Chapter 6: Periphyton Protocols

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Benthic algae (periphyton or phytobenthos) are primary producers and an important foundation of many stream food webs. These organisms also stabilize substrata and serve as habitat for many other organisms. Because benthic algal assemblages are attached to substrate, their characteristics are affected by physical, chemical, and biological disturbances that occur in the stream reach during the time in which the assemblage developed.

Diatoms in particular are useful ecological indicators because they are found in abundance in most lotic ecosystems. Diatoms and many other algae can be identified to species by experienced algologists. The great numbers of species provide multiple, sensitive indicators of environmental change and the specific conditions of their habitat. Diatom species are differentially adapted to a wide range of ecological conditions.

Periphyton indices of biotic integrity have been developed and tested in several regions (Kentucky Department of Environmental Protection 1993, Hill 1997). Since the ecological tolerances for many species are known (see section 6.1.4), changes in community composition can be used to diagnose the environmental stressors affecting ecological health, as well as to assess biotic integrity (Stevenson 1998, Stevenson and Pan 1999).

Periphyton protocols may be used by themselves, but they are most effective when used with one or more of the other assemblages and protocols. They should be used with habitat and benthic macroinvertebrate assessments particularly because of the close relation between periphyton and these elements of stream ecosystems.

Presently, few states have developed protocols for periphyton assessment. Montana, Kentucky, and Oklahoma have developed periphyton bioassessment programs. Others states are exploring the possibility of developing periphyton programs. Algae have been widely used to monitor water quality in rivers of Europe, where many different approaches have been used for sampling and data analysis (see reviews in Whitton and Rott 1996 , Whitton et al. 1991). The protocols presented here are a composite of the techniques used in Kentucky, Montana, and Oklahoma ( Bahls 1993, Kentucky Department of Environmental Protection 1993, Oklahoma Conservation Commission 1993).

Two Rapid Bioassessment Protocols for periphyton are presented. These protocols are meant to provide examples of methods that can be used. Other methods are available and should be considered based on the objectives of the assessment program, resources available for study, numbers of streams sampled, hypothesized stressors, and the physical habitat of the streams studied. Examples of other methods are presented in textboxes throughout the chapter.

The first protocol (6.1) is a standard approach in which species composition and/or biomass of a sampled assemblage is assessed in the laboratory. The second protocol (6.2) is a field-based rapid survey of periphyton biomass and coarse-level taxonomic composition (e.g., diatoms, filamentous greens, blue-green algae) and requires little taxonomic expertise. The two protocols can be used together. The first protocol has the advantage of providing much more accuracy in assessing biotic integrity and in diagnosing causes of impairment than the second protocol, but it requires more effort than the second protocol. Additionally, the first protocol provides the option of sampling the natural substrate of the stream or placing artificial substrates for colonization.

Periphyton samples should be collected during periods of stable stream flow. High flows can scour the stream bed, flushing the periphyton downstream. Recolonization of substrates will be faster after less severe floods and in streams with nutrient enrichment. Peterson and Stevenson (1990) recommend a three-week delay following high, bottom-scouring stream flows to allow for recolonization and succession to a mature periphyton community. However, recovery after high discharge can be as rapid as 7 days if severe scouring of substrata did not occur (Stevenson 1990).

Two sampling approaches are described for natural substrate sampling. Multihabitat sampling best characterizes the benthic algae in the reach, but results may not be sensitive to subtle water quality changes because of habitat variability between reaches. Species composition of assemblages from a single habitat should reflect water quality differences among streams more precisely than multi-habitat sampling, but impacts in other habitats in the reach may be missed.

The length of stream sampled depends upon the objectives of the project, budget, and expected results. Multihabitat sampling should be conducted at the reach scale (30-40 stream widths) to ensure sampling the diversity of habitats that occur in the stream. Ideally, single habitat sampling should also be conducted at the reach scale. A shorter length of stream can probably be sampled for single habitat samples than multihabitat samples because the chosen single habitat (e.g., riffles) is usually common within the study streams.

The following procedures for multihabitat sampling of algae have been adapted from the Kentucky and Montana protocols (Kentucky DEP 1993, Bahls 1993). These procedures are recommended when subsequent laboratory assessments of species composition of algal assemblages will be performed.

