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BIOLOGICAL METHODS As a rule of thumb, concentrate samples when phytoplankton densities are below 500 per ml; approximately 6 liters of sample are required at that cell concentration. Generally, 1 liter is an adequate routine sample volume. The following three methods may be used for concentrating preserved phytoplankton, but sedimentation is preferred. 3.1.1 Sedimentation Preserved phytoplankton samples can often be settled in the original storage containers. Settling time is directly related to the depth of the sample in the bottle or settling tube. On the average, allow 4 hours per 10 mm of depth. After settling, siphon off the supernatant (Figure 1) or decant through a side drain. The use of a detergent aids in settling. Exercise caution because of the different sedimentation rates of the diverse sizes and shapes of phytoplankton. 3.1.2 Cen trifugation During centrifugation, some of the more fragile forms may be destroyed or flagella may become detached. In using plankton centrifuges, many of the cells may be lost; modern continuous-flow centrifuges avoid this. 3.1.3 Filtration To concentrate samples by filtration, pass through a membrane filter. A special filter apparatus and a vacuum source are required. Samples containing large amounts of suspended rnaterial (other than phytoplankton) are difficult to enumerate by this method, because the suspended matter tends to crush the phytoplankters or obscure them from view. The vacuum should not exceed 0.5 atmospheres. Concentration by filtration is particularly useful for samples low in plankton and silt content. 3.2 Zooplankton The zooplankton in grab samples are concentrated prior to counting by allowing them to settle for 24 hours in laboratory cylinders of appropriate size or in specially constructed settling tubes (Figure 1). 6 50.8 eM Figure 1. Plexiglas plankton settling tube with side drain and detachable cup. Not drawn to scale. Take care to recover organisms (especially the Oadocera) that cling to the surface of the water in the settling tube. 4.0 SAMPLE ANALYSIS 4.1 Phytoplankton 4.1.1 Qualitative analysis ofphytoplankton The optical equipment needed includes a good quality compound binocular microscope with a mechanical stage. For high magnification, a substage condenser is required. The ocular lens should be 8X to 12X. Binocular eyepieces are generally preferred over a monocular eyepiece because of reduced fatigue. Four turret-mounted objective lenses should be provided with magnifications of approximately 10, 20, 45, and
100X. When combined with the oculars, the following characteristics are approximately correct. Maximum working Objective Ocular Subject distance between Depth of lens lens magnification objective and focus, J1 cover slip, mm lOX lOX lOOX 7 8 20X lOX 200X 1.3 2 45X lOOX lOX lOX 450X 1000X 0.5-0.7 0.2 1 0.4 I An initial examination is needed because most phytoplankton samples will contain a diverse assemblage of organisms. Carry out the identification to species whenever possible. Because the size range of the individual organisms may extend over several orders of magnitude, no single magnification is completely satisfactory for identification. For the initial examination, place one or two drops of a concentrate on a glass slide and cover with a No.1 or No. 1-1/2 cover slip. Use the lOX objective to examine the entire area under the cover slip and record all identifiable organisms. Then examine with the 20 and 45X objectives. Some very small organisms may require the use of the 100X objective (oil immersion) for identification. The initial examination helps to obtain an estimate of population density and may indicate the need for subsequent dilution or concentration of the sample, to recognize characteristics of small forms not obvious during the routine counting procedure, and to decide if more than one type of counting procedure must be used. When identifying phytoplankton, it is useful to examine fresh, unpreserved samples. Preservation may cause some forms to become distorted, lose flagella, or be lost together. These can be determined by a comparison between fresh and preserved samples. As the sample is examined under the microscope, identify the phytoplankton and tally under the following categories: coccoid bluegreen, filamentous blue-green, coccoid green, filamentous green, green flagellates, other pigmented flagellates, centric diatoms, and pennate diatoms. In tallying diatoms, distinguish be- 7 PLANKTON COUNTING tween "live" cells, Le., those that contain any part of a protoplast, and empty frustules or shells. The availability of taxonomic bench references and the skill of the biologist will govern the sophistication of identification efforts. No single reference is completely adequate for all phytoplankton. Some general references that should be available are listed below. Those marked with an asterisk are considered essential. American Public Health ASSOCIatIOn, 1971. Standard methods for the examination of water and wastewater. 13th editIOn. Washington, D.C. Bourrelly, P. 1966-1968. Les algues d'eau douce. 1966. Tome I-III, Boubee & Cie, Paris. Fott, B. 1959. Algenkunde. Gustav Fischer, Jena. (2nd reVIsed edition, 1970.) Prescott, G. W. 1954. How to know the fresh-water algae. W. C. Brown Company, Dubuque. (2nd editIon, 1964.) *Prescott, G. W. 1962. Algae of the Western Great Lakes Area. (2nd edition), W. C. Brown, Dubuque. *Smith, G. M. 1950. The freshwater algae of the Umted States. (2nd edition), McGraw-Hill Book Co., New York. Ward, H. B., and G. C. Whipple. 