Biological field and laboratory methods for measuring the quality of ...

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Water quality may vary greatly with time and location in lakes, impoundments and streams. If meaningful data are to be obtained, therefore, the sampling program must take these variations into account. 2.3 Apparatus and Test Conditions 2.3.1 Glassware Use good-quality borosilicate glassware. When studing trace nutrients, use special glassware such as Vycor or polycarbonate containers. Although container size is not critical, the surface to volume ratios are critical because of possible carbon limitation. The recommended sample volumes for use in Erlenmeyer flasks are: 40 ml in a 125 ml flask; 60 ml in a 250 ml flask; and 100 ml in a 500 ml flask. Use culture closures such as loose-fitting aluminum foil or inverted beakers to permit good gas exchange and prevent contamination. 2.3.2 Illuflnination After inoculation, incubate the flasks at 24 ± 2°C under cool-white fluorescent lighting: 200 ft-c (2152 lux) ± 10 percent for blue-green algae and diatom test species, and 400 ft-c (4304 lux) ± 10 percent for green algae test species. Measure the light intensity adjacent to the flask at the liquid level. 2.3.3 pH To ensure the availability of carbon dioxide, maintain the pH of the incubating cultures below 8.5 by using the sample volumes mentioned above and shaking the cultures at 100 oscillations per minute. In samples containing high concentrations of nutrients, such as highlyproductive surface waters or domestic waste effluents, it may be necessary to bubble air or an air/carbon dioxide mixture through the culture to maintain the pH below 8.5. 2.4 Sample Preparation Two alternate methods of sample preparation are recommended, depending upon the type of information to be obtained from the sample: • membrane filtration (0.45 pore diameter) remove the indigenous algae by filtration if 3 Prepare test flasks in triplicate. ALGAL ASSAY you wish to determine the growth response to growth-limiting nutrients which have not been taken up by filterable organisms, or if you wish to predict the effect of adding nutrients to a test water at a specific time. • autoclaving -- autoclave samples if you wish to determine the amount of algal biomass that can be grown from all nutrients in the water, including those in the plankton. Autoclaving solubilizes the nutrients in the indigenous filterable organisms and releases them for use by the test organisms. 2.5 Inoculum The algal test species may be one of those recommended in the Bottle Test or another that has been obtained in unialgal culture. Grow the test species in a culture medium that minimizes the intracellular carryover of nutrients in the test species when transferred from the stock culture to the test water (Table I.) When taken from the stock culture, centrifuge the test cells and discard the supernatant. Resuspend the sedimented cells in an appropriate volume of glass-distilled water containing 15 mg sodium bicarbonate per liter and recentrifuge. Decant the supernatant, resuspend the algae in fresh bicarbonate solution, and use as the inoculum. The amount of inoculum depends upon the algal test species used. The following initial cell concentrations are recommended: Test organism Selenastrum capricornutum Anabaena [los-aquae Microcystis aeruginosa lnitial cell count!ml IOOO/rol SOOOO/ml SOOOO/rol 2.6 Growth Response Measurements The method used to determine growth response during incubation depends on the equipment available. Cells may be counted with a microscope, using a hemacytometer or a Palmer-Maloney or Sedgwick-Rafter plankton counting chamber. The amount of algal biomass may be determined by measuring the optical density of the culture at 600 -750 nm with a colorimeter or spectrophotometer. The amount of chlorophyll contained in the algae may be
BIOLOGICAL METHODS TABLE 1. STOCK CULTURE AND CONTROL NUTRIENT MEDIUM MACROELEMENTS : Final Element Element Compound concentration furnished concentration (mg/!) (mgil) NaN03 25.500 N 4.200 Kz HP04 1.044 P 0.186 K 0.468 MgClz 5.700 Mg 1.456 MgS04'7HzO 14.700 Mg 1.450 S 1.911 CaClz'2Hz° 4.410 Ca 1.203 NaHC03 15.000 Na 11.004 (If the medium is to be filtered, add the following trace-element-iron-EDTA solution from a single combination stock solution after filtration, With no filtration, K zHP04 should be added last to avoid iron precipitation. Stock solutions of individual salts may be made up in 1000 x's final concentration or less.) H3B03 MnClz ZnClz CoClz CuCiz Naz Mo0 4'2HzO FeCl3 NazEDTA'2H z O measured either directly (in vivo) by fluorometry or after extraction by fluorometry or spectrophotometry. If available, an electronic particle counter will provide an accurate and rapid count of the cells. All methods used for determining the algal biomass should be related to a dry weight measurement (mg/l) determined gravimetrically. (See the Plankton Section of the manual for analytical details.) 