DOCUMENT RESUME
.., ED 159 028 - SE 024 838
AUTHOR Reber, Cornelius I., Ed.. 4 . . TITLE Biological Field and Laboratory MethoaS for Measuring
. the Quality of Surface 'Waters and Effluents. Program . Element 1BA027. NSTIT Environmental Protection Agency, Cincipnati", Ohio.
,.., REPORT NO EPA-670/4-73-001 . PUBS' DATE Jul 73 ,,.
NOTE qv, 196p.; Contains occasions small, light and broken, ." / type; Pas 3-8,of of Weight and M4Wsure missing from docuient prior to being shipped to : EDRS 4r. EDRS PRICE ar-s0.83 HC-$10.03 Plus Poste. DESCRIPTORS Biological S *ences; *Biology; Environmental Technicians; Field. Studies; *Laboratory Techniques; *Sampling; *Statistical Analysis; *Water Pollution
. Control; Zoology IDENTIFIERS Environmental Protection Agency
ABSTR*T -) This Environmental Protection Agency manual was developed to provide pollution biologigts with the most recent methods for measuring the effects of environmental contaminants on freshwater and marine organisms. The sections of this manual include: (1) Biometrics;(2) Plankton;(3) Periphyton;(4) Macrophyton; (5) Macroinvertebrates; (6) Fish; and'(7) Bioassay. Each of.these sections is divided up into chapters. The Bicmetrics section deals primarily with introductovtstatistics. The section discussing Plankton covers sample col/ection,.preservation, prepartion, analysis and biomass determination. Sample collection, preservation, aialysiS, and preparation for Periphyton and Macrophyton are covered in those respective sections. In the section on Macroinvertebrates, selection of sampling sites, sampling method and data evaluation are described. Also in this section, a, axonomibibliography for the
Aicroinvertebrate groups is.A..neIif" . In th Fish section, sample collection, preservation and an is is discussed among other things. In the final section, bioassay techniques for Phytoplankton, Periphyton; Macroinvertebrates, and Fish are described and the
Flathead Minnow and Brook Trout Chronic Tests are included. , References are given in all of theabove sections. (MR)
a r *******************vit************************************************* )* Reproductions supplied by EDRS are the best thalf can be made *
* i fr-Om the original document. * ******************************************************************* U.S. DEPARTMENT OF REALM1. EDUCATION &WELFARE --APA-670/t-73.001 NATIONAL INSTITUTE OF EDUCATION July 1913 THIS,, MENT HAS BEEN REPRO. oute CTLY AS TIECEIVE0 FROM THE r R ORGANIZATION ORIGIN- ATI TS OF VIEW OR OPINIONS STA E T NECESSARILY REPRE: SENT 0 NATIONAL INSTITUTE OF EDUCA POSITION OR POLICY
, (.1 BIOLOGIQAL FIELD AND LABORATORY METHODS
y)c:; 'FOR 'MEASURING THE QUALITY .OF SURFACE WATERS AND EFFLUENTS
Edited-by Cornelius I. Weber, Ph.D. Chief, Biological Methods Analytical Quality Control Laboratory National Environmental Research Center-Cincinnafi
Program Element 1BA021
J!,
NATIONAL ENVIRONMENTALRESEARC1fCEHTER OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTALPROTECTION AGENCY C) CINCINNATI, OHIO 45268 e\ Review Notice
. . This report' has been reviewed by the National Environmental. Research Center, Cincinnati, and, approved for publication. Mention of trade namesor commercial products does riot constitute endorsement or recommendation for use.
,e FOREWORD
Man and his environment must be protected from the adverse effects of pesticides, radiation, noise and other forms of pollution, and the uhwise management of solid waste. Efforts to protect the environment re uire a focus that recognizes the interplay bqitween the components of our hysical environmwt air, water, and land. The National Environmentalesearch centers pvide this multidisciplinary focus through programs engaged in studies on the effects of envirdiimental contaminants on man and t
bibsphere, and' . da search for w4ys to prevent contamination and to recyclevaluable . resources. This manual was within the National Environmental Research Center Cincinnati to provide pollution biologists with the most ,recOtt 'methods for measuring the effects of environmental contaminants on fresh-, water and marine organisms in field and laboratory studies which arecarried out to establish water qualiW criteria for the recognized beneficial uses of water and to.monitor surface quality.
Andrew W. Breidenbach, Ph.D. Director National Environmental Research Center, Cincinnati, Ohio
1 PREFACE
This manual was published under Research Objective AchievementPlan IBA -05AEF, "Methods for Determining Biological Parametersof all Wat rs," as part of the National Analytical MethodS.DevelopmenkResearch Pro am. The manual was prepared largely by a standing committeeof senior Agency biologists organized in 1970 to assist.the Biological Methods Branch in the selectmen of methods for, use inroutine field and laboratorwork in fresh and marine waters arising curing short-term enforcement stud es,'swater quality trend monitoring, effluent testing and research projects. The methods contained in this manual are considered by theommittee to be the best available at this time. The manual will be revisean3 new methods will be recommended as the need arises. The, Committee attempted to avoid duplicating field and labOratory methods already adequately described for Agency use in StandardMethods for the Examination Water and Wastewater, 13th edition, and frequent reference is made to this source throughout the manual. Questions and comments regarding the contents of this manual should be directed to: Cornelius I. Weber, Ph.D. Chief, Biological Methods Br rich Analytical Quality Control Laboratory National EnVironmental Research Center U.S. Environmental Protection:Agency, Chicinnati, Ohio 45268
3 V BIOLOGICAL ADVISORY COMMITTEE. January 1, 1973
CHAIRMAN:.Corif(eliusI. Weber,' Ph. D.
Name Program Ndme Program Anderson, Max Indiana Office, Region V, Evansville, Maloney, Thomas Natl. Eutropkication Research Pro- IN gram, Co iris, OR Arthur, John W. Natl. Water Quality Lab, Duluth, MN Mathews, John Region VI, Dallas, TX Bugbee, Stephen L. Region VII, Kansas City, MO Murray, Thomas Office of ti & Water Programs, De Ben, Wally Natl?(toastal PollutionResearch Washingto , DC Program, Corvallis, OR Nadeau, Dr. Royal Oil Spill Research Progrart, Edison ), Duffer, Dr. William R. Natl. Water. uality Control Research NJ Program, Ada, OK Nebeker, Dr. Alan V. Western Fish Toxicology Lab, Cor- Gakstattcr, Dr. Jack H. Nat I.Eutrophication Survey Pro- vallis, OR - gram, Corvallis; OR Oldaker, %%Tien Region I, Needham Heights, MA Harkins, Dr. Ralph Region VI, Ada; OK Parrish, Ldys Region VIII, Denveri1C0 Horning,William Newtown Fish Toxicology Lab, New- Phelps, Dr. Donald K. town, OH, -'Natl. Marine Water Quall/y Labora- tory, Narraginett, RI Ischinger, Lee Field Natl. Investigations c'sa Prager, D Cincinnati, OH Natl. Marine Watr Quality Labora- r tory, Narragansett, RI .Jackson, Dr. Herbert W. Natl. Training Center, Cincinnati, OH Pest Karvelis, Ernest Natl.FieldInvestigations Center, Wheeling Office, RegionIII, Cincinnati, OH /". Wheeling, WV SLinsbur, John c Kerr, Pat Natl.Fate of Pollutants Research Region X, Seattle, WA Program, Athens, GA Tebo, Lee Region IV, Athens, GA Keup, Lowell E. Officeof Air & Water Programs, Tljpmas, Nelson A. Large Lakes Research Program, Washington, Df Grosse Jle, MI Kleveno, Conrad Region V, Chicago, IL Tunz r. Milton Region IX, Alameda, CA LaB uy , James Region III, Charlottesville; VA Wagner, Richard A. Region X, Seattle,W A Lassiter, Dr. Ray Region IV, Squtheast Water Lab, Wapher, Richard W. Natl.FieldInvestigati s-tOCen ter, Athens, GA Denver, CO
Other personnel who were former members of the Advisory-Committeeor assisted in thereparation of the manual:
Austin, R. Ted NatI.Eutrophication Survey, Corvallis, McKim, Dr. James Natl. Water Quality 14, Duluth. MN
OR 1 Tckenthun, Kenneth Office of Air and Water Programs, Boyd, Claude E. Savannah River Ecology Lab, Aiken, EPA, Washington, DC SC Mason, William T. Jr. Analytical Quality Control Lab, Collins, DS.-gerY Analytical ,Quality Control Lab, Cincinnati3OH s - Cincinnati, Oil -- 'Lewis, Philip Analytical QualityControl Lab, Garton, Dr. Ronald Western Fish Toxicology Lab, Cor- 72' Cincinnati, OH vallis, OR Schneider, Robert Nol. FieldInvestigationsCenter, Hegre, Di. Stanley Natl.Marine Water QualityLab, Denver, COti arragansett, RI Seeley, Charles Region IX, San Francisco, CA Katko, Albert Na . Eutrophication Survey, Cor- Sinclair, Ralph Natl. Training Cent6"r, Cincinnati, vallis, OR ° OH McFarland, Ben Analytical Quality Control Labora- Stephan, Charles Newtown Fish Tvicology Lab, New- tory, Cincinnati, OH town, OH
r / PERSONNEL CONTRIBUTING TO THE BIOLOGICAL METHODS MANUAL r SUBCOMMITTEES:
Biometrics Macroinvertebrates Lassiter, Dr! Ray Chairman Tebo, Lee'Chairman/. Harkins, Dr. Ralph Garton, Dr. Ronald Tebo, Lee \\(,. Lewis, Philip A. Mackenthun, Kenneth Plankton Mason, William T., Jr. Maloney, ThomasChairman Nadeau, Dr. Royal Collins, Dr. Gary Phelphs, Dr. Donald De Ben, Wally Sthneider, bert Duffer, Dr. William Sinclair, R ph Katko, Albert Fish Kerr, Pat ' McFarland, Ben LaBuy, JamesChairman Prager; Dr. Jan Karvelis, Ernest Seeley, Gliarles Preston, ROnald Warner, Richard Wagner, Richard' Periphyton-MacrophYton Bioasspy Anderson, Max Chairman Arthur, John .2-- Chairman Boyd, Dr. Claude, E. Hegrej,:Dr. Stanley Bugbee, Stephen L. IsOinger, Lee Keup, Lowell Jackson, Dr. Herbert Kleveno, Conrad ,Maloney, Thomas McKim, Dr. James Nebeker, Dr. Allan Stephan, Charles Thomas, Nelson
a4 . ..
, .1 INTRODUCTION
The role of aquatic biology in the water car, to an actualeld-study in which samples are pollutioncontrolprogramoftheU. S. collected for the' purpose of characterizing the Environmental Protection Agency includes field physical bounddries of the various habitat types. and laboratory studies carried out to establish (substrate, current, depth, etc.) and obtaining waterquality criteriafortherecognized cursory infotmation mi, the flora and fauna. , ' beneficialusesof water resources andto Although they may, bean' 'end in themselves, monitor water quality. reconnaissance surveys are generally conducted/ Field studies are employed to: measure the with a view to obtaining information adequate toxicity of specific pollutants or) effluents to io design more conibrehensive studies. They' individual species or communities of aquatic, may be quantitative or :qualitative in approach. organismsunder. naturalconditions;detect As discussed in the bioinetricS-section, quant. i ta- violations of water quality standards; evaluate tivyeconnaisiance samples are wery.useful for the trophic status of waters; and determine evaluatingtheamount of samplingeffort long-term trends in water quality. required to obtain the desired level of precision Laboratory studies are employed to: measure in more detailed studies. 0 the effects of known or potentially deleterious Synoptic surveys generally involve an attempt substances on aquatie organiSms" to estimate to determine the kinds and relative abundance "safe" concentrations; and determine environ- of organisms, present in the environment being mental requirements (such as temperature, pH, studied. This type of study may be expanded to v dissolved oxygen, etc* of the mare important include quantitative estimates of standing crop and sensiii6e .speciei of aquatic organisms. Field or production of biomass, but is generally more surveys,. and- waterqualitymonitoringare qualitativedn approach.- Systematic sampling, ifi con ducted principally by the regional which a deliberate attempt- is made to collect surveillance and arialY'sis and national enforce- specimens fromall recognizable habitats,is ment programs. Laboratory studies of water generally utilized jp synoptic surveys. Synoptic qualityrequirements,toxicity.testing,and surveys provide useful background data, are methods development are conducted principally valuableforevaltiatiflg )changes in by the national research programs. species present; Ind: prbvide useful infOrthation 1 The effects of pollutants are reflected in the r for long-term surveillance programs. 4 population,density, species composition arid * The more usual type of field studies involve diversity, physiological condition and metabolic. comparative evaluations, which may tateTiarious roes of natural aquatic communities. Methods forms including: comparisons of the flora and for field surveys and long-term water quality fauna _in different areas of the sameody of monitoring described in this manual, therefore, water, such asconventional'"dpstream- are directed primarily 'toward sample collection downstream" studies; comparisons of the flora and processing, organism identification, and the and fauna at a given location in a body of water measurement of bi6mass and Metabolic rates-. overtime,such fisisthecaseintrend GUidelines'are also provided for data evaluation monitoring; and compsons of the flora and and interpretation. . fauna in different boles of water. / There are three basic types of biolfgical field Coniparative studies frequently involve both/. studies; reconnaissance surveys, synoptic quantitative and qualitative approaches. HoW=, surveys, and comparative evaluations. Although ever, as preyiously pointed out, the choice its there is a considerable amount of overlap, each often dependent upon such factorsias available of the aboPe types has specific requirements in resources, tithe limitations, and characteristids of terms of study design. the habitat to be studied. The latter Pactoi' may :Reconnaissance surveys may range from a btluite important because the habitat/ to be brief perusal of the study area by boat, plane, or died may not by amenable to the use/Of guan-
- , ,. w 8 4 C, IS titative sampling'clevices. SAFETY A special field method that warrants abrief notation is scuba (Self ,ContainedlUnderwater The hazards associated with work on or near Breathing Apparatus). ScUba enables the biolo- water require special consideration. Personnel gist to observe, first hand, conslitions thatwther- should not be assigned to duty alone in boats, j.yvise could be described only. from sediment, and should be competent in the use of bOating chemical, physical, and biological samples taken equipment (courses are offere,d by the U. S. with various surfacg-oRerated equipment, Equip- Coast Guard): Field training should also include ment .modified fromtandard sampling e_quip- instructions on the proper rigging and handling-' ment or prefabricated; installed. and /or operatedof biological sampling gear. Life preservers (jacket type work vests) should by'ScutijA divers has proven very valuable in_, sessing the environmental conditions wheie sur- be worrat all times when on or near deep water. face. sampling gear 'Was ina'clectiate. Underwater Boats should have air-tight or fct4m-filled com- photography presents visual evidence of existing partments for flotation and be equipped "with' conditions Sand peymits the monitoriAg of long- fireextinguishers,runninglights,oars, and term changes in an aquatic environment.* anch'or. The use of inflatable plastic or rubber By utilizing Itch underwater habitatsas boats is discouraged. Tektite' and Sublimnos, biologists can observe, All boat trailers should have fivo rear running collect; and analyze sample's olvitlikout leaving the and. strip lights and turn signals and a license aquatic environment. Scuba is n very effectiN?e plate illuminator. Trailers 80 inches (wheel to toolavailabletothe aquaticbiologist, and wheel) or more wide should be equipped with methods incorporating scuba should be . con- amber marker lights on the front and rear of the sidered for usp in situations where equipment frame on both sides. operated at pie surface does not provide suffi- Laboratories should be iprovided with fire extinguishers, fume hoods, and eye fountains. cient information. ',Safety glasses should be worn when mixing T , P.E. Gehring, and C.O. Kleveno.Biological dangerous-,chemicals and preservatives. studies relatedto oxygen depletion and nutrient regeneration A copy of the;EPA Safety Manual is available processes in th Erie -Basin. Project Hypo-Canada Centre for Inland Waters, Paiier No. 6, U. S. Environmental Protection from the Office of Administration, Washington, Agency Technical Report TS05-71-208-24, Febrilary%1972. D.C.