Table 6-1. Summary of collection techniques for periphyton from wadeable streams (adapted from Kentucky DEP 1993, Bahls 1993).

Variability due to differences in habitat between streams may be reduced by collecting periphyton from a single substrate/habitat combination that characterizes the study reach (Rosen 1995). For comparability of results, the same substrate/habitat combination should be sampled in all reference and test streams. Single habitat sampling should be used when biomass of periphyton will be assessed.

Most monitoring groups prefer sampling natural substrates whenever possible to reduce field time and improve ecological applicability of information. However, periphyton can also be sampled by collecting from artificial substrates that are placed in aquatic habitats and colonized over a period of time. This procedure is particularly useful in non-wadeable streams, rivers with no riffle areas, wetlands, or the littoral zones of lentic habitats. Both natural and artificial substrates are useful in monitoring and assessing waterbody conditions, and have corresponding advantages and disadvantages (Stevenson and Lowe 1986, Aloi 1990). The methods summarized here are a composite of those specified by Kentucky (Kentucky DEP 1993), Florida (Florida DEP 1996), and Oklahoma (Oklahoma CC 1993). Although glass microslides are preferred, a variety of artificial substrates have been used with success (see #2 below and textbox entitled "Field Equipment/Supplies Needed for Periphyton Sampling--Artificial Substrates").

The Methods summarized here are a modified version of those used by Kentucky ( Kentucky DEP 1993), Florida ( Florida DEP 1996), and Montana ( Bahls 1993). For more detail or for alternative methods, see Standard Methods for the Examination of Water and Wastewater ( APHA 1995).

Many algae are readily identifiable to species level by trained personnel who have a good library of literature on algal taxonomy (see section 6.3). All algae can not be identified to species because: the growth forms of some algal species are morphologically indistinguishable with the light microscope (e.g., zoospores of many green algae); the species has not been described previously; or the species is not in the laboratory's literature. Consistency in identifications within a laboratory and program is very important, because most bioassessment are based on contrasts between reference and test sites. Accuracy of identifications becomes most important when using autecological information from other studies. Quality assurance techniques are designed to ensure "internal consistency" and also improve comparisons with information in other algal assessment and monitoring programs.

Palmer counting cells are excellent for identifying and counting soft-algae in most species assemblages. When samples have many very small blue-green algae or a few, relatively important large cells, other slide preparation techniques may be useful to increase magnification and sample size, respectively. Because accurate diatom identification is not possible in Palmer cells, we have recommended counting cleaned diatoms in special mounts. However, if the taxonomy of algae in samples is well known, preparation and counting time can be reduced by mounting algae in syrup. In syrup, both soft algae and diatoms can be identified, but resolution of morphological details of diatoms is not as great as in mounts of diatoms in resins (e.g., Naphrax®).

Assemblages with many small cells: We recommend a simple wet mount procedure when samples contain many small algae so samples can be observed at 1000X. A small volume of water under the coverglass prevents movement of cells when adjusting focus and using oil immersion. These preparations usually last several days if properly sealed (see below).

Wet mounts:

Assemblages with a few large cells: Sedgewick-Rafter counting chambers, which are large modified microscope slides with 1.0 mL wells, increase sample size. Counts in Sedgewick-Rafter counting cells should be done after counts in Palmer cells or wet mounts so that the relation between sample proportions with the two methods can be determined. While keeping track of the proportion of sample observed, identify and count large algae in transects at 200X or 100X magnification in the counting cell.

Syrup mounts:

Note: Preserve color of chloroplasts by keeping samples in dark.

Special Note: If slides get too warm in storage, syrup will loose viscosity and become runny. Algae and medium may then escape containment under coverglass. Store slides in a horizontal position.

The periphyton metrics presented here are used by several states and environmental assessment programs throughout the US and Europe (e.g., Kentucky DEP 1993, Bahls 1993, FL DEP 1996, Whitton et al. 1991, Whitton and Kelly 1995). Each of these metrics should be tested for response to human alterations of streams in the region in which they are used (see Chapter 9, Biological Data Analysis). In many cases, diatom and soft algal metrics have been determined separately because changes in small abundant cyanobacteria (blue-green algae) can numerically overwhelm metrics based on relative abundance and because green algae with large cells (e.g., Cladophora) may not have appropriate weight. However, attempts should be made to integrate diatoms and soft algae in as many metrics as possible, especially in cases such as species and generic richness when great variability in relative abundance is not an issue.

