arrow) attached to stems of Lobelia dortmanna in Great Pond, Wellfleet. Artificial substrates (hereafter referred to as periphytometers) are commonly used as a way to sample attached algae (Barbour et al. 1999). Although there are many different periphytometer designs and materials that can be used, the basic methodology is the same. Periphytometers are placed in the waterbody and left in place for a certain period of time. The duration is dependent upon the rate at which periphyton accumulates on the substrate. As a rule, they should be removed just short of the point that the attachment surface becomes “saturated” and the algae begins sloughing off. At the end of the collection period, the periphyton is collected simply by scraping it off the surface. The material can then be dried and weighed for an estimate of biomass per unit area per unit time (i.e., g/cm2/d). Other measurements include chlorophyll a concentration (typically used as a surrogate for biomass), taxonomic composition, and tissue nutrient content. One advantage of using periphytometers is that they are very easy and inexpensive to use. In addition, they provide a highly standardized environment with respect to light. The physical and chemical properties of the attachment surface are also important in that various plant exudates influence the growth of algal communities (Hootsmans and Blindow 1994, Mulderij et al. 2003). Thus, while communities growing on artificial substrates may differ somewhat from those growing on natural vegetation (although often they are the same), there is no confounding effect of disparate surface types (i.e., different macrophyte species). Another advantage of this method is that periphyton integrates water quality conditions over time, which may provide a better representation of pond water quality status than do instantaneous measures of specific constituents. Although periphyton is widely used as a bioindicator of water quality in many different kinds of aquatic environments, it is not currently part of CACO’s Kettle Pond Monitoring Program. Thus, the main objective of this project was to develop field procedures for a periphyton pilot study and to collect baseline data on biomass and taxonomy in a number of different Kettle Ponds. Preliminary analyses of these data were then used to evaluate whether periphyton may be a good parameter to include in the Kettle Pond Monitoring Program. Methods Periphytometer design - Periphytometers were fashioned using plates cut out of clear Plexiglas. Aluminum wire was run through holes drilled through the tops of four Plexiglas plates (10 x 50 cm) and Styrofoam floats were fastened to the ends (Figure 2). The surface of one side of one plate in each unit was designated for collection of material for taxonomic analysis (not included with biomass determinations). 
Figure 2. Periphytometer design. Study sites - Five Kettle Ponds showing a gradient in trophic status (Roman et al. 2001) were sampled (Great (Truro), Ryder, Long, Duck, and Herring). In each Pond, two periphytometers were positioned in open areas (i.e., in full sunlight away from emergent or overhanging vegetation) of the littoral zone on the north (south-facing) shoreline. The units, each tethered to a wooden dowel by fishing line, were placed at least 10 m apart along a 50 cm depth isopleth. The periphytometers were deployed for ~ 1 month (July 7-August 9, 2004). Sample collection and analysis – At the end of the incubation period, all attached algae were scraped off the slides, composited, and placed in a scintillation vial. Material from the plate face designated for taxonomic sampling was scraped and put in a vial with 2% Lugol’s solution for preservation. At the laboratory, the biomass samples were dried in a convection oven at 60 for 24 hrs. and subsequently weighed. Samples collected for taxonomic analyses were sent to Water’s Edge Scientific LLC for enumeration by species (Baraboo, Wisconsin). Results Periphyton biomass - Periphyton biomass did not show large differences among ponds, with the exception of Duck Pond which had a much lower value than the rest (Figure 3). There are, however, some potential flaws in the data. The Herring Pond periphytometers were colonized very quickly and it is possible that biomass accumulation reached an asymptote before the end of the incubation period. In other words, biomass could not continue to accumulate as the entire surface was already covered and the existing periphyton could not support additional increases in thickness without sloughing. A shorter deployment period (for this Pond at least) may solve this problem. In Ryder pond, near the end of the incubation period, one of the periphytometers broke free from its tether and was washed into shallow water where part of the plates were exposed to air. As such, the biomass value is probably a little low. In Long Pond one unit was lost during the incubation period, probably due to human interference. As a result, there is no replication within this pond. 
* 
Figure 3. Mean periphyton biomass accumulated on periphytometers during July 7-August 9, 2004 (* denotes possible bad datum due to sloughing). Taxonomy – In total (all ponds), 63 species were found, representing blue-green algae, cryptomonads, diatoms, golden brown algae, and green algae (see Appendix I and II). The number of species in each pond ranged between 11 (Duck Pond) and 31 (Great Pond). Shannon-Weiner diversity was low in Duck and Long Pond compared to Great, Herring, and Ryder (Figure 4a). Principle Components Analysis (PCA) (Primer ver 5.0) of species composition showed that Ryder and Herring Ponds were distinctly different from each other and from Long, Duck, and Great Ponds, which were similar to each other (Figure 4b). Eigenvector values revealed that the variability was principally due to the relative abundance of 5 species: Achnanthes sp. (diatom), Anabaena cylindrical (blue-green), Aulacoseira sp. (diatom), Helicodictyon planctonicum (green), and Mougeotia sp (green).  
a. b.
Relationships between biotic and abiotic variables - Periphyton biomass was positively correlated with molar N:P ratios calculated from total (organic + inorganic) nutrient concentrations in August 2004 (Figure 5a). The relationship was weaker for biomass vs. inorganic N:P or TN (Figure 5b, c). Relationships between total vs. inorganic nutrient concentrations were essentially non-existent (Figure 5d, e).    a. b. c.