4
or' CONTENTS
FOREWO PREFACE.t.
BIOLOGICAL ADVISORY COMTl'EE
PERSONNEL CONTRIBUTINP,TO THE BIOLOGICAL METHODS MANUAL
Its TROD
BIOMETRICS
PLANKTON
PERIPHY-i'ON.
MACROPflYTON MACROINVERTEBRATES t FISH
BIOASSAY .
AProEND.IX
J
'
BIOMETRICS
Page INTRODUCTION., :3.1 Terminology 2.0 STUDY DESIGN 1 2.1 Randomization ...... 2' 2.2 Sampl Size ...... 4 " 23 Subsampling . t . . . 6 .0 GRAPHICEXAMI,D4TION OF DATA . 6 3.1 Raw Data 6 3.2 Freiluenc:yliistograms 6 3.3 Frequency Polygon Ns 7 3:4 Cumulative Frequency'' . 7 3.5' ,Two-dimensional graphs 8 SAMPLE MAN AND VARIANCE 9 .1 Genera) Application' A 9 .2 Statistics for Stratified Random Samples .E 10 .3 Statistics for Subsamples . . . tinf- A Rounding TESTS OF. HYPOTHESES, 11
5.1 T-teSt `." . .: ...... 11 . . Chi Square Test 13 5.3 ,.F-)est 15 5.4 Analysis Of Varianc,w 15 6.0 CONFIDENCE'-INTERVALS FORMEANS AND VARIANCES 18 7.0 LINEAR REGRESSION AND,CORREIATION 19 7.13 Basic.Concepn, 19 ComPUtations 20 7.3 Tests of Hypotheses 24. 7.4 Regression-for Bivariate -Data ,. 26, s1.5 Lillear"Correlatio'n 27 BIBLIOGRAPHY 27
v.- BIOETR-ICS .41
1.0 INTRODUCTION 1.1.3Characteristics of interest Field and laboratory studips shoutld be weli- In any experiment of sampling study, many planned in advance to assure the collection of types of observations or measurements could be unbiased and precise data which are technically made. Usually, however, there are few types of defensible and amenable to statistical evaluation. measurements that are related to the purpose of The purpose of this chapter is,to present some the study. The measurement of chlorophyll or of the basic concepts and techniqiies of sampling ATP in a plankton haul may .:be of interest, design and data evaluation.;AW can be easily whereas the cell count or detritus content may applieglby biologists. , not be of-interest. Thus, the characteristic of An attempt has been made to present the interest is the characteristic to be observed or - -material in a format comfortable to the non- measured, the measurements recorded, analyzed statistician, and examples are used to illustrate and interpreted in order to draw an inference most of the techniques., abut the real world. cit4 1.1Terminology 1.1.4Universe and experimental unit To avoid ambiguity in -the following discus- The experimental unit is-the object upon sions, the basic terms must be defined. Most of which an observation is made. The characteristic the terms are widely used in everyday language, of interest to the study'is observed and recorded lipt in biometry may be used in a very restricted for each unit. The experimental unit may be , sense. referred- to in some cases as the sampling unit. ' FOr example, a fish, an entire catch, a liter of 0.1 Experiment pond water, or a square meter of bottom may Anexperiment is oftenconsidered to be a each be an experimental unit. The experimental rigidly controlled laboratory investigation, but/ 'unit must be clearly defined so as to restrict in this chapter the terms experiment, study, and measurements to only those units of interest to field study are used interchangeably as the the study. The set of all experimental units of context seems to require. A general definition interest to the study is termed the "universe." which will usually fit either of these terms is' "any scientific endeavor where .observations or 1.1.5 Population and sample measurements are made inorderto draw In biology, a population is considered to be a inferences about the real,world." group of individuals of the same species. The statistical use of the term population, however, 1.1.2Observation refers to the set of values for the characteristic This term is used here in much the same of interest for the entire group of experimental manner as it is in everyday language. Often theunits about which the inferences are to be made context will suggest using the term "measure- (universe). ment" in place of "observation." This will imply WhenstudiLl;e made, observationsare not aquantified observation.Forstatistical usually taken for all possible experimental units. purposes, an observation is a record representing Only a sample is taken. A sample is a set of obser- some property or characteristic of a real-world vations, usually only a small fraction of the total object. : number of observations that conceivably could This-may be a numeric value representing the be taken, and is a subset of the population. The weight of a fish, a check mark indicating the term sample is often used in everyday language presence- of some species in a bottom quadrat to mean a portion of the real world which has in short, any type of observation. been selected for measurement, such as a water BIO GICAL METHODS
-4. sampleor a plankton haul However: in this Thus our efforts to select a "good" sample sectionthe term "sample" willbe, used to should include an appropriate effort -to ;efine denote set of observations" the written the problem in such a way as to allow tus to 'records emselves. estimate the parameter of interest usinga sample, of MOivn probability; i.e., a random sample. 1.1.6 Parameter and statistic The preceding discussion should lehve little ., When we attempt to characterize a popula- doubt-that there is a fundamental distinction tion, we realize that we can never obtain a per- betwen a "haphazardly-selected',' sample and a fect answer, soe settle for whatever. accuracy, "randbinly-se ated" sample. The distinction is and precision that s required. We try to take an that a haPEazardly-selected sample is one where adequately-sized sa and conipuie-a number there is no conscious bias, whereas a randomly- from our sample that is representative ''a he selected sample is one where filet ig.cOnscious. ly Population. For example; if we are interested in riro bias. There is consciously 4tb bias because the the population mean, we take a sample and com- randomization ,is, planned, -and therefore bias is pute the sample mean. The sample mean is-.planned out of the study. This is usually accom- referred to as a statistic, whereas the population plished with the aid of a table of randoni mean is referred to as a parameter. In general, nUmbers. A sample,selected according to a plan the -statistic is related to the parameter in much that includes rantiorniselection of-aperimental the same way as the sample ig related to the pO nits is the .only sample validly called a random ulation. Hence, ws, speak of population param sample. eters and sample statistics. Reference, to the definition of the term, Obviously many samples may be selected sample, at the beginning of the chapter. will- from most populations. If thete is variability in remind us that a sample consists of a set, of the population, a statistic computed from one observations, each-made uRon an experimental, sample willdiffer somewhat from the same or sampling unit. To saniple randomly, the statistic imputed from another sample. Hence, entire set of sampling units (population) must be whereaS a parameter such as the population identifiable and. enumerated. Sometimes the task. mean is fixed, the statistic or sample mean is a of enumeration may be considerable, but often variable, and there is uncertainty associated with it may be minimised by such conveniences as it as an estimator of the population parameter maps,thatalloweasier access to adequate which derives from the variation among samples. representation of the ehtity to be sampled. f The comment has frequently been made that 2.0 STUDY DESIGN random sampling causes effort to .be put into drawing samples.of little meaning or utility to 2.1 'Randomization the study. This need not be the case. Sampling In biological studies, the experimental units units should be defined tip the investigator so as s (sampling units or sampling points) must be to eliminate those units which are potentially, of selectedwith known probability.Usually, no interest. Stratification can be used to place _random selection is the only feasible means of less emphasis on those units which are of less satisfying the "knownprobability"criterion. interest. The question -of why known probability is re, Much of the work done in biological field quired is.a valid one. The answer is that only,by studiesis aimed 'at' explaining spatialdistri- knowing-the probability of selection of a sample butions of _populationdensities or of -some caii we ,extrapolate from the sample- to the paiameter related to population densities and population in an objective way. The probability the measurement of rates of change which allows us to place a weight upon an observation permit prediction of some future course of a in making our vtrapolation to the population. biologically-related parameter. In these cases the There is no other quantifiable measure of "how sampling unit is a unit of space (volume, area). well" the -selected sample representsthe Even in cases where the sampling unit is not a population. unit of space, the. problem may often be stated
2 BIOMETRICS RANDOM SAMPLING
in such-a manner that a unit of space may be statistical population is needed in advance of the used, so that random sampling may be more fullscalestudy.This information may be easily carried out. obtained from prior related studies, gained by For example,. suppose the problem isto pre-study reconnaissance, or if no{direct in- estimate the chlorophyll content of algae in af formationisavailable,professional opinion pond at a particular' time of year. The measure- about the characteristics of the population may ment is upon algae, yet the sample 'consists of a be relied upon. volume of water. We-kould use our knowledge of the way the algae are spatially distributed or 2.1.1Simple random sampling - make some seasonable assumptions, men . Simple(or unrestricted) rando- m sampling is construct a random sampling scheme based upon used when there is no reason to subdivide the a unit of volume (liter) as the basic sampling population from which the sample is drawn:The unit. sample is drawn such that every unit of the Itis always a simple or straightforward population has anequal chance of being matter to define sampling units, because of the selected. This may be accomplished by using the dynamic nature of living populations. Manykandom selection scheme already described. aquatic organisms are mobile, and even rooted orsessile forms change with time, so that 2.1.2 Stratified random sampling changes occurring during the, study often make If any knowledge of the expected size or, data interpretation difficult. Thus/the benefit to vkriation of the observations is available, it can be derived from any attempt to considensuchoften be used as a guide "in subdividing the factors in the planning stage will be consider- population into iiibpopulations (strata) with a, able. resulting increase in efficiency of estimation. Random sample selection is a subject, apart Perhaps the most profitable means of obtaining from the selection of the study site. It is of use information for stratification is through a pre- onlyafterthestudyobjecti ebeen study reconnaissance (a pilot study). The pilot - definid,the type of measur; nts ha e been study planning should be done carefully, selected, 'and the sampling its haVe been perhaps stratifying based upon suspected varia- defined. At this point, rand sampling pro- bility. ,The results of the pilot study may. be used vides an objective means of obtaininginforma- toobtainestimates of -variances needed to ,tiOn to-achieve the objectives of the study. establish sample size. 'Other advantages of the One satisfactory method of random sample pilot. study are that it accomplishes a detailed selectio.p is described. First, number the universe reconnaissance, and it provides the opportunity or entire set of sampling units from which the to obtain experience in the actual field situation sample, will be selected. This number is N. Then where the final study' will be Made. Information from a table of randomnumbers select as Manyobtained and difficulties encountered may often _random numbers,, n, as there will be sampling be used to set up a more realistic study and units selected kir the sample. Random numbers avoid costly and needles§ expenditure's. To maxi- tables are available in most applied statistics mize precision,strata should be constructed texts or books'. of mathematical tables. Select a such that the observations- are most alike within starting point in the table and read the numbers strata and most different among strata,i.e., consecutively in any direction (across, diagonal, minimum. variance- withi.p_strata an0 maximum down, up). The ...tiumber of observations, n variance among strata. In practice, the informa- (sample size), must be determined prior to tion used to form strata' will usually be from sampling.For example, if n isatwo-digit previously obtained data, or information about number, select two -digit numbers ignoring a y characteristics correlated with the characteristic number greater' than n "or airy number tha as of interest. In aquatic field situations, stratifica- already been selected. These number 114be the tion, may be based upon depth, bottom type, numbers of the sampling units to beelected. 'isotherms, and numerous other variables sus- To obtain reliable -data, informatin about the pected of being correlated with/ the character- 3 BIOLOGICAL METHODS