Many metrics can be calculated based on presence/absence data or on relative abundances of taxa. For example, percent Pollution Tolerant Diatoms can be calculated as the sum of relative abundances of pollution tolerant taxa in an assemblage or as the number of species that are tolerant to pollution in an assemblage. Percent community similarity can be calculated as presented below, which quantifies the percent of organisms in two assemblages that are the same. Alternatively, it can be calculated as the percent of species that are the same by making all relative abundances greater than 0 equal to 1. The following metrics can also be calculated with presence/absence data instead of species relative abundances: % sensitive taxa, % motile taxa, % acidobiontic, % alkalibiontic, % halobiontic, % saprobiontic, % eutrophic, simple autecological indices, and change in inferred ecological conditions. Although we may find that metrics based on species relative abundances are more sensitive to environmental change, metrics based on presence/absence data may be more appropriate when developing metrics with multihabitat samples and proportional sampling of habitats is difficult. In the latter case, presence/absence of species should remain the same, even if relative abundance of taxa differs with biases in multihabitat sampling.

The metrics have been divided into two groups which may be helpful in developing an Index of Biotic Integrity (IBI). Metrics in the first group are less diagnostic than the second group of metrics. Metrics in the first group (species and generic richness, Shannon diversity, etc.) generally characterize biotic integrity ("natural balance in flora and fauna." as in Karr and Dudley 1981) without specifically diagnosing ecological conditions and causes of impairment. The second group of metrics more specifically diagnoses causes of impaired biotic integrity. Metrics from both groups could be included in an IBI to make a hierarchically diagnostic IBI. Alternatively, an IBI could be constructed from only metrics of biotic integrity so that inference of biotic integrity and diagnosis of impairment are independent ( Stevenson and Pan 1999).

Autecological information about many algal species and genera has been reported in the literature. This information comes in several forms. In some cases, qualitative descriptions of the ecological conditions in which species were observed were reported in early studies of diatoms. Following the development of the saprobic index by Kolkwitz and Marsson 1908, several categorical classification systems (e.g., halobian spectrum, pH spectrum) were developed to describe the ecological preferences and tolerances of species (see Lowe 1974 for a review). Most recently, the ecological optima and tolerances of species for specific environmental conditions have been quantified by using weighted average regression approaches (see ter Braak and van Dam 1989 for a review). We have compiled a list of references for this information in Section 6.4. These references will be valuable for developing many of the metrics below.

Metrics of Biotic Integrity

Diagnostic Metrics that Infer Ecological Conditions

The ecological preferences of many diatoms and other algae have been recorded in the literature. Using relative abundances of algal species in the sample and their preferences for specific habitat conditions, metrics can be calculated to indicate the environment stressors in a habitat. These metrics can more specifically infer environmental stressors than the general pollution tolerance index.

Table 6-2. Environmental definitions of autecological classification systems for algae (as modified or referenced by Lowe 1974). Definitions for classes are given if no subclass is indicated.

Measurement of periphyton biomass is common in many studies and may be especially important in studies that address nutrient enrichment or toxicity. In many cases, however, sampling benthic algae misses peak biomass, which may best indicate nutrient problems and potential for nuisance algal growths (Biggs 1996, Stevenson 1996).

Biomass measurements can be made with samples collected from natural or artificial substrates. To quantify algal biomass (chl a, ash-free dry mass, cell density, biovolume cm^-2), the area of the substrate sampled must be determined. Two national stream assessment programs sample and assess area-specific cell density and biovolume (USGS-NAWQA, ; and EMAP, Klemm and Lazorchak 1994). These programs estimate algal biomass in habitats and reaches by collecting composite samples separately from riffle and pool habitats.

Periphyton biomass can be estimated with chl a, ash-free dry mass (AFDM), cell densities, and biovolume, usually per cm² (Stevenson 1996). Each of these measures estimates a different component of periphyton biomass (see Stevenson 1996 for discussion).