2.7 Data Evaluation Two parameters are used to describe the growth of a test alga: maximum specific growth rate and maximum standing crop. The maximum specific growth rate (J..Lmax) for an individual flask is the largest specific growth rate (J..L) occurring at any time during incubation. The J..Lmax for a set of replicates is determined by MICROELEMENTS: (jJ.g/l) 185.64 B 264.27 Mn 32.70 Zn 0.78 Co 0.009 Cu 7.26 Mo 96 333 Fe 4 (jJ.g/l) 33 114 15 0.35 0.003 2.88 33 averaging the J..Lmax of the individual flasks. The specific growth rate,J..L,is defined by: where: In = log to the base "e" X2 = biomass concentration at the end of the selected time interval Xl =biomass concentration at the beginning of the selected time interval t 2 - t I =elapsed time (days) between selected determinations of biomass Because the maximum specific growth rate (J..Lmax) occurs during the logarithmic phase of growth (usually between day 3 and day 5), the biomass must be measured at least daily during the first 5 days of incubation.
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... EPA-610/4-13 -001 July 1973 BIO
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• FOREWORD Man and his environmen
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Name Anderson, Max Arthur, John W.
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The role of aquatic biology in the
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FOREWORD 1 PREFACE CONTENTS BIOLOGI
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BIOMETRICS 1.0 INTRODUCTION 1.1 Ter
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BIOLOGICAL METHODS sample or a plan
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TABLE 1. RAW DATA ON PLANKTON COUNT
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BIOLOGICAL METHODS y POSITIVE INTER
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PLANKTON 1.0 INTRODUCTION . 2.0 SAM
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sampling is usually sufficient. In
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Several sampling methods can be use
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100X. When combined with the ocular
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float, examine the underside of the
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of counts to numbers per ml is quit
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pipet with distilled water into a c
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BIOLOGICAL METHODS Calculate the ch
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PLANKTON REFERENCES Holmes, R. W. 1
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PERIPHYIOI
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BIOLOGICAL METHODS Diatom Species P
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BIOLOGICAL METHODS Patrick, R. 1957
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MACROPHYTON 1.0 INTRODUCTION . 2.0
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3.0 REFERENCES MACROPHYTON Blackbur
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I '" MACROINVERTEBRATES 1.0 INTRODU
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1.0 INTRODUCTION The aquatic macroi
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the limitations of available sampli
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Because of the extreme spatial and
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predetermined depth. Grabs with spr
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BIOLOGICAL METHODS Because of the s
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BIOLOGICAL METHODS fauna and hydrol
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Page 107 and 108: TABLE 7. (Continued) MACROINVERTEBR
Page 109 and 110: MACROlNVERTEBRATE REFERENCES 33. Ll
Page 111 and 112: MACROINVERTEBRATE REFERENCES Hauber
Page 113 and 114: MACROINVERTEBRATE REFERENCES Robert
Page 115 and 116: FISH
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Page 137 and 138: BIOASSAY 1.0 GENERAL CONSIDERATIONS
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Page 157: Appendix A Test (Evansville, Indian
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Page 177 and 178: Item Source Cat. No. Unit Approx. C
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To Minims _ Liquid Ounces _ Gills _
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