istic of interest. Stratification is often done on other hand, a sample is too small, the question other bases such as convenience or administra- of importance to the study may not be properly tive imperative, but except where these cor- answered. respond withcriteriawhich minine the variation within strata, no gain in precision may Case 1 Estimation of a Bill'enzial Proportion be expected. An estimate of the proportion of occurrence of the two categories must be available. If the Number of Strata categories, are presence and absence, let the In aquatic biological field studies, the use of probability of observing a presence be P (0 < P knowledge of biological cause-and-effect may < 1) and the probability of observing an absence help define reasonable strata (e.g., thermoclines, be Q (0 < Q < 1, P + Q = 1). The second type of sediment ty'pes, etc:, may markedly affect the infOimation which is needed isan acceptable . organisms so that the environmental.featrire mayJmagnitude of error, d, in estimating P (and. be the obvious choice for the strata diviOons). hence Q). With this information, together with Where a gradient is suspected and where stratifi- the size, n, of the population, the formula for n cation is based on a factor correlated to an as an initial approximation (rio), is: unknown degree withthecharacteristicof t2PQ . interest, the- answer to the question of how no = 2 many strata to form and where to locate their d boundaries is not clear. Usually, as many strata - The value fortis obtained from tables of are selected as may be handled in the study. In "Student's t" distribution, but for the initial practice, gains in efficiency due ,to 'stratification computation (he value 2 may be used to obtain usually. become negligible after only a few dicoisk,,a sample size, no, that will ensure with a .95 sions unless -the characteristic used as the basis probability, that P is within d of its true value. If of stratification is very highly correlated with noisless than 30, use a second calculation' the characteristic of interest. where t is obtained from a table of "Student's 2.1.3 Systematic random samnling with no-1 degrees of free'clOm. If the calculation Infieldstudies,thebiologistfrequently results in an no, where < .05, no further wishes to use some sort of transect, perhaps to calculation is warranted. Use no as the sample be assured of including an adequate cross section while maintaining relative ease of sampling. The size. If 112 > .05, make the following, computa- use of trartsectsis an example of systematic tion: sampling. However, a random starting point is chosen along thetransect to introduce the n= noncl randomness needed to guarantee freedom from (2) bias and allow statistical inference. 4 The method of placement of the transect Case 2Estimation of a Population Mean for should be given a great deal of thought. Often Measurement Data -itransects are set up arbitrarily, but they should In this case an estimate of the variance, s2, not be. To avoid arbitrariness, randomization must be obtained from some source, and a state- should be employed in transect placement. ment of the margin of error, 'd, must be ex- 2.2S'ample Size pressed in the same units as are the sample observation. To, calculate an initial sample size: 2.2.1Simple random sampling .. t2 S-2I (3) In any study, one important early question is d that of the size of the sample. The question is important because if, on the one hand; a, sample is too large, theieffort is wasteful, and if, on the a
4
1. "0: ikr BIOMETRICS RANDOM SAMPLING I If ii < 30, recalculate using' t from the tables, where t = the entry for the desired probability level front a table of "Studentt" (use 2 for a -> -,05; a further- Calculation is in order: rough estimate); Nk = the number of sanipli units in stratum k;.sk2aE the variance of stratu n=no" (4) ,k; N = the total. number of sampling units in al 1 + strateand, d = the acceptable error expressed in After a sample of size, n, is obtained from the the same units as the observations., population, the basic sample statistics may be For optimal allocation, the calculation is: calculated. The. calculations are till same as for-. t3( ksk)2 N2 d2 equations (11) through (15) unless the sample n - (7) size n, is greater than 5 percent of the popula- t2 INN ksk2 4 1 +N.d2 ty. tion N. If n > .05, a correction factor is used so that the calculation for the sample variance is: where the symbols are the same as above and -- Exi2 a. 2 where Sk =1/7k-r, the standard deviation of s2 N-n) n (5) stratum k (see Equations (16) to (19)i . (N/ n - 1 .Having established sample size, it remains to. determine -the portion of the sample - to be' The other calculations' mace use l' of,52,,as ;calculated above, wherever s2appears in the 2allocated to each stratum. For proportional allocation: formulas. . nNk nk = (8) 2.2.2Stratified random sampling 4 To compute the sample size squired towhere nk = the number of observationsto be obtainanestimate of the mean withina made in stratum k. specified acceptable error, computations can be For optimal allocation: made similartothoseforsimple .random nNksk nk sampling: a 'probability level must be specified; ksk (9) _an estimate of the'variancejWithin each stratum must be available; and the number of sampling Sample selectionwithin each stratumis units in each stratum must be known. Although performed in- the same manner as for simple this involves a good deal of work, it illustrates random sampling. the need for a pilot study and indicates that we 2.2.3 Systematic random sampling Rust know something about the phenomena we o are studying if we are to plan an effeCtive Afterthelocationofatransectlineis sampling, program. Selected, the'number of experimental units (the, , If the pilot study or other sources of infonba- number of possible sampling points) along this tion have resulted in what are considered to be line must be determined. This may, be done in reliable estimate's of the variance within strata,many ways depending upon the particular situa- e sampling can be optimally .allocatedto tion. Possible examples are the number of square str a.Otherwise proportional allocation should meter plots., of bo,ttom centered along a 100 besed. Optimal allocation,'properly used, will meter transect = 100); or the meters of res Itin more precise estimates for a given distance along a 400-meter transectias points, of sample size. departure for making a plankton haul of some For proportional allocation thecalculation for predetermined duration perpendicularf to the sample size is: transect, (In theirs eonexample, a question of-F t2ENksk2 subsampling or s me assumption about local, Nd2 homogeneous distribution might arise since the n = (6) plankton net has a_pdius les5 than one meter). 1 N- d- The interval of sampling, C, determines sample
5 1
BIOLOGICAL METHODS
size: n j N/C. The mean is estimated as usual; 3.0 GRAPHIC EXAMINATION OF DATA the variance asor a simple random sample if Often the most elementary techniques are of. there are no trehds, periodiCities, or other non- the greatest use in dateinterpretation. Visual random effects. examination of data can point the way for more 2.3Subsampling discriminatory analyses, or on the other hand, interpretations may become sookyious that S.ations often arise where it is natural' or_further analysis is superfluous. Ineitherther case, imrative that the sampliiitunits are defined in graphical examination of data is often the most a two-step manner. For ekample: colonies ofeffortless 'way to obtain an initial 'examination benthic organisms might be, the first step, and of data and affords the chance' to .organize the the measurement of some Characteristic on the data. Therefore, it is often done as a first step. individuals within the colony might be the Some commonly useti techniques ai presented second step; or streams might be thefirst below. Cell-counts (algal cells per Villiliter) will (primary) step, and reaches, riffles or poo as serve as the, numeric example (Table 1 ). the second step (or eIent) within the un v3.1 When a sample of ,prinrary units is selected, and Raw Data then for each ry unit a sample is selected As brought, out in other vhapters of thii by observing ome element of the primary unit,7 nual, it is ofAtinost importance that raw data ,:ttheisampling scheme is known as subsampling or, be recorded in. a careful, logical, interpretable,'1 two'-stage sampling. The computations are manner together with appropriate, but not super- straight forward, but somewhat more involved.. fluous, annotations. Note, that although some The method of selection Of the primary' units annotations may be considered superfluous to must be established. It may be a simple random the immediate intent of the data, they may not sample (equal probabilities), a stratified random be so for. other purposes. Any note that might sample (equal -probabilities within strata), or aidin (determining whether the other scheme such as probability proportional to data are size (or estimated size) of primary unit. In any comparableqo other similar data, etc.,rshould be recorded if possible. ease, let, us call the probability, of selection of ,the ithprimary unit, For simple random 3.2 " Frequ ency 'His tograms sampling, N' where N is the number of , To construct a frequency histogram from the data of Table 1, ,examine the raw data to deter- . primary units: in. the universe. For stratified mine the range, then establish intervals. Cifoose 1 the intervals with care so they will be optimally random sampling, Zk Iv where k signifies the k integrative and differentiative. If the intervals 0 1' stratum. For selection in w ich the primary area oo wide, too many observations will be units are selected with probabili poitionalintegr ted into one interval and the picture will' to their size, the probability of selection of the be 'hidden; if too narrow,,too few will fall into Jt hprimary -- unit is one interval and a confusing overdifferentiation Lj or overspreading of the data will. result.Itis Zj = (10) often enlightening if thv same data are plotted with the Use of several interval sizes. Construct =1 the intervals so that no doett exist as to which where L equals the number of elements in the interval an observation belongs, i.e.,,the end of primary unitindicated byitssubscript.If one interval must not be the same number as the stratificationis used with the latter scheme, beginning of the next. . merely apply the rule to each stratum. Other The* algal count data in Tables 2 and 3 were methods of assigning probability.robability of selectiongrouped by two interval 'sizes (10,000 cells/ml may be used. The important thing is toestablisl....-and 20,000 cells/mg It istasytosee that the, data the probability of selection for each primaryare grouped largely 'n the range 0 to 6 x 104 unit. cells/ml and that t frequency of occurrence is 6 BIOMETRICS 6RAPI-11C EXAMINATION
TABLE 2. FREQUENCY TABLE FOR DATA TABLEll W DATA ONPJANKTON' IN TABLE 1 GROUPED AT AN INTERVAL COUNTS WIDTH OF 1°0,000 CELLS /ML
Date Date Count Date Count Colt Interval interval Frequency _ Frequency .16 .te July 23,077 7,692 11 44,231 0-10 6 200 - 210 a 12 50,000- 36,5,38 26\ 23,077 10 -20 7 210 - 220 0 26,923, -27 134,615 13 26,923 20 -30 9 220 - 230 28 44,231 11 83,077 32,692 14 ) 30 -40 2 .230 1240 0 13,462- 29 25,000 15- 46,154 12 40 -50, 6 240- 250 19,231 30 146,154 16 55,768 13 50 -60 5 250 -26b 0 14 '-- 21,154 July 17 9,615 60 -70 1 260 - 270 15 61,538 1 107,692 18 13,462 `80 1 270 280 19 3,846 16 96,154 2 13,462 80 -90 280 - 290 a 17 23,077 3 9,615 20 , 3,846 -90 - 100 1 290 - 300 1 21 11,538 18 46,154, 4 148,077 100 - 110 2 300 - 310 5 7,692 19 48,077 53,846 22 110 - 120 0 310 - 320 o 20 51,923 6 103,846 23 13,462 120 - 130 0 320 - 330 0 24 421,154 21 50,090 78,846 130 - 140 2 330- 340 25 , 17,308 22 292,308 8 132,692 140 - 150 2, 340 - 350 0 23 165,385 9 228,846 150 - 160 0 3501: 360 0 24 I 10; 307,692 42,308 160 - 170 1 360 - 370 0 170 - 180 0 370 - 380 0 1 180 - 190 0 380 - 390 0 190:200 0 390 - 400 0 .0 lesser, the larger the value. Closer ipspection will reveal that with the finer interval width (Table illustrate the frequency polygon in Figure 3. o 2); the frequency of 'occurrence .does not in- 3.4 Cumulative Frequency crease monotonically ascell.count decreases. Ra ther,the frequency peak is foiled in the Cumulative frequency plots are often useful in interval 20,000 to-30,000 cells/.ml. This observa- data interpretation. As an example, a cumulative tion was not possible using the coarser interval frequency histogram (Figure 4) was constructed width; the frequencies were "overintegrpted" using the frequency table (Table 2 or .3). The' and did. not reveal this part of the Pattern. Finer height of a bar (frequency) is the sum of all interval widths could further change the picture frequencies up to and including the one being presented by each of these groupings. plotted. Thus, the first bar will be the same as ,.. Although a frequency table, contains all the the frequency histogram, the second bar eMuals info,rmation that a comparable histogram con- the sum of, the first and second bars of the frequency histogram, etc., and the last bar is the, tains,the graphical value of a histogramis usually worth the small effort required for its suit i of all frequencies. ° construction. Figures 1 and 2 are frequency histograms corresponding to Tables2 and /3, respectively. It can be seen that the histograms are more immediately interpretable. :The height 10- of each bar is the Ifrequency of the interval;the width is the interval width. L./Z6 - 17' ...7 L./ ' 3.3Frequency Polygon c 2- to ;,;presentessentially the same Another way P P ,1 information as tat in a frequency histogram is. 40 80 120 160 . 200 240 280 320 the use of a fre uency",',1polygon.,t Plot 'points at ALGAL CELLS/N1 THOUSANDS the'height of the frequency and at the midpoint of the, interval,/ and /connect the points with Fig ire 1. Frequency histogram; interval'width is straight lines: The data of Table 3 are used to 10,000 cells/ml,
7
_ 7 BIOLOGICAL METHODS' 21
TABLE 3. FREQUENCY TABLE FOR DATA smooth out s nie of the variation noticed-in the w. IN TABLE 1 GROUPED AT AN INTERVAL frequency. histogram. In addition, the phrase WIDTH OF 0,000 CELLS/ML "fraction of observations less than or equal to some \chosen value" can easily be,read -fraction Interval Interval Freqaency Frequency of time -the 'observationis less than or equal, to some chosen value." It is temptingio generalize 0- 20 13 200 - 220 0 20- 40 11 220 - 240 1 fromthisreiding and extend these results 40- 60 11 240 - 260' 0 beyond then ..urge of applicability. 2 260 - 280 0 ' 80 -100 1 280 - 300, 1 109 - 120 - 2 300 - 320 1 120 - 140 .2 320 - 340 0 14 -6. r 140 - 160 2 340 - 360 0 160 - 180 361:k 380 0 180 - 200 0 380 - 400, 0
Closely related to the cumulative frequency histogram is the cumulative frequency distribu- tion graph, a graph of relative frequencies. Tos obtain the cumulative graphs, merely 'change the