Chlorophyll a ranges from 0.5 to 2% of total algal biomass (APHA 1995), and this ratio varies with taxonomy, light, and nutrients. A detailed description of chlorophyll a analysis is beyond the scope of this chapter. Standard methods (APHA 1995, USEPA 1992) are readily available. The analysis is relatively simple and involves:

Ash-free dry mass is a measurement of the organic matter in samples, and includes biomass of bacteria, fungi, small fauna, and detritus in samples. A detailed description of analysis is beyond the scope of this chapter, but standard methods (APHA 1995, USEPA 1995a) are readily available. The analysis is relatively simple and measures the difference in mass of a sample after drying and after incinerating organic matter in the sample. We recommend using AFDM versus dry mass to measure periphyton biomass because silt can account for a substantial proportion of dry mass in some samples. Ash mass in samples can be used to infer the amount of silt or other inorganic matter in samples.

Cell densities (cells cm^-2) are determined by dividing the numbers of cells counted by the proportion of sample counted and the area from which samples were collected. Cell biovolumes (mm³ biovolume cm^-2) are determined by summing the products of cell density and biovolume of each species counted (see Lowe and Pan 1996) and dividing that sum by the proportion of sample counted and the area from which samples were collected.

High algal biomass can indicate eutrophication, but high algal biomass can also accumulate in less productive habitats after long periods of stable flow. Low algal biomass may be due to toxic conditions, but could be due to a recent storm event and spate or naturally heavy grazing. Thus, interpretation of biomass results is ambiguous and is the reason that major emphasis has not been placed on quantifying algal biomass for RBP. However, nuisance levels of algal biomass (e.g., > 10 µg chl a cm^-2, > 5 mg AFDM cm^-2, > 40% cover by macroalgae; see review by Biggs 1996) do indicate nutrient or organic enrichment. If repeated measurements of biomass can be made, then the mean and maximum benthic chl a could be used to define trophic status of streams. Dodds et al. (1998) have proposed guidelines in which the oligotrophic-mesotrophic boundary is a mean benthic chl a of 2 µg cm^-2 or a maximum benthic chl a of 7 µg cm^-2and the mesotrophic-eutrophic boundary is a mean of 6 µg chl a cm^-2 and a maximum of 20 µg chl a cm^-2.

Semi-quantitative assessments of benthic algal biomass and taxonomic composition can be made rapidly with a viewing bucket marked with a grid and a biomass scoring system. The advantage of using this technique is that it enables rapid assessment of algal biomass over larger spatial scales than substrate sampling and laboratory analysis. Coarse-level taxonomic characterization of communities is also possible with this technique. This technique is a survey of the natural substrate and requires no laboratory processing, but hand picked samples can be returned to the laboratory to quickly verify identification. It is a technique developed by Stevenson and Rier⁽²⁾.

A great wealth of taxonomic literature is available for algae. Below is a subset of that literature. It is a list of taxonomic references that are useful for most of the United States and are either in English, are important because no English treatment of the group is adequate, or are valuable for the good illustrations.

Camburn, K.E., R.L. Lowe, and D.L. Stoneburner. 1978. The haptobenthic diatom flora of Long Branch Creek, South Carolina. Nova Hedwigia 30:149-279.

Collins, G.B. and R.G. Kalinsky. 1977. Studies on Ohio diatoms: I. Diatoms of the Scioto River Basin. Bull. Ohio Biological Survey. 5(3):1-45.

Cox, E. J. 1996. Identification of freshwater diatoms from live material. Chapman & Hall, London.

Czarnecki, D.B. and D.W. Blinn. 1978. Diatoms of the Colorado River in Grand Canyon National Park and vicinity. (Diatoms of Southwestern USA II). Bibliotheca Phycologia 38. J. Cramer. 181 pp.

Dawes, C. J. 1974. Marine Algae of the West Coast of Florida. University of Miami Press.

Dillard, G.E. 1989a. Freshwater algae of the Southeastern United States. Part 1. Chlorophyceae: Volvocales, Testrasporales, and Chlorococcales. Bibliotheca, 81.

Dillard, G.E. 1989b. Freshwater algae of the Southeastern United States. Part 2. Chlorophyceae: Ulotrichales, Microsporales, Cylindrocapsales, Sphaeropleales, Chaetophorales, Cladophorales, Schizogoniales, Siphonales, and Oedogoniales. Bibliotheca Phycologica, 83.