 
d. e.
In terms of specific functional groups, the diatom fractions of the communities were strongly related to TN concentrations (R2 > 95%) (Figure 6a). On an overall community basis, Shannon-Weiner diversity also was positively correlated with TN and N:P (R2 > 65%) (Figure 6b, c).   
a. b. c.
When species composition and environmental data were compared to each other as multivariate datasets, there was good correspondence between the two. This can be demonstrated visually with side by side comparisons of the datasets in ordination space (Figure 6a, b). Separate PCAs of algal community composition - normalized so that all species are of equal importance in determining principle components (Clarke and Warwick 2001) - and water quality variables are remarkably similar (see Figs below). According to BIOENV correspondence analysis (Primer ver. 5), where similarity matrices of species composition and environmental data are compared, 96% of the variance in taxonomy can be explained by August 2004 water column concentrations of Cl, SO4, Ca, TP, and TN
Figure 6. a) PCAs of species composition and b) log-transformed water quality variables. Discussion Although this data is preliminary and represents a small number of observations, it suggests that periphyton may be a good tool with which to monitor Kettle Pond water quality. The data further suggest that some phytoplankton, particularly certain species of diatoms, are not saturated with N, even at N:P ratios well above 16. Although this result is somewhat surprising, a number of studies (Elser et al. 1990, Maberly et al. 2002, Maberly et al. 2003) suggest that nitrogen limitation of algal growth may be far more common in freshwater lakes than is generally believed. In fact, molar ratios of both total and dissolved nitrogen to phosphorus in the water column of several CACO Kettle Ponds show potential N limitation during August (Table 1, 2), as do C:N ratios in sediments (Table 3). Furthermore, many of CACO’s ponds are very near or below the reported pH tolerance of cyanobacteria (Garcia-Pichel et al. 2003), which otherwise provide N to aquatic systems through atmospheric N fixation. Table 1. Molar N:P calculated from total (organic + inorganic) N and P (values < 16 indicate potential N limitation). 
Table 2. Molar N:P calculated from dissolved (inorganic) N and P (values < 16 indicate potential N limitation). 
Table 3. Molar C:N calculated from sediment total C and N (values > 6 indicate potential N limitation). 
Optimal N:P ratios are different for different kinds of algae. For example, chlorophytes (green algae) generally have higher internal N:P and would therefore still experience N limitation at higher N:P ratios (i.e., > 16). Finally, it is pertinent to note that TN concentrations in four out of the five ponds discussed in this study were < 350 ug/L, which classifies them as oligotrophic (Nurnberg 1996). As such, N inputs could still have a substantial effect on algae growth, especially for those species with higher optimal N:P ratios. This is significant in that anthropogenic sources of N, either in groundwater or precipitation, could be altering algal communities. Given that algae and bacteria comprise the base of the food web, changes in these communities could impact the entire pond ecosystem. Recommendations for future monitoring In 2005, periphytometers will again be used to collect periphyton. The following changes and additions to sampling are proposed:
Figure 7. Example of nutrient diffusion design.
Literature Cited Barbour, M.T., J. Gerritsen, B.D. Snyder, and J.B. Stribling. 1999. Rapid Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic Macroinvertebrates and Fish, Second Edition. EPA 841-B-99-002. U.S. Environmental Protection Agency; Office of Water; Washington, D.C. Clarke, K.R. and R.M. Warwick. 2001. Change in marine communities: an approach to statistical analysis and interpretation, 2nd edition. Plymouth Marine Laboratory. Plymouth, UK. Elser, J.J., E.R. Marzolf, and C.R. Goldman. 1990. Phosphorus and nitrogen limitation of phytoplankton growth in the fresh-waters of North America – a review and critique of experimental enrichments. Canadian Journal of Fish and Aquatic Science 47:1468-1477 Fairchild, W.G., R.L. Lowe, and W.B. Richardson. 1985. Algal periphyton growth on nutrient-diffusing substrates: an in situ bioassay. Ecology 465-472 Garcia-Pichel, F. 2000. Cyanobacteria. In: Encyclopedia of Microbiology, 2nd Edition, Lederberg J (ed.) Academic Press, San Diego Hootsmans, M.J.M., Blindow, I. 1994. Allelopathic limitation of algal growth by macrophytes. Geobotany 21: 175-192. Maberly S.C., King L., Dent M.M., Jones R.I. and Gibson C.E. 2002. Nutrient limitation of phytoplankton and periphyton growth in upland lakes. Freshwater Biology 47: 2136-2152 Maberly S.C., King L., Gibson C.E., May L., Jones R.I., Dent M.M. and Jordan C. 2003. Linking nutrient limitation and water chemistry in upland lakes to catchment characteristics. Hydrobiologia 506: 83-91 Mulderij, G., E. van Donk and JGM Roelofs. 2003. Differential sensitivity of green algae to allelopathic substances from Chara. Hydrobiologia 491: 261-271. Nurnberg, G. 2001. Eutrophication and trophic state. Lakeline (Spring 2001):29-33. Roman, C.T., N.E. Barrett, and J.W. Portnoy 2001. Aquatic vegetation and trophic condition of Cape Cod (Massachusetts, USA) kettle ponds. Hydrobiologica 443:31-42.Appendix I. 
Appendix II. 
Appendix III. Alternative design for periphytometers. 
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