scale of the frequency :axis on the cumulative 40 80 120 , 160 200 240 280 320 frequency histogram. The scale change is made ALGAL CELLS/ML, THOUSANDS by dividing all values on the scale by the Littlest value on the scak (in .this case the hurner _of Figure-3. Frequency polygon; interval width is observations or 48). 20,00p cellstml. The value of the cumulative frequency distri- bution graph is to allow relat1frequency to be4 read, i.e., the fraction of observations less than 50 or equal to some chosen value. Exercise caution in extrapolating from a cumulative' frequency .40 distribution to other situations. Always bear in mind that in spite of a planned lack of bias, each 30 sample, or restricted set of samPles; is subject to
influences not accopted for and is therefore 20
unique. This caution is all the more pertinent for ca cumulative frequency plots because they tend to 10
40 80 120 160 200 240 280 320 360 400 14 'ALGAL CELLS/ML, THOUSANDS
10 Figure 4. Cumulative frequency histogram; in-
La.1 terval width is 10,000 cells/ml. Le 6
3.5 Two-dimensional Graphs 1111.1111111111111= Often data are taken where the observations 0 40 80 120 160 200 240 280 320 are recorded as a pair (cell count and time), ALGAL CELLS/ML, THOUSANDS' (biomass and nutrient concentration). Here a quick plot of the set of pairs will usually be of Figure 2.requency histogram; interval width is value. igure 5 is such a graph of data taken 20,000 cls/mt. fromable 1. Each point is plotted at a height r, 8
0.4 1
BIOMETRICS SAMPLE MEANAND VARIANCE A
- corresponding =to cell count and ata distance The data values are labeled with consecutit_ from the ordinate axis corresponding to ,the numbers (recall from the defigitionsthat these _number of days since the beginning obsrati9in. numeric values are observations), and there-are n The peaks and troughs, their frequency; tog Cher values in the set of data. A typical Obs rvation is with intimate -knowledge' of the conditions of Xi, where i may take any value betwe n 1nd n,
the study,_pight suggest something-of biological inclusive: and the subscript indicate's ch )t is r. interest, further statistical analysis; or further being referenced. field or labdratory work. <.k The sum ofthe numbers in a data set, such as In summary, carefully preyed tables add our sample, is indicated in statistical computa-, graphs may be important /and informative steps tions by capital- sigma, E. Associated with E arie .fi' in data analysis. The_added__effoft--is_ usuajly an operand (here, Xi), a subscript (here, i = 1% small, whereas gains-in interprqive insight may be large. Therefore, graphic examination of.dataind a _superscript (here, n)ZnXi. The sub- is a recommended procedure in the coursof )1-1 scripti = 1 - ttalue most-investigations. yr indicates that the of k. operand Xis to be the number labqlecf Xi tin our data set and-that this is to be,the first observa-, tion of the sum. Th ?superscript n indicates that the last number of the summation is tOrbe the 30(r- value of X, the last X in our data set. ComputatiAs- forthe mean, variance, 7.` standard deviation; variance of the mean, and x200- standard deviation of the mean (standard error) are presented below.. Note that these are compu- tations for a sample of n observations, i.e., they are statistics. Mean (X.): n EXi 1=1, n to 20 3d 40 DAYS . \ Variance (52): Figure5, An example of a two-dimensional graph plotted from algal-count data in Table 1. Xi2 E Xi) 2 1=1 1=1 (12) n s2 4.0 \AMPLE MEAN AND VARIANCE \ Note: The Xi's are squared, then the summation 4.1General Applicaon his performed in the first term of the numerator; Knowledge of 'certaincomputations and in the second term, the sum of the _Xi's is first computational notations is essential to the use formed, then the sum is squared, as indicdted b_ y of statistical techniques. Some Of the more basic. the Arentheses. Standard deviation (s): C -of these .will be briefly reviewed here. . 4 To illustrate the computations, let us assume S=747- (13) we have a set of data, i.e., a list of numeric values written down. Each of these values can be V rianceof the mean (q(- labeled by a set of numerals \beginqing with 1. Thus, thefirsi of these values 'can -be called X1 2 -52 (14) the second -X2, etc., and the last one we tall Xn X n --.)
r")
%.° BIOLOGICAL METHODS
Standard deviation of the mean or standard sampling): ,9 error (s- ): For the sample mean:
1 fljri (15) '(20) n Ij 1=1 , 4. y is the aver ge, comptiteaover sub- Statistics for Stratified JRaridom &imp' 'samplesamples Awells for hiwsample Ttecal,culations of the, sample statistics for stxatified random sqmplin& are as follows (see yi 1=1 (21) , 2.2.2 Stratified samples): Yi = n For -the mean df Aratvm k: where yiequals the observation for the b nk element in the ith primary unit, and Li is the E Yid i =1 (16).number of. observations upon elemen0, for primaw unit i. For the variance of the sample mean: i.e., simply comilute an arithmetic 'average for the measurements of stratum k. 2 = n- -A L ) 1 For the variance of stratum n 2 n(n-1) (E 14)2 1=1 (23)
Jtk nkt 2 E yki - EYk1) 2 A 1=1 i =1 (1. 7) where Yi is computed as nk 52 A LLT,i nk -1 (23) a. Yi Z1
7 A i.e., simply Equation 12 applied to the data of .whereYn is computed as the kth- stratum. A n A- n For the Mean of the stratified sample: Yn = Yi =Y E (24) ni =1 i. ni E NkYk or alternatively k=1 (18) A.., 1 4 Yist fE yo s2 (Y) EYi n(n71) ( E L02 (25) for either ,type allocation or alternatively for i=i proportional allocation:
m 4.4-7Rounding PkT'k k =1 (19) The questions of rounding and the number of ist-= digits to carry through, the calculations always arisein making statistical' computations. Note thatEquations 18) and(19)are Measurement data are approximations, since identical only for proportional allocation. they are rounded when the measurements were taken; count data;. and binomial data are riot 'subject to this type of approximation. r'4.3Statistics for Subsamples Observe the following rules when working with measurement or continuous data. If simple random sampling is used to select a subsample, the following formulas are used to )Vhen rounding numbers to some number
calculate the sample statistics(see 2.3 Sub- . of decimal- places; .first look- at the digit to the -e
BIOKTRICSTESTS OF HYPOTI4SES
k ° right or the last place to be retained. If this C Retaining Significant Figuras\s. number is greater than 5, the last place to: be Retention of signifieant figures in statistical 'retailed is rounded.tip by 1; if it is less than 5-; compUtations can be summarized in three rules: do not change last place merely 'drop the Never use more signi cance for a taw-data extra places. To round to 2 decimal places: value than is warranted. During intermediate , t -Unrounded omputatidlis keep all Rotinded ,..§ignificant 'figures for each data .valuel and carry 1'. 239 L24 the computations out in full. 28.5849 28.58 Round the final Jesuit to the accuracy set by the least accurate data value. If the digit tothe right Of tlie last place to be retained is 5, then look at'the second digit to 5.0 TESTS OF HYPOTHESES the right of the last place to be kept, provided Often ih biological field studies some aspeet tfiat the unrounded number is recorded withof the study'is directed to answering a hypothet- that 'digit as a significant digit. If the second iC-af question abouta population.If the hy- digit to the right is greater than 0, then round.pothesis is quantifiable, such as: "At the time of the number tup by 1in the last place to be kept; sampling, the standing crop of plankton biomass if the second digit is 0, then look at the third per liter in lakb A was the same as the standing digit, etc. To rounto I place: crop per liter in lake B," then the hiPothesis can beVested statistically. The question of drawing a Unrounded ounded sample in such a way thatthere is freedom from 13.251 13.3. bias, Co that such a test may be made, was dis- 13.2'5)001 13.3 cussed in the section .on sampling (2.0). Three standard types of tests of hypotheses If the number is recorded to only one place will be described: the "t-test," the "x2 - test," to the right of the last place to be kept, and that and the "F- test.." digit is 0, or if the'ignificant digits two or more 5.1T.test places beyond the last place to be kept are all 0, a special rule (odd-even rule) is followed to en- . The t-test is. used to compare a sample statistic sure that, upward rounding occurs as frequently (such;,as the mean) with some'value for the as downward rounding. Tile 'rule is: if the digit purpose of making a judgment about the popula- to the right of the last place to be kept is 5, and tion as indicated by the sample. The comparison is the last digit of significance, or if all following value may be the mean of another. sample (in significant digits are round up when the last which case we are using the two samples to judge digit to be retained is odd and drop the 5 when whether the two populations are the same). The the last digit to-be retained is even. to round to form of the t-statistic is I place: 0- 8 I. (26) Unrounded Rounded where 0 = some sample4istic; So = the 13.2500 13.2 standard deviation of the sample statistic;-and 0.3500 13.4 0 = the value to which the sample statistic is compared (the value of the null hypothesis). The /Use of the t-test requires the use ;'of Caution: all rounding must be made in 1 step t-tables. The t-table4s_ a two-way table usually to avoid introducing bias, For example the arranged with the column headings being, the uumber 5.451 rounded to a whole number is probability, a, of rejecting theXill hypothesis clearly5-; but if the rounding viere done in two when it is true, and the row headings. being the, steps it would first be rounded to 5.5 then to 6. degrees of freedom; Entry of the table at the
11 (-) BIOLOGICAL METHODS
correct probability requires a discussion of.and the table is for one-tailed tests, use this same i two- types of hyppteses testable using the column for..05 as two-tailed test(dokible any t-statistic. one-tailed test heading to get'lke proper two-- The null hypothesis- is- hypothesis, of no,tailed lest ha-ding; or conversely, halve the two - difference .betweena population parameter and tailed (test heating to obtain proper headings for'.( !another value. (Suppose -the' hypothesis to be one-taited tests). tested is that the mean, bt, of sonipopulation ,Testing Ho: µ = .M (the. population mean als10. Thep we would' write thenull ehuals some Valiie M): ° hypothesis (rhcilized ) as dr x 1`14 (27). H: p = - 1 sY( - ' Here 10 is the value of 0 in the general form for wheptie' is given 15y' \equation (1.1) /. ottier4 thet-statistic.An alternativetO e appropriate equation; M = the hypothesWed hypothesis -is' now required. The NCes igator, population mean; and sx is given by°. equation .viewing the experimental SItuation, determines (15).. The t-table en tf red 'at the chosen proba- the way in which this stated. If the investi- bill level (often) 5f and n-1 degrees of free- gatormerely wants to answer ,whether the dom, where n. ishe number of observations In sample indicates that f 10 or 'not, then the the sample. alternatethypothesis, Ha, is , *hen' the eompUted t-statistic exceeds the tabular value there is said to be a rt-.probal \H a :1.1 10' bility that Ho is false.\. :
If it isknown,foi(Vampleithal://cannot be ,Testing : '= 14; (the mean of the popula- than JO, then Ha s tion .front vihich sample 1 was taken equal's" the Ha : A>10 mean o1 the population from which sample 2 ee was taken): .'and reasoninethe other possible 14a is X1 a:/.1<10 t= 3C2 (28) 'RI k2 Hence, there e ty0s of alternate hy- ' -,potheses: where the alternative is simply wheres -x2= the pooled standard=or that the .null .hypothesisis false (Ha :// -10); obtained, by addingthecorrected sums bf the other, that the null hyPothesiS is' false and, squares for sample 1to thcorrected sums of t on, that Othe population parameter lies ivi,ding by the sum of irfilkaddi squares for sample 2,and to one side or the other cot' the hypothesized the degrees of freedom, foach times the sum value[ Ha : (> <) 104. In the caseof : A of the numbers of observatiOns, i.e., 10, the 'test is called a two-tailed test; in the alternate case of either of the second types of I X 2(1 X;)2+'E X 2(E X2) hypotheses, the t-tekt is.called a one-tailed te5. 1 ni '2(29)* (%1 +;122 ) Uoi 1)+ (o2 1)] ...Co use a° t-4atile,itmust be determined whether the column headings (probability of a An alternative and frequently useful form is
larger valu, br percentage point, or other 1.- 1)s12 (n2 1)522 5 V(n1 means -of expressing a) are set for one-tailed or R1 x2 (ni + ni)(oi + n2 2) two-tailed tests* Some tables are presented with both Iladings, and the terms "sign ignored" and .where S1 2 and s2 2 are each computed according "zignconsidered':areused."Sign ignored" to equation (12). 7 implies a two-tailed test, and "sign considered" For all condition's to be met whey the t-test is implies a one-tailed test. Where table's are given applicable, the sample should have been selected forone-tailedtests,thecolumnfor any isthecolumn probability(orpercentage) *I sign, whpn unsu1scripted, will indicatesuminationtlall appropriate to twice the probability for a two - observations, hence /XI means sum of all observations in tailed test. Hence, if a column heading is .025 sample 1. ( 12 a )3IQMETRIC CHITSQUARE TEST
from a population distributed as a normal distri- bution. Even if the pophlatiOn is not distributed Note that = a2 for the P'OiSson,Ibus the ,normally, however,as sample size increases, the t-test ap achesto appOcability.Iftitis/Kandard.cipyiation of the.mean, sR: suspected tthe populationdeviates 'too drastically fro the normal, exercise care in the 5.2Chi Square Test ( x2-test) use of t etest. One methOd of eoking Like\ t, X2 values_may ba. found n mathe- whether the data are rr6rrhally .distributedjs to matcal and_staristictIllables tabu ated hi a two- . plot the observations on- normal pbability\wayrrangement. UsuallYas With t, the column graph paper. If the plot approximatesstraialt' he,ittings probabilities oobtaining a larger line, using the t-test is acceptable. value.c.vheri Hvis 'tru'e; an` DOW heading, --The t-test is used in certain' cases where it- is'are= degrees, or freedom. If the calculated. known thatthe' parentdistribution tie not".ceeds -the tabular value,, then 'the null. hy'13Othesis ) One case commonlycomonly incount ;is rejected. The chi square Cast is'ttfte,n,used, yith. field studies the binomial. The binorriiaI may . the 4ssumoion (4 approximate narthait-3,-in the describe presence or absence, dead or a, male population.