Dillard, G.E. 1990. Freshwater algae of the Southeastern United States. Part 3. Chlorophyceae: Zygnematales: Zygenmataceae, Mesotaeniaceae, and Desmidaceae (Section 1). Bibliotheca Phycologica, 85.

Dillard, G.E. 1991. Freshwater algae of the Southeastern United States. Part 4. Chlorophyceae: Zygnemateles: Desmidaceae (Section 2). Bibliotheca Phycologica, 89.

Drouet, F. 1968. Revision of the classification of the oscillatoriaceae. Monograph 15. Academy of Natural Sciences, Philadelphia. Fulton Press, Lancaster, Pennsylvania.

Hohn, M.H. and J. Hellerman. 1963. The taxonomy and structure of diatom populations from three North American rivers using three sampling methods. Transaction of the American Microscopal Society 82:250-329.

Hustedt, F. 1927-1966. Die kieselalgen In Rabenhorst's Kryptogamen-flora von Deutschland Osterreich und der Schweiz VII. Leipzig, West Germany.

Hustedt, F. 1930. Bacillariophyta (Diatomae). In Pascher, A. (ed). Die suswasser Flora Mitteleuropas. (The freshwater flora of middle Europe). Gustav Fischer Verlag, Jena, Germany.

Jarrett, G.L. and J.M. King. 1989. The diatom flora (Bacillariphyceae) of Lake Barkley. U.S. Army Corps of Engineers, Nashville Dist. #DACW62-84-C-0085.

Krammer, K. and H. Lange-Bertalot. 1986-1991. Susswasserflora von Mitteleuropa. Band 2. Parts 1-4. Bacillariophyceae. Gustav Fischer Verlag. Stuttgart. New York.

Lange-Bertalot, H. and R. Simonsen. 1978. A taxonomic revision of the Nitzschia lanceolatae Grunow: 2. European and related extra-European freshwater and brackish water taxa. Bacillaria 1:11-111.

Lange-Bertalot, H. 1980. New species, combinations and synonyms in the genus Nitzschia. Bacillaria 3:41-77.

Patrick, R. and C.W. Reimer. 1966. The diatoms of the United States, exclusive of Alaska and Hawaii. Monograph No. 13. Academy of Natural Sciences, Philadelphia, Pennsylvania.

Patrick, R. and C.W. Reimer. 1975. The Diatoms of the United States. Vol. 2, Part 1. Monograph No. 13. Academy of Natural Sciences, Philadelphia, Pennsylvania.

Prescott, G.W. 1962. The algae of the Western Great Lakes area. Wm. C. Brown Co., Dubuque, Iowa.

Prescott, G.W., H.T. Croasdale, and W.C. Vinyard. 1975. A Synopsis of North American desmids. Part II. Desmidaceae: Placodermae. Section 1. Univ. Nebraska Press, Lincoln, Nebraska.

Prescott, G.W., H.T. Croasdale, and W.C. Vinyard. 1977. A synopsis of North American desmids. Part II. Desmidaceae: Placodermae. Section 2. Univ. Nebraska Press, Lincoln, Nebraska.

Prescott, G.W., H.T. Croasdale, and W.C. Vinyard. 1981. A synopsis of North American desmids. Part II. Desmidaceae: Placodermae. Section 3. Univ. Nebraska Press, Lincoln, Nebraska.

Prescott, G.W. 1978. How to know the freshwater algae. 3rd Edition. Wm. C. Brown Co., Dubuque, Iowa.

Simonsen, R. 1987. Atlas and catalogue of the diatom types of Friedrich Hustedt. Vol. 1-3. J. Cramer. Berlin, Germany.

Smith, M. 1950. The Freshwater Algae of the United States. McGraw-Hill, New York, New York.

Taylor, W. R. 1960. Marine algae of the eastern tropical and subtropical coasts of the Americas. University of Michigan Press, Ann Arbor, Michigan.

VanLandingham, S. L. 1982. Guide to the identification, environmental requirements and pollution tolerance of freshwater blue-green algae (Cyanophyta). EPA-600/3-82-073.