1965. Fresh-water bIOlogy. 2nd edition edIted by W. T. Edmonson. John WIley and Sons, New York. *Weber, C. 1. 1966. A gUIde to the common diatoms at water pollution surveillance system stations. USDl, FWPCA, Cin· CInnati. West, G. S., and F. E. Fritsch. 1927. A treatise on the British freshwater algae. Cambridge Univ. Press. (Reprinted 1967; 1. Cramer, Lehre; Wheldon & Wesley, Ltd.; and Stecherthafner, Inc., New York.) Specialized references that may be required for exact identification within certain taxonomic groups include: Brant, K., and C. Apstem. 1964. Nordisches Plankton. A. Asher & Co., Amsterdam. (Reprint of the 1908 publicatIOn publIshed by Verlag von Lipsius & Tischer, KIeI and Leipzig.) Cleve-Euler, A. 1968. Die diatomeen von Schweden und Finn· land, I-V. Bibliotheca Phycologica, Band 5, J. Cramer, Lehre, Germany. Cupp, E. 1943. Marine plankton diatoms of the west coast of North America. Bull. Scripps lnst. Oceanogr., Univ. Calif., 5:1-238. Curl, H 1959. The phytoplankton of Apalachee Bay and the Northwestern Gulf of Mexico. Univ. Texas lnst. Marine Sci., Vol. 6, 277-320. *Drouet, F. 1968. RevIsion of the claSSIficatIon of the Oscillatoriaceae. Acad. Natural Sci., Philadelphia. *Drouet, F., and W. A. Daily. 1956. Revision of the coccoid Myxophyceae. Butler Univ. Bot. Stud. XII., Indianapolis. Fott, B. 1969. Studies in phycology. E. Schweizerbart'sche Verlagsbuchhandlung (Nagele u. Obermiller), Stuttgart, Germany.
Page 1 and 2: ... EPA-610/4-13 -001 July 1973 BIO
Page 3 and 4: • FOREWORD Man and his environmen
Page 5 and 6: Name Anderson, Max Arthur, John W.
Page 7 and 8: The role of aquatic biology in the
Page 9 and 10: FOREWORD 1 PREFACE CONTENTS BIOLOGI
Page 11 and 12: BIOMETRICS 1.0 INTRODUCTION 1.1 Ter
Page 13 and 14: BIOLOGICAL METHODS sample or a plan
Page 18: TABLE 1. RAW DATA ON PLANKTON COUNT
Page 33: BIOLOGICAL METHODS y POSITIVE INTER
Page 40: PLANKTON 1.0 INTRODUCTION . 2.0 SAM
Page 43 and 44: sampling is usually sufficient. In
Page 45: Several sampling methods can be use
Page 49 and 50: float, examine the underside of the
Page 51 and 52: of counts to numbers per ml is quit
Page 53 and 54: pipet with distilled water into a c
Page 56: BIOLOGICAL METHODS Calculate the ch
Page 59 and 60: PLANKTON REFERENCES Holmes, R. W. 1
Page 61 and 62: PERIPHYIOI
Page 66 and 67: BIOLOGICAL METHODS Diatom Species P
Page 68 and 69: BIOLOGICAL METHODS Patrick, R. 1957
Page 70: MACROPHYTON 1.0 INTRODUCTION . 2.0
Page 73 and 74: 3.0 REFERENCES MACROPHYTON Blackbur
Page 75 and 76: I '" MACROINVERTEBRATES 1.0 INTRODU
Page 77 and 78: 1.0 INTRODUCTION The aquatic macroi
Page 79 and 80: the limitations of available sampli
Page 81 and 82: Because of the extreme spatial and
Page 83: predetermined depth. Grabs with spr
Page 86 and 87: BIOLOGICAL METHODS Because of the s
Page 88 and 89: BIOLOGICAL METHODS fauna and hydrol
Page 90: BIOLOGICAL METHODS technique, or co
Page 94: BIOLOGICAL METHODS Equitability "e,
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BIOLOGICAL METHODS TABLE 7. CLASSIF
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BIOLOGICAL METHODS TABLE 7. (Contin
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TABLE 7. (Continued) MACROINVERTEBR
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MACROlNVERTEBRATE REFERENCES 33. Ll
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MACROINVERTEBRATE REFERENCES Hauber
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MACROINVERTEBRATE REFERENCES Robert
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FISH
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Deepwater seInIng usually requires
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BIOLOGICAL METHODS (Lonchocarpus ni
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BIOLOGICAL METHODS Figure 4. Gill n
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BIOLOGICAL METHODS the major univer
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BIOLOGICAL METHODS 7.0 BIBLIOGRAPHY
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Freshwater: Northeast FISH REFERENC
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FISH REFERENCES U.S. Department of
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BIOASSAY 1.0 GENERAL CONSIDERATIONS
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BIOLOGICAL METHODS When making wast
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BIOLOGICAL METHODS TABLE 1. STOCK C
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BIOLOGICAL METHODS the concentratio
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BIOLOGICAL METHODS Dimick, R. E., a
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Laboratory in Newtown, Ohio. Groups
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taining some yellow pigment, coarse
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Appendix A Test (Evansville, Indian
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2.3 Food Use a good frozen trout fo
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BIOLOGICAL METHODS 3.5 Methods When
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APPENDIX
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Item Source Cat. No. Unit Approx. C
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2.3 Fish Sources of information on
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Definitions In its original concept
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To Minims _ Liquid Ounces _ Gills _
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