or female, 'etc,: .42. i, I Chi square appears in two forini6fhar differ Testing. Ho : P = K (the population propOlison-not only in aPpearahce, but that provide'forrhats'. equals some value K),: / ,..-- for different applications. 'tfo ,.. , One form: PIt - 1 t rm \ (31) (33) ' , .1- . where P = the synSolfor. the po-pulation propor- is useful in tests- regarding hypotheses about at The other form: , tion (e.g., proportion of males in the popula-,. tion); K 7 a constAnt positive fraCtion as \ the 2 (0E)2 (34) hypothesized proportion; p = the proportion E obseYved in the sample; q = the complementary where 0 = an observed value, ancl.E = an ex-- .proportion (egg., the proportion of femal' in pected (hypothesized) value, is especially useful the sample or 1 - p); and n = the numbe ofin samplirig from binomial and multinomial observations in thesample. Dote that since p is distribution, i.e., where the data may be classi- computed as (number of males in the sarnple.) / fied into two or more categories. (total number of individuals ih the Sample)',, it Consider first a binomial situation. SupRose will always be a positive number less thanne;the data from fish collections from three lakes/- and hence, so will q. Again 0 must be chosen kla)are to be ppoied and the hypothe'sis of an-equal can be anY..of the ,types 'previously discus ed; sex ratio tested. (Table 4). and the degrees of freedom are n -1. Count data, where the :objects:counted are cTABLE 4. POOLED FISH SEX _. distributed randomly, follow a Poisson distribu-. .3 DATA FROM 3 LAKES tion..If the Poisson can be used as antadequate . description of the distribution, of the popula- No. males No. fernags Total tion, an approximate t. may be computed. 892* (919)t 946 (919) 1838 Testi-Ho:. /1 = glfor the Poisson (the mean *Observed values. of the, p&p ption distrilited as a Poisson equals/ . tExpected, or hypothesized, values. some hypoth ized value M)°:
To compute the hypothesizedvalues. (919 - X M above), it is necessary to haye fOrmulated a null (32) hypothesis: In this Case, it was Ho : No. males = No. 4emalcs = (.5) (total)
13 * BIOLOGICAL METHODS
Expected varies are always computed based AnAer X2 test, may be illustrated' by the fupon the null hypothesis. Thecomputation for following ekample. Suppose that Comparable X2 is techniques were usedto cbllect from four eains-.--Witir -the-use of three spedes common (892- 919)2 + 919)2_ X2 919 to all streams, it is desired to test the hypothesis that the three species occur in the same ratio *n.s. = not significant , regardless of stream, that thetr ratiois There is one aegee of freedom- for this test.'independent oistream (Table 6). Sinde 9mputed X2 is not greater than tibulated _X2 (3.84),,the nullhypothegis is not rejected. TABLE O. OCCURRENCE OP THREE This test, of Course, applies equally wql to data SPECIES OF FISH has,110 bgen pooled, i.e., where' the values Number of organisms Stream_ Frequency are5 from two impo6led,categories, Species 1 Species 2' Species 3
The informatidn. contained in each of the 1 24* (21.)t 12(12.5)-30(31.7) 66 collections is partially obliterated by 'pooling:If 2 15, 08.5) 14(10.6) 27(26.9) 56 the'identityp of th'eollections is maintained, two 3 28(27.4j 15(15.7) 40(39.9) 83 ' 4 .20 (19.4) 9(11.2) 30(28.4) 59' type's test may be made: a -test or the null : Total 87 ' 50 127 . 264 hyp6thesis- for, each collection separately; gnd a Expected .? 4 test %of': interaction,i.e.,Whetherthe ;ratio ratio 87/264 50/44 127/264 deperids upon thlake frond: which, the sample *Observed values. was obtained (Tableable , tExpected, or hypothesized TABLE'S. FISH SE(k DATA FROM 3 LAKES To disCgss the table above, 0;= the observa; . tion for theit-h.stream and thejt-1-' species. Bice No Males No Females Total X 2 Hence,.02 3is'the observation for stream two 6.._., , . 1 346(354)j 362 (354). ,,,.''708 .36 n.s. and speeles three, or 27., A similar indexing 2 302(288) 2Z4 (288) 576 1.30 n.s. scheme applies to the expecte'd values, For 3 244(277) 310 (277) 554 7.88 P =.005 the totals, a subscript replaced- by a dot (.) Total 892(919) 940(919) .858 1.59 n.s. symb\olizes that slmunation haS occurred for the observitions indicated by that subsCript. Hence, Observed values. tExpected, or hypothesized valuei. 0.2 is the total for species tvo:(, 5 0); 03, is the total for stream three (83); and O. is the grand With the use of the same null hypothesis,.the total (464). .following results are obtained. Coniputations of expected values make use of The individ4al X2"; were' -computed in the the null hypothesis that the ratios are the smile same manner. as equation (34), in separate tests regardless of stream. The best estimate. of this of the hypothesis for each lake. Note that the 0.j fist two are not significant whereas the third is ratio for any species is zT,the ratio of the sum ,significant. This points to probable ecological for species j to the total of all species. This ratio differences among lakes, a possibility that would multiplied by the. total for stream i gives the not have been discerned by pooling the data: expected number 'of organisms of species j in Theitest for interaction (dependence), is made stream i: by suming the individual X2,'s and subtracting. the X 'obtained' using, totals, i.e.; E; i =O":7/ ) (35) X2 (interactions) = EX2 (individuals)'- X2 (total) = .36 + 1.30 + 7.881.59 = 7.95, For example,. O.2 The degyees of freedom for the interaction X2 E12 = (-0 ) (0/ ) "are the number of individual X2 's minus,ofie; this case, two. This interaction X 2 is significant 50 (P > .025), which indicates that the sex ratiois 264 "'"/ 'indeed dependent upon the lake. = 12.5 14 - \-) BIOMETRICS F-TEST
2 X _si computed as.- f2 = n2 -I. The table is entered at the chosen , (0 fj probability level, a, and if F exceeds the tabu- X 2 2 .6 9 (n s.) lated value,it 1 j : E j is said that there is a probability that at 2 exceeds a2 2. For this type of hypothesis, there are (rows 1) 5.4Analysis of Variance (colums1) degrees of freedom in this case. (3) (2) = 6 Twsimple but potentially -useful examples of thanalysis Of variance' are presented to In the example, x2 is nonsignificant. Thus, there illustrat use of this technique. The analysis is' no evidenee;Tithat the ratiownionE, the organ- of variance is a powerful and general technique isms are different for different's.ttgaiiis.7 applicable to data from virtually any experimen- Tests of two types of :hypotheses by X' have tal or field study. There are restrictions, however, been illustrated. The first type of hypothesis was in the use of the technique. Experimental errors one where there was a. theoretical ratio, i.e., theare assumeeto be normally (or approximately ratio of males to females isI :1. Thebsecond type normally) distributed about a mean of zero and of hypothesis .was one where equal ratios were have a common variance; they are also assumed hypothesized,butthevalues of theratios to be,independent (i.e., there should be no cor-' themselves were computed from the data..To relations among responses that are unaccounted . draw the proper _inference, itis important to, for by.,,,the identifiable factors of the study or-by' make a distinction between these two types ofthe model). The effects tested must be assumed hypotheses. Because the ratios are derived from to be linearly additive. In practice these assump- the data .in the later case, a better fit to these tions are rarely completely fulfilled, but the ratios (smaller X2) is expected. This is compen- analysis of variance can be used unless signifi- sated for by loss of -clegrees.Aireedom,, Thus, cant departures- from noormality, or correlations smaller computedtX2 's may be judged:signifi-among adjacent obFrvations, or other types of cant than would be in the case where the ratios Measurement bias are suspected. It would be are hypothesized independently of the data. prudent, however, to check with a statistician regarding any uncertainties about the appli- 5.3F-test cability of the test before issuing final reports or publications. The F distribution, is used for testing equality of variance. Values Of F are found in books of5.4.1 Randomized design mathematical and statistical tables as well as in The analysis of varianceforcompletely most statisticstexts. Computation of the. F randomized designs provides a technique often statistic involves the ratio of two variances, each useful in field studies. This test is commonly With associated degrees of freedom. Both of usedfor data derived from- highly-contr011ed these are used to enter the table. At any entry of laboratory 'orfield experiments where treat- the F tables for (ni I) and (n2 I) degrees of freedom, there are usually two or more entries. ments are applied randomly to all experimental units, and the interest lies in whether Or not the These entries are for various levels of probability treatments significantly affected the response of of rejection of the null hypothesis when in fact the experimerital,tinits. This case may be of use it is true. The simplest F may be computed by formingin water quality studies, but in these studies the the ro of two variances. The null hypothesis is treatmentsare the conditions found, or are H(, v 2 = q2 2 . The F statistic is blassificatiorls. based upon ecological criteria. Here the desire is to detect any differences in si 2 F, . (36) some type of measurement that might exist in conjunctionwith thefieldsituationor the where s12is computed from n1observations classifications-or criteria. and s2 2from n2. For simple variances, the For example, suppose itis desired to 'test degrees of freedom, f, will be fl = n1 I and whether the biomass of organisms attaching to 4
- BIOLOGICAL METIIODS
slides suspended in streams varies from stream to If the null hypothesis (Ho) ) is true, thp ratio of stream. A simple analysis such as this couldthese values is expected to equal one. If Ho' is precede a more in-depth biological study of the not true, i.e., if there are real differences d e to comparative productivity of the' streams. Data the effect of streams, then the mean squa e for from such a study are presented in table 7. streams is affected by these difference and is expected to be the larger. Thea io inthe TABLE 7. PERIPHYTON second case is expected to be greater than one. PRODUCTIVITY DATA The ratio of these two variances forms an F-test. The analysis of variance is presented in Table Biomass Stream Slide (mg dry wt.) 8. The corhputations are: 1 \J I 26 2 20 ' (85 + 85 + 134)28401:45 3 14 C 11 4 25
2 1 34 E Xi j2 = 262 + 20 2 + + 4024+ 282= 8936 2 28 i j 3 Lost 4 23 Total SS = 89367 8401.45 = 534.55 3 , 31 Xj .2 852 852 1342 + = 8703.58 2 35 E 3 40 i " 28 Streams SS = 8703.588401.45 = 302.13 ti Slides w/i streams SS = Total SSStreams SS Intesting with the analysis of Aariance, as = 534.55302.13 with other methods, a null hypothesis should be = 232.42 ntrIlhypothesis formulated.Inthis case the The mean ,squares (MS column) are computed could be the by dividing the sums of squares (SS column) by I-10 :, There are nodifferencesin itscorresponding degrees of freedom(df bioniass of organisms attached to the column). (Nothingis usually learned inthis slides that may be attributed to differ- Context by computing a total MS.) The F-test is ences among streams. In utilizing the anasis of variance, the test TABLE 8.F-TEST USING PERIPHTON DATA for whether therear differences among streams is made by comparitt two types of variances; Source - df SS Total N-1* E xii2 -c most often called "mean squares" in this con- j text. Two mean squares are computed: one 3c; .2' based upon the means for streams; and'one that Streams ri. In our i isfree of the effect of the means. Slides w/i streams E(ri-D Total SS - Stream SS example, a mean square for streams is computed with the use of the averages (or totals) from the *The symbols are defined as: N = total riumber of observations streams. The magnitude of this mean squareis (slides); t = number of streams; ri = number of slides in stream i; affected both by differences among the means ).Cj j = an obserillation (biomassfslide); Xi . = sum of the and by differences among slides of'the same observations for stream i; and C = rection for mean = (E j)2 stream. The mean square for slides ,is. computed it that has no contribution due to stream differ- N ences. If the null hypothesis is true, thendiffer- ences among streams do not existand, there f6re, Source df SS MS they make no contribution to the mean square Total, 10 534.55 Thus, both mean squares (for Streams 2 302.13 151.065 5.20* for streams. Slides w/i streams and for slides) are estimates of the same streams 8 232.42 29.04 variance,and, with repeatedsampling, they would be expected to average to the same value. *Significant at tile (3.05 probability level.