Whitford, L.A. and G.J. Schumacher. 1973. A manual of freshwater algae. Sparks Press, Raleigh, North Carolina.

Wujek, D.E. and R.F. Rupp. 1980. Diatoms of the Tittabawassee River, Michigan. Bibliotheca Phycologia 50:1-100.

Beaver, J. 1981. Apparent ecological characteristics of some common freshwater diatoms. Ontario Ministry of the Environment. Rexdale, Ontario, Canada.

Cholnoky, B. J. 1968. Ökologie der Diatomeen in Binnegewässern. Cramer, Lehre.

Fabri, R. and L. Leclercq. 1984. Etude écologique des riviéres du nord du massif Ardennais (Belgique): flore et végétation de diatomeées et physico-chimie des eaux. 1. Station scientifique des Hautes Fagnes, Robertville. 379 pp.

Fjerdingstad, E. 1950. The microflora of the River Molleaa with special reference to the relation of benthic algae to pollution. Folia Limnologica Scandanavica 5, 1-123.

Hustedt, F. 1938-39. Systamatische und ökologische Untersuchungen über die Diatomeen-Flora von Java, Bali und Sumatra nach dem Material deter Deutschen Limnologischen Sunda-Expedition. Allgemeiner Teil. I. Ubersicht über das Untersuchungsmaterial und Charakterisktik der Diatomeenflora der einzelnen Gebiete. II. Die Diatomeen flora der untersuchten Gesässertypen. III. Die ökologische Faktoren und ihr Einfluss auf die Diatomeenflora. Archiv für Hydrobiologie, Supplement Band, 15:638-790 (1938); 16:1-155 (1938); 16:274-394 (1939).

Hustedt, F. 1957. Die Diatomeenflora des Flusssystems der Weser im Gebiet der Hansestadt Bremen. Abhandlungen naturwissenschaftlichen. Verein zu Bremen, Bd. 34, Heft 3, S. 181-440, 1 Taf.

Lange-Bertalot, H. 1978. Diatomeen-Differentialarten anstelle von Leitformen: ein geeigneteres Kriterium der Gewässerbelastung. Archiv für Hydrobiologie Supplement 51, 393-427.

Lange-Bertalot, H. 1979. Pollution tolerance of diatoms as a criterion for water quality estimation. Nova Hedwigia 64, 285-304.

LeCointe C., M. Coste, and J. Prygiel. 1993. "OMNIDIA" software for taxonomy, calculation of diatom indices and inventories management. Hydrobiologia 269/270: 509-513.

Lowe, R. L. 1974. Environmental Requirements and Pollution Tolerance of Freshwater Diatoms. US Environmental Protection Agency, EPA-670/4-74-005. Cincinnati, Ohio, USA.

Palmer, C. M. 1969. A composite rating of algae tolerating organic pollution. Journal of Phycology 5, 78-82.

Rott, E., G. Hofmann, K. Pall, P. Pfister, and E. Pipp. 1997. Indikationslisten für Aufwuchsalgen in österreichischen Fliessgewässern. Teil 1: Saprobielle Indikation. Wasserwirtschaftskataster. Bundesminsterium für Land- und Forstwirtschaft. Stubenring 1, 1010 Wein, Austria.

Slàdecek, V. 1973. System of water quality from the biological point of view. Archiv für Hydrobiologie und Ergebnisse Limnologie 7, 1-218.

Van Dam, H., Mertenes, A., and Sinkeldam, J. 1994. A coded checklist and ecological indicator values of freshwater diatoms from the Netherlands. Netherlands Journal of Aquatic Ecology 28, 117-33.

Vanlandingham, S. L. 1982. Guide to the identification, environmental requirement and pollution tolerance of freshwater blue-green algae (Cyanophyta). U. S. Environmental Protection Agency. EPA-600/3-82-073.

Watanabe, T., Asai, K., Houki, A. Tanaka, S., and Hizuka, T. 1986. Saprophilous and eurysaprobic diatom taxa to organic water pollution and diatom assemblage index (DAIpo). Diatom 2:23-73.

1. David Beeson is a phycologist with Schafer & Associates, Inc.
2. S.T. Rier is a graduate student at the University of Louisville.