( 16 ,-' BIOMETRICS ANALYSIS OF VARIANCE
performed by computing the ratio, (meansquare This hypothesis is not stated in statistical terms for streams)/(mean square for slides),in this and, therefore, only implicitly tells us'what test 151.065 case, 5. to make. Let us took further at the analysis 29.055 before attempting to statea null hypothesis in When the calculated F value (5.20) iscom- statistical terms. pared with the F values in the table (tabular F Inthis study two factors are identifiable: values) where df = 2 for the numerator and df= 8 for the denominator, we find that thecalcu- times and positions. A study could have been lated F exceeds the value of the tabular F for done on each of the two factors separately\zi.e., an attempt could have been made to distinguish probability .05. Thus, the experiment indicatesa whether there was a difference associated with high probability (greater than 0.95) of there times, assuming all other factors insignificant, being a difference in biomass attached to the and likewise with the positions7The example, slides, a difference attributable to differencesin streams. used here, however, includes bothfactors simultaneously. Data are given for times atrd for Note that this analysis presumes good biologi- positions but with the complication thatwe cal procedure and obviously cannot discriminate cannot assume that one. is insignificant when differences in streams from differences arising, studying the other. For the purpose of this for example, from the' slides having been placed study, whether there is a significant difference in a riffle in one stream and a p of in the next. with times or on the other hand with positions, In general, the form of any analy 's of variance are questions that are of littleinterest Of derives from a model describing al observation interest to this study is whether the upstream- in the experiment. In the exam ,the model, downstream difference varies with times. This although not stated explicitly, assumed only two type of contrast is termed a positions-times inter- factorsaffectrAgabiomass- measurement action. Thus, our null hypothesis is, in statistical, streams -and slides within streams. If the model had included other factors, a more complicated analysis of variance'would have resulted. TABLE 9. POUNDS OF FISH CAUGHT PER 1.0 HOURS OVERNIGHT SET dF A 5.4.2 Factorial design 125-FOOT, 1'/2 -INCH -MESH GILL NET Another application of a simple analysis of variance may be made where the factorsare Times Positions arranged factorially. Suppose a field study where Upstrearit (PI) Downstream (P2) the effect of a suspected toxic effluentupon the Before 28.3 29.0 (T1) 33.7 28.9 fish fauna of a river was in question (Tables 9 38.2 20.3 ' and 10). Five samples were taken aboutone- 41.1 36.5 quarter mile. upstream and five, one-quarter mile 17.6 29.4 downstream in August of the summer before the After 15.9 19.2 (T2) 29.5 22.8 plant began operation, and -the sampling scheme 22.1 24.4 was repeated in August of the summer after 37.6 16.7 operations began. 26.7 11.3 Standard statistical terminology refers to each of the combinations PI T1, P2 T,, P, T 2, .and P2 T2 as treatments or treatment combinations. TABLE 10. TREATMENT TOTALS FOR Of use in the analysis is a table of treatment THE DATA OF TABLE 9' totals. In plann for -this field study, a null and Total Positions Times totals alternate h pothesis should have been formed. Upstream Downstream In faCt, whether stated explicitly or not, the null Before 158.9 144.1 303.0 After 131.8 "94.4 hypothesis was: 226.2 Positions Grand total I-10: The toxic effluent has no effect upon totals 290.7 238.5 529.2 the weight of fish caught 17 BIOLOGICAL METHODS terminology: Times Sum of Squares (SST): Ho : There is no significant interaction effeft E x j .2 Computations for testing this hypothesis *ith sp (226.2)2 the use of an analysis of variance table are (303.0)2 14002.63 = 294.91 presented below. '1. 10 10 Symbolically, an observation must have three Interaction of. Positions and Times Sum of indices specified tobe toMpletely identified: Squares (SSPT): position, time, and sample number. Thus there are three subscripts: Xi jk is anobservation at SSTMT SSPSST position i, time j, and from sample k. A value of 456.69136.24294.91r=21.54 1 for i. is upstream; 2, downstream; 1for j is Error Sums of Squares; "1 before;, after. A particular example is X123, E xi2 -SSTMT CT 0 the third sample upstream after the plant began operation, or 22.1 -pounds. A total (Table 10) is 15?08.24456.6914002.63 = 848,92 specified by using the dot notation. For/ the Although not iMportant to this example, the value of Xi j, then the individually, sampled main effects, positions and times, are tested for values for position i, time j are totaled. It is a significance. The F table is entered with df = "1 total for a treatment combination. Forexample, for effect tested, and df = 16 for error. Theposi- the value of X11, is 158.9, and the value of X1.. tions effect. is not significant at anyprobability where' samplings and times are both totaled to usually employed. The times effect is significant give the total for upstream-, is 290.7. with probability greater. than '.95. The inter- For. a slight advantage in generality, let the action effect, is not significant, and we, there- following additional symbols apply: t = number fore, conclude that no effect of the suspected of times, of sampling (in this case t = 2); p = toxic effluent can be distinguished in this,data. number of positions samples (in this case p = Had the F value for Interaction been large s = number of samples pertreatment combi enough, we would hay& rejected the null hy- tion; and n = the total number of observations: pothesis, and cdncludedthat the effluent had a The amputations are: significant effect (Table 11). Correction for mean (Cr): TABLE 11. ANALYSIS OF VARIANCE .,(E Xi jk)2 (529.2)2 TABLE FOR FIELD STUDY DATA n = 717 OF TABLE 9 14002.63, Source df SS MS F Treatment Sum of- Squares (SSTKT),, Treutments 3 456.69 4 \ I, PcFOions. 136.24 136.24 2.56 '.11nle,,a ' 294.91 294.91 5.55 s. POsitions 158.9)2. :' (14,8) : L1114.212 ''A0421)1' X times 25.54 ' 25.54 <1 53.05 1" 5.r, 7.7r"....449q'P.=1456.9 Z1101' .16 848.92 (Note that the diViSOr,,(5);r1i4;-iierfacipred Out here, if desired, but where of..6.0 CONFIDENCE INTERVALS FUR MEANS samples is taken for lifirefi frattriOr eOrIbirr'ation AND VARIANCES it should be left as above.) ' When means ar(Coinputed in field studies, the desire often is to eport them as intervals rather Positions Sum of Squares (SSP): than as fixed numbers. This is entirely reason- ablebecause computed means are virtually Exi ..2 CT always derived from - samples arid are subject to, St the same uncertainty that is associated with ihe (238.5)2 (250.7)2 11002.63 = 136.24 10 10 sample. 183Q
Ne BIOME4RICSCONFIDENCE INTERVALS
The correct computation of confidence this section the 'confidence limits for the true intervals requires that the distribution of the variance, a2. The information needed Mere is observations be known. But very often approxi- similar to that needed for the mean, namely, the mations are close enough to correctness to be of estimated variance, s2 ; the degrees of freedom, use, and often are, or may bekipade to be, con- n- k; and values from X2 tables. The values'from servative. For computation of confidence inter- X depend upon the degrees of freedom and vals for themean, the normal distribution istupon the probability level, a. The confidence usually assumed to apply for severalreasons: the -,interval is central limit theorem assures us that with large spies the mean is likely to be approximately (n-1)s2 <.2 (n-1)s2 normally distributed; the required computations X a Ia are well known and are easily applied; and when i / the normal distribution is known not to apply, suitable transformation of the data often is avail-, This will be illustrated for a = .05; (n- I)= 30; able to allow a valid' application. and s2 = 5. Since, a = .05; 1 = 0.975; the The confidence interval for a mean isan inter- val within which the- true mean is said to have associated X2.975, = 16.8, and the X2'0.0 = some stated probability of being found. If the 47.25. Thus, the probability statement for the probability of the mean not being in the interval variance in this case is is a (a could equal .1, .05, .01 or any probability < < value), then the statement may be written P 02 =8.93]= .95 P (CL1 < I.L.CL2)= 1 a This is read, "The probability that the lowercon- 7.0 LINEAR REGRESSION AND CORRE- LATION fidence limit (CLI ) is less than the truemean (p) and that the upper confidence limit (CL2) is 7.1Basic Concepts greater than the true mean, equals Ia." How- ever; we never know whether or not the true It is .ofteii" desired to investigate relationships mean is actually included in the interval.-So the between variables, i.e., rate of change of biomass confidence interval statement is reallya state- and concentration of some nutrient; mortality ment about our procedure rather than about p: per unit of time and concentration of some It says that if we follow the procedure for toxic substance,; chlorophyll and biomass; or re- growth- rate arid temperature. As biologists, peated experiments, a proportion of thoseex- we periments equal to a will by chance alone, fail appreciate the incredible complexity of the real- to include the true mean between our limits. For world relationships between, such variables, but, example, if a = .05, we can expect 5 of 100 simultaneously, we may wish to investigate the confidence intervals to fail to include the true,desirability df approximating these relationships mean. with a straight line. Safi an approxiniation may prove invaluable, if used judiciously within the To compute, the limits, the sample mean, X; limits of the conditions where the relation holds. the standard error, sk ;and the degrees ofIt, is important to recognize That no matter how freedom, n-4...4nust be known. A ta,n-value well the straight line describes this data,a causal from tables of Student's t is obtained corre-relationship between theVariablesisnever sponding to n-1degr4s of, freedom andimplied: Causality is much more difficult to probability a: The computation is establih than mere description by a statistical relation. cL, = (ta)(sk) When studying 'the relationship between two CL2= X + (ta) (sk) variables, the data -maY be taken in one of two ways. One way is to measure two variables, e.g., Other. confidence limits may be compute measure dry weight biomdss and an. associated and one additional confidence' limit is giv chlorophyll measurement. Where tFo'variables 19 BIOLOGICAL METHODS
are measured, the data are termed bivariate. The TABLE41 PERCENT SURVIVAL other way is to choose the level of one variable TO Flt TAGE OF EGGS OF GOGGLE-EYED WYKE VERSUS . and measure the associated magnitude of the other variable. CONCENTRAIJON OF Straight line equations may be obtained for SLIFERCHLOR'OKILL IN - each 'of these situations by the technique of PARENTS' AQUARIUM WATER linear regression analysis, aIrd if the object is to Concentration, ppb (X) other,itis Percent survival (Y) predict one variable from the 74. desable.i to obtain such a relation. When the 82. degree of (linear) association is to be examined, 68. line need be derived only a 65. n9 straight 60. 2. measure of the strength of the relationship.This 72. 2. measure is the correlation coefficient,and the 64. 3. analysis is termed correlation analysis. 60. ) 3. 57. 3. Thus, linear regression analysis and linear cor- 51. 4. relation analysis are two ways in which linear 50. 4. relationships between two variables may be ,55. 4. 24. examined. 28. 6. 6. 7.2 Basic Complitations -4 o. 10. 10. 10. 7.2.1Regression equation 4. 10. The regression equation is the equation for a straight line, (4) ZX2 = the total of the squared X's, Y=a+bx (5 ZY2 = the total of the squared Y's, , (6) ZXY.= the total of the products of theX,Y A. tgaphic,representation of this funotion is a pairs, straight line plotted on a two-axis graph. The (7) (EX)2 = the square.of quantity(2), line -intercepts the y-axis a distance, a, away (8) (ZY)2 = the square of quantity (3), from the origin and has a slope whose value is b. (9) (ZX)(ZY) = the product of quantities(2) Both a and b can be negative, zero, or positive. and (3), / Figure 6 illustrat'various possible graphs of a (10) CTX = quantity (i) divided by quantity(1),- regression equation. (11) CT,, = quantity (8).divided by quantity (1), The regression eq ation is obtained by "least- (12) CT, , quantity (9) divided by quantity squares," a technique -en9ring that a "best" line (1). will be objectively obtained. The applicationof With the calculation of these quantities, most least-squares to the simple case of astraight line of the work associated with using linear regres- is extremely relationbetween 'two -.variables sioniscomplete. Often calculating machine simple. chaiacteristics may be so utilized that-when one In Table 12 is a set of data that are usedto quantity is calculated the calculation ofanother illustrate the use of regression analysis.Figure 7 is partly accomplished.' Modern calculatorsand is a plot of these data along with fitted lineand computers greatly simplify this task. cOnfidence bands. In Table 13 are the computedvalues of In fitting the regression equation, it is con- quantities (1) thrOugh (12) for the dataof Table venient to compute at least thefollowing quan- 12. . tities The estimated value for the slope of the line, of observation of X (1)n = the number of pairs b, is computed using and Y, ,ZXY CTxy(6) -(12 (37) (2) EX = the total for X, "r)--.717. Crx (4)(10 (3) ZY = the total for Y, 20 3^ BIOMETRICS 1- LINEAR REGRESSION
4/ C\ jpr the example, this is-. subjeOt to some uncertainty, and a statement of that `uncertainty should invariably accompany 2453 3726.67 b the use of the predicted y. 498. - 338 = -8 7.2.2 anfidence intervals rounded to the nearest dole number. The proper statement of the uncertainty is an Computation of the estimated; intercept,a, is interval estimate,the same .typeas,. those as follows: . previously discussed for means and variances. The probability statement for a predicted y a=y bit" (38) depends upon, the type of prediction, being .Ey Ex = made. The regression equation is perhaps most n n often used to predict the mean y to be expected .(a)_b(2) when x is some value, but it may also be used to ,) (1) oy predict the value of a particular observation of y which for the &le when x is some value. These two types of predic- 860 78 tions differ only in the width of the confidenceI \18 ""18 intervals,, A-confidence interval for a predicted \ =82 observation will bt the wider of the two types because of uncertainty associated with variations rounded to tike nearest whole number. among observations of y4or a given x. Thus, the reessiOn equatiofor this data is To compute the, co6f ence intervals, first A Y = 82 - 8X compute a variance estimate. 's is the variance, due to deviations of the observed values from where = the percent survival, and X = con- centration of pesticide. the regression line. This computation is: Figure 7 shows the regression line, plotted (ExY- CTxy:)2 al9ng with the data points. Note that this line /Y2crY(Ex2 CTx) si.x appears to be a good fit but that an eye fit might 'n-2 (39\ . have 'Veer-Nightly different and still appear to be For this example: a "good fit." This indicates that some unce?- (2,453 3,727)2 tainty is associated with the line. If a value for y 51,67641,089 -; (498 338) is obtained with the of the regression equ 4.x 28 tion with, a given x, another experiment, how- ever well controlled, could easily produce a 'dif- This statistic is useful in other computationsas ferent value. The predicted values for y are will become apparent. 4For the confidence intervals the Kilda root of TABLE 13. COMPUTED VALUES the 'above statistic, or the "..st6rderror of OF QUANTITIES (I) THROUGH deviations from regression is required, i.e., (12) FOR THE DATA OF TABLE 12 sy.x ="1:Fc=5 (40) Quantity Value The onfidence limits are computedas follows (1) n *018 for a pdicted mean: (2) EX 78 (3) EY 860 A - R)2 .( 4) EX2 498 CL(Y) =a + bXp ±( (sy.x) 1 ( 5) EY2 51,676 a (a2_crx)(41) ( 6) EXY 2,453 (7) (DO; 6,084 ( 8) (EY)- 739,600 where ttils chosen from a table of t values Using iris ( 9)(EX) (EY) 67,080 n-2 degrees of freedom and oprobability le 'el a; (10) Clic 338 (11) CTy 41,088.89 Y = the computed Y for which the confidence (12) CTxy 3,726.67 intervalis sought, a mean 'Y predicted to be
21
L._ g..) BIOLOGICAL METHODS .
POSITIVE INTERCEPT POSITIVEItiERCEPT- NEGATIVE SLOPE POSITIVE SIOPE
f
4 NEGATIVE INTERCEPT ZERO INTERCEPT POVTIVE SLOPE POSITIVE SLOPE
,NEGATI E (INTERCEPT NEGATI E LOPE NOTE: A SLOPE OF ZERO IMPLIES NO RELATIONSHIP.
Figure 6. Examples of straight-linegraphs illustrating regression equations.
`""? 22..), o BIOMETRICSLINEAR REGRESSION j
,
95% CONFIDENCE BANDS (PREDICTED MEAN) V I
Y AND CL FOR Xp=3 95% CONFIDENCE, BANDS
.NE (PREDICT,ED VALUE) 71.
20L
PREDICTED SINGLE X VALUE AND CL
FOR Y=40 1,
\ I I i ,1 1 I I I 1 2 V 4 '6 8 10
CONCENTRATION OF SUPERCHLOROKILL (PPB) 0
Figure 7. Regression analysis of data in Table12.
23 . BIOLOGICAL METHODS observed on the average when the X value isX toobtai stioideconcentration from egg R survival. is may be done graphically from a Xp = the particular X value used to computeY; plot suc asthatillustratedinFigure7. intervals X = the mean of theX's used in theecoMputa- PredicteX's and associated confidence may be ead fromthe plot (see horizontal line E X (2)EX2 = relation (.4) in the tions4 =(1)' coerespoing to y, = 40 and notation). amputations; and C-1', = relation (104 in the Calibration curves and confidenceintervals (41) may also be workedalgebraically. Where the computations. Note that?in usinrEquation the where the, signs (±) are shown, the minus(- ) sign problem has fixed X's, as in the example, ekgation for X should be obtainedalgebraically, is used when computing the lowerconfidence 71. limit and the plus (+) for the upper. i.e., Y If a confidence interval for a particular 46= (Y-a) (43) A b the (given, a particular X, i.e., Y) is' desired, A confidence limits are computed using fcliapredicted X (X) given' a mean valueYm frdm a sample of m observations, theconfidence A 1 (XpR)2 (42) cl...(v)= a 1- 6Xp ±(t&(sy,x41; (Ex2- CTx) limits may be computed as follows: compute the quantity Note that Equation (42)diffks from Equation 22 (41) only by the addition of 1 underthe radical. A = b2 --tasY'x All the symbols are the same as'for Equation will be (41). Again the'se confidence intervals Compute the confidence limits as (44) wider than those forV. . rn- V) (vm_ s/-)2 If a graphical representationof the confi- CL(X) + A ( +)+ , A A mn -crx) A A of Xis deuce interval for Y or Y over a range where Ym = the average)pf m newlyobserved Y desired, merely compute theconfidence interval Crx, and n = values values of X, plot values; X, b, Y, sy Ex2, for several (usually about 5) obtained from the original set of dataand whose them on the same graph asthe regression line, meanings are unchanged. Note that m mayequal through them.. The and draw a smooth curve one, and)7,'would therefore be 'asingle intervals at the extremesof the data will be observation. . . wider than the intervalsnear the mean values. This is because the uncertaintyin. the estimated 7.3 .Tests of Hypotheses 1- slope is greater kn. the extremevaluesthan for If itis not clear that a relationship exists the central ones. between Y and X, a test should "be made to ith such a plot, the predicted valueOf Y and determine whether the slope differs from zero. its associated confidenceinterval for a given X The test is a t-test with n-2 degrees offreedom. can be read(see Figure 7, vertical line cone- The t value is computed as sponding to X = 3 and notation). b - Po t = (45), 7.2.3 Calibration curve sb Often with. data such as that givenin Table where 12, a calibratidn-curve is neededfrom which to sb predict X when Y is,given.That is, the linear Va2 CTx relationis established from thedata where fixed and then Y Since the null hypothesis is values of X (say pesticide) are 110:0 =0 (survival of eggs) is observed,where this relation predicts y given X; then unknownconcentra- set 130 = 0 in the t-test and it becomes b tions of the pesticide areused," egg survival t = measured, and the relation is Workedbackwards sb 4 24 4.
BIOMETRICS tINEAR REGRESSION
If the computed t exceeds the tabular t, then the level .of X; inthiscase always3..For the null hypothesis is rejected and the estimated example, slope, b,, is tentatively accepted. Other values of , Ti2 po may be tested in the null hypothesis and in E 51341 t1 at-test stVistic. With data such as those in Table 12, another With this, the analysis of variance table (Table hypothesis may be testedthat of lack of fit of 15) may be constructed.. In the first 'part of the model, to the data, or bias. This idea must be Table 15, the sums of squares and degrees of distinguished from random deviations from the freedom are given symbolically to relate to the straight line. Lack of fit impliesa nonlinear computations of Table13 and to the above trend as the true model, whereas random devia- computations. The mean squares (MS)are always tions from the model imply that the model°obtained by dividing SS by df. adequately, represents the trend. If more than , When the data for Table 12are arialyzed one Y observation is available for each.{ (3 in''(secondpart of Table15),thereis a -very the example Table 12); random fluctuationscan Unusual coincidence in thevalues of MS for be Separated from deviations from the model; deviations from regression, lack of fit, anderror. i.e., a random error may be computed at eachNote that this is coincidence and theymust point so that deviations from regression may be always be computed separately. partitioned into random error and lack of fit. As already known from the graph, t-test, The test is in the form of an analysis of vari- the regression is highly significant. A negative ance and is illustrated in brief form symbolically result from the teat for nonlinearity (lack of fit) in Table 14. Here, the F ratio MSL/MSE tests was also suspected from the visually-satisfactory linearity, i.e., whether a linear model is suffi- fit of Figure 7. Therefore, for thisrange of data, cient; the ratio MSR/MSD tests whether the we can conclude that a linear. (straight line) rela-, slope is significantly different from zero. TABLE 15. ANALYSIS OF VARIANCE OF TABLE 14. ILLUSTRATION OF ANALYSIS THE DATA OF TABLE 12; TESTS FOR OF VARIANCE TESTING LINEARITY OF LINEARITY AND SIGNIFICANCE OF REGRESSION AND SIGNIFICANCE OF . REGRESSION* REGRESS ION 'Source df ss Source df MS Total . n-1 Ey2-cry Total n-1 ).Regsession (EXY- Cr xir)2 "., ..Regression , 1 MSR MSRA1"3 Dey,iations from ( EX -cIx) regression M5b., Deviations from LaclCof fit m-2 MSL MSL/MSE regression n-2 Total SS - Regression SS Error n-m. MSE Lack pf fit m-2 Deviation SS - Error SS
Error n-m Ey2.ZT? To use this analysis,,,one set of computations must be made in addition to those of Table 13. *Syjpbols refer to quantities of. Table 13 of to sy`gbols de- The computation is the same as that for treat- fined in the text immediately preceding this table. ment sums of squares in the analysis of variance previously discussed; in this case, levels of Xare For the data of Tabl 12: comparable to treatments. First compute the sum of the Y's, Ti, for each level of X. For Source df SS Total 17 10;587 X = 1, Ti = 224, etc. Then compute: Regression 1 10,139 10.;11.9 362** Deviations from Ti-2 E regressi 16 448 28 Lack of 4, 113 028 1 n.s. Error 12 335 28 where ki = the number,fobservations for the **Significant at thp0.01 probability level. 25
17) t-y BIOLOGICAL METHODS
, tionship exists, with estimated slope and inter- analgebrtc rearrangement of the regression of cept as computed. Y on X. For the bivariate case, this approach is . not appropriate. If- a regression of Y on X is 7.4Regression for Bivariate Data fitted for bivariate data, and subsequently a pre- *, As mentioned, where two associated mease- diction of X rather than Y is ,desired, a new ments are taken without restrictions on either, regression must be computed. 's is a' simple the data are called bivariate. Linear regression is task, and all the basic quantities re tained in sometimes used to predict one of the variables a set ofcomputations similar to computa ns in by using a value from the other. Because no Table. 13. A summary of the .types of computa- tions for univariate and bivariate data is given in ,attempt is usually made to test bivariate dat*for _.lack of fit, a test for deviation from regression is Table 16. as far asan analysis of variance table istaken. Sincethe computationsforthe bivariate Linearityisassumed. Large deviations from regression of Y on X are the same as those for linearity will appear in deviations from regres- the univariate case, they will not be repeated. sion and cause the Flvalues that are used to test Wh re Xis to be predicted, all computations for the significance of regression to appear to be pr ceed simply by interchanging' .X andYin the nonsignificant. no ation. The computations for b and a are', Computations for thebivariate case exactly for the slze: ,follow those for the univariate case [quantities CTxy a (46) (1) to (12) and as illustrated for the univariate x. y2cfy case, Table 13rThe major operatingdifference is that, for bivariate data, the dependent variable (6) (12) is chosen as the variable to be-p edicted, whereas (5) (11) forunivariate data, the depe dent variable is for the intercept: fixed in'advance. For example, \,if the bivariate (IX) O ax.y bx.), ' (47)' data are pairs of observations onalx,a1 biomass n. and chlorophyll, either could be considered the ,(2) (3) isbeing' dependent variable.If biomass T1T 'Y(1) predicted, then it is dependent. For the uni- variate case, such as for the data of Table. 12,,7.5Linear Correlation percent survival- is the dependent variable by or virtue of the nature of the experiment. If a linear relationship is knOwn to 'exis In the, preceding section, it was seen that X can be assumed; the degree of associationof two and its confidence interval could be predicted'variables can be examined br linear correlation from Y for univariate d to (Equations 43, 44, analysis. The data must be bivariate. and 45). But note that E cation (43) is merely The correlation coefficient, r, is.compu' ted by -the following: TABLE 16. TYPES OF EXY CTxy COMPUTATIONS ACCORDING (48) -TO VARIABL PREDICTED AND 1/(1X2CTx) (tY2 7 CTy) DATA TYPE* A pepfect correlation (all points falling on a Predicted. Bivariate Univariate data straight line with a nonzero slope) is indicated variable data (fixed X's) by a correAtion- coefficient of, r = 1, Or. r Y y.= Ri (X) Y = R1 (X) The riegatkvalue implies a decrease in one X x= (Y) X = (y) the .vatiables'!with an increase in the of *RI' symbolizes tiar),..regressioilusir3g Y as Correlation coefficients of r = 0 implies ar dependent variable, R2 a regression cdmputed relationship between the variables.Arty Teal data using X as dependent variable, RI-t. is aalge- braic rearrangement solving for X when tlya- twill result in correlation coefficients between regression was RI. the extremes. ,26 O BIOMETRICSLINEAR CORRELATION
1.1 ;- If a ;correlation coefficient iscomputed and is The computed t is compared withal tabular of low magnitude, test it to determinewhether with n-2 degrees of freedom andchosen proba- it is significantly different frorri zero.The test, a bilitylevel.If the computed t-test, is computed as follows: t exceeds the tabular t, the null hypothesis that thetrue corre- r = lation coefficient equalszero is rejected, and the V( 1-r2) (49). computed r may be used. (n-2)
8.0 BIBLIOGRAPHY Cochran, W. G. 1959. Sampling techniques. John Wiley and Sons, New York. 330pp. Li, J. C..R. 1957y-Intro diiction to statistical inference. Edward Brothers, Inc., Ann Arbor.53 pp. Natrella. M: G. 1963. Experimental statistics. National Bureau of Standards Handbook Nol.1, U.S. Govt. Printing Office. Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods, 6th edition. lowa,State Uni.. Press, Ames. Southwood, R. T. E. 1966. Ecological methodswith particular reference to the study of, i London. 391 pp. sect populations. Chaplian,and Hall, Ltd. Steele, R. G. D., and J. H. Torrie: 1960. Principlesandprocedures of statistics with special Hill, New York. 481 pp. erence to the biological sciences. McGraw 4r Sirt, A. 1962. Basic ideas'of scientific sampling. HaTher,New York. 99 pp.
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I PLANKTON
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1.0 INTRODUCTION 1 Li 2.0 SAMPLE COLLECTION AND PRESERVATION 1 2.1 -General Considerations 1 2.1.1Ipfluential factors 1 . 2 2.1.2 Sampling frequency , 2.1.3 Sampling locations 2 2.1.4 Sampling depth 2 2.1.5 Field' notes 3 3 2.1,6 Sample labellirig v . 2.2 Phytoplankton . 2.2.1 Sampling equipment "---* 3 2.2.2 Sample yolume 4 2.2.3 54inple servation 4 2,3 Zooplankton 4 2.3.1 ampli g equipment* 4 2.3.2 Sample volume - 5- 2.3.3 Sampleeservation 5 3.0 SAMPLE PREPARA N 5 3.1 Phytoplankton 3.1.1 Sediment-0i n 6 3.1.2 Centrifugati , 6 6 . 3.1.3 Filtration 3.2 Zooplankton 6 4.0 SAMPLE ANALYSIS 6 4.1 Phytoplankton , 6 4.1.1 Qualitative analysis of phytoplankton 6 8 4.1.2 Quantitative analysis of phytoplanktoni 4.2 Zooplankton .12 4.2.1 Qualitative analysis of zooplankton 12 4.2.2 Quantitative analysis`f zooplankton 12 5.0 BIOMASS DETERMINATION 13 '5.1 Dry and Ash-Free Weight c .1'3 5.1.1 Dry weight 14
_ 5.1.2 Ash-free weight 14 5.2 , Chlorophyll 14. 5.2.1 In vitro measurements 4. , 14 5.2.2 In vivo measurement 15 5.2.3, Pheophytin correction 15 5.3 Cell Volume 16 /*. 5.3.1 Microscopic (algae and bacteria) 16 5.3.2 Displacement (zooplankton) 16 5.4 Cell Surface Area of Phytoplank'ton 16 6.0.PHYTOPLANKTON PRODUCTIVITY ,,, 16 6.1 Oxygen Method 16 , 6.2 Carbon-14 Method , 17 -.1 '7.0 REFERENCES 17 S. PLANKTON
L 1.0 INTRODUCTION the plankton move with the water currents;" Plankton are defined here as organisms sus- thus, the origin of the plankton may be obscure pended in a body of water and because of their and the duration of exposure to pollutants may, physical characteristics or size, "areyinpapable of be unknown. sustained mobility in directions c tyhter to the ,The quantity of phytoplankton occurring ata water currents. Most' of the planktonre micro- particular statio. depends upon many factors , including sampling depth, time of day, season of scopic and or essentially neutral buoys .All of them drift with the currents. year, nutrient content of water, and the pres- Plankton consists of both plants (phytoplank- ence of toxic materials. ton) and animals (zooplankton), and complex 2.0SAMPLE COLLECTION AND PRES- interrelationships exjgeamong the varidus com- ERVATION ponents of, these groups. Chlorophyll-bearing plants suchasalgaeusually constitutethe 2:1- General Considerations greatest portion or the biomass of the plankton. before plankton samplesare collected, a study, Phytoplankton use the energy of sunlight to design must be formulated. The objectives must metabolize inorganic nutrients and convert them be ,clearly defined, and the scope of the study to.,...,9omplex organic materials. Zooplankton and must remain within the limitatiOns of available other herbivores graze upon the phytoplankton manpower, time, and money. Historical, biqlo- andrin turn, are preyed upon by other organ- gical, chemical, and physiCal (especially hydro- isnis, thus passing the stored energy along to logical) data should be examined wheri, planning larger and usually Inore 'complex organ4ms. In a study. Examination of biological and 'chemical this manner16trients become available to-large data often reveals areas th'at warrant intensive organisms s.h as macroinvertebtates and fish..sampling and other areas where lQeriodic or \ Organic materials excreted by plankton, and seasonal sampling will suffice. I . 1 products of plankton decomposition, provide Physical data are extremely' useful inthe (.\- nutrients forheterotroplfic. microorganisms design of plankton studies; of particular impor- ,(many of which are also members of the plank- Lance are data concerning volume of flow, cur- ton assemblage)!. Th .heterotrophs break down rents,prevailing wind _direction, temperature, organic matter itnrelease inorganic nutrients turbidity (light penetration), depths of reservoir which become a'valable again for use by the penstock releases, and estuarinesalinity "primary produce . In waters severely pol- "wedges." luted by organic mat -r, such as sewage, hetero- After historical daia hayZ1been examined, the tro-ph-s--may -be- extremely abundant,- sometimes study site should' be visited for reconnaissance having"a mass exceeding that of the algae, As a and preliminary sampling. Based on the results result of heterotrophio metabolism, high con- of this "reconnaissance and on the preliminary centrations of inorganic nutrients beco'me avail- plankton data, the survey plan can be modified able and- massive algalbloornsIrray develop. to better fulfill study objectives and to facilitate Plankton may form thee base ,of the food efficient sampling. pyramid and drift with,the pollutants; therefore, data- concerning them may be particularly signif- 2.1.1Influential factors icant.to the pollution biologist. Plankton blooms In pliwning an conducting a plankton survey, often cause extreme fluctuations of the ,dis- a number of f ctors ,influence . decisions and solved oxygen content of the water, may be oneoften alter colleion routines. Since water cur-,,.)-- of the causes of tastes and odors in the water rentsdetermine of plankton and, if present in large numbers, are aesthetically movements, Icitowing the directions, intensity, objectionable. In some cases, plankton may be and complexity of currents in the sampling area of limited value as indicator organiSms because is important. Some factors that influence cur- s. BIOLOGICAL METHODS
I rents are winds, flow, solar,-heating, andlides.- of limitations in time arid manpower. Sunlight -influences, hOth. the moveihents of The physical nature of the water greatly plankton and prii 2 ,I -0; PLAliTONCOLLECTION sampling is usually sufficient. In deeper areas, , Total depth at station take samples at regular intervals with depth. If A list of all types of ''mples taken at only phytoplankton are to be examinedi samples station. may be taken at three depths, evenly spaced General descriptiv,e information(e.g., from the surface to the thermocline. When col- direction, distance, and descriptionof lecting zooplankton, however; sample at1- effluents.in thevicinity).Sampling Mete!: intervals from the surface to the lake stations should be plotted on a map. bottom. Because many factors influence the nature 2.1.6 Sample labelling and distribution of plankton in estuaries, in -" Both labAis and marker should be water proof tensive sampling is necessary. Here, marine and (a soft-leld pencil is recommended). Insert the freshwater plankton may be found along withlabels into sample containers immediately as brackish-water, organisms that are neither strictly plankton samples are collected. Record the marine nor strictly freshwater' inhabitants. In following information on all labels: addition to the influences of the thermocline and light penetration on plankton depth distri- Location bution, the layering of waters of different sa- name of river, lake, etc. linities may inhibitthe complete mixing of distance and direction to nearest city freshwater plankton with marine forms.In state and county estuaries 4with....e.x.tremetides,,,the dimensions of river mile, latitude, and longitude, or these layers may change considerably during the other description course of .the tidal cycle. However, the natural e.Date and time buoyancy of the plankton generally facilitates Depth the mixing of forms. Estuarine plankton should Type of sample (e.g., grab, vertical plank- be .sampled at regular intervals from the surface ton net haul, etc.) to the bottom three or four times during one or Sample volume, tow length more tidal cycles. . Preservatives used and concentration In deep marine waters orlakes, collect plank- Name of collector ton' samples at 3- to 6-meter intervals through- out the euphotic zone (it is'neither practical nor2.2 Phytoplankton profitable to sample the entire water coluinn in ,very deep waters). The limits of sampling depth 2.2.1Sampling equipment in these waters may be an arbitrary depth below The type of samping equipment used is highly the thermocline or the euphotic zone, or both. dependent upon where \and how the sample is Perform tow' or net sampling at 90° to the wind being taken (i.e., from a small lake, large deep direction.":" lake, small stream, large stream, from the shore, from a bridge, from a small boat, or from a large 2.1.5 Field notes boat) and how it is to be used. -Keep a record book containing all information The cylindrical type of sampler with stoppers written on the' sample label,plus pertinent that leave the ends open to allow free passage ofr_ additional notes. These additional notes may water through the cylinder whileit'sbeing include, but need not be restricted to: lowered is recommended. A messenger is re- leased at the desired depth to close the stoppers Weather information especially di- in the ends. The Kepimerer, Juday, and Van rection and intensity of wind Dorn samplers have such a design and can be etained in a, variety of sizes and materials. Use Cloud cover only nonmetallic samplers when metal analysis, Water surface condition smooth? Is algal assays, or primary productivity measure- plankton clumping at surface? ments are? being performed. In shallow waters Water color and turbidity and when surface samples aredesired,the 3 /° BIOLOGICAL-METHODS sampler can be held in a horizontal position and cluniping of settled organisms. One part of operated manually. For sampling in, deep waters; surgical detergent to five parts of water makes a the Nansen reversibig water bottle is often used convenient stock solution. Add 5 ml of stock and a boat equipp d with a winch is desirable. solution per liter of sample. Do, not use deter- Take caution when sampling from bridgeslith a'gent when dialorn slides are to be made. Kemmerer type water bottle; if the messe ger is Merthiolate is less desirable as a Rreservative, dropped from the height of a bridge, it can)but offers the advantage of staining cell parts batter and, destroy the trigglz device. To and simplifyingitlentifiFation.It also causes avoid this, support' a messengerr a ew feet above some of the algae, such as blue - greens, to lose the sampler by an attached string and drop itgas from their vacuoles and, therefore, enhances when the sampler is in place. settling. Samples preserved with merthiolate are Netcollection of phytoplanktonisnot not sterile, and should not be stored for more recommended fora quantitative work. Nanno- than 1 year. After that time formalin should be plankton and even larger algae, such as someused. Merthiolate solution is prepared by eis- pennate diatoms,arethinenoughtopass solving the folloWing in 1 liter of distilled water. through the meshes of the netif oriented properly. Using a pump also presents problems: when the water is stratified, the tubing must be 1.0 gram of merthiolate (sodium, ethyl- flushed between samplings and delicate algae mercury thiosalicylate). ,, 1.0 ml ofcitreous saturated iodine may be harmed. - . , potassium dide solution prepared by 2.2.2 Sample Vtgu,rne .z. dissolving 40 grams of iodine and 60 No fixed rule can. be followed concerning the grams of cotassitirn-iodide in 1ipter of volume of sample to be taken sampling per-, distilled water.- sonnel must use their own judgment. The vol- 1.5 gram of Borax (sodium borate) ume of the sample nee.fir depends on the numbers and Icindsof analyses to be carried out, Dissolve each of the components separately in e.g., cell counts, chlorophyll, dry weight. When approximately 300 ml of distilled water, corn - phytoplankton densitieare lest than 500 per bine, and make up to 1 liter with distilled watei.. ml, approximately 6 lits of sample are required Add the resulting stock solution to samples to for Sgdgwick-Rafter atilt; diatom species pro- give a final concentration (V/V) of 36 mg/liter portional counts. In most cases, a1- to 2-liter (i.e., 37.3'ml added to 1 liter of sample). sample will suffice for mare productive waters. 2.3Zooplankton 2.2.3 Sample preservation Biologists use a variety of preservatives, and 2.3.1Sampling equipment each has advantages. If samples are to be stored Zooplankton analyses require ,larger samples for more than 1 year, the preferred preservative than those needed for phytoplankton analyses. is formalin (40 percent formaldehyde = 100 pets. Collect quantitative samples with a messenger- cent formalin), which has been neutralized with operated water bottle, plankton trap, or metered sodium tetraborate (pH 7.0 to 7.3). Five milli- plankton net. Obtain semi-quantitative samples literstof the neutralized formalin are added for by filtering surface water samples through nylon each 100 ml of sample. This preservative will netting or by towing an unmetered plankton net cause many flagellated forms to lose flagella. through the water. In moderately, and highly Adding saturated cupric sulfate solution to the productive waters,a6-liter water sample is preserved -samples maintains the green color of usually sufficient. In oligotrophic, estuarine and phytoplankton samples and aidsindistin- coastal waters, remove zooplankters from sc,v_eral guishing phytoplankton from detritus. One milli- hundred liters of'the waters being sampled with liter of the saturated solution per liter of sample the use of towed nets. Take duplicate samples if is adequate. Adding detqrgent solution prevents chemical analyses are desired. ' 4 PLANKTON PRESERVATION Several sampling methods can be used. water, allow to dry, and lubricate all moving parts with light machine oil. Clean nylon netting Towing material thoroughly, rinse with clean water, and An outboard motor boat fitted witha small-allow to .dry (out of direct sunlight) before davit, meter wheel, wire-angle indicator, and storing. hand-operated winch is desirable. A 3- to 5-kg ) weight attached to the line is used to sink the 2.3.2 Sample volume net. Maintain speed to ensure a wire angle near The sample volume varies with the specific 60° for easy calculation of the actual sampling purpose of the ',study.Twenty-liter surface depth of the net. The actual sampling depth samples obtained by bucket and filtered through equals the amount of wire extended times the No. 20 net are large enough to obtain an cosine of the wire angle. .estimate of zooplankton present in 'flowing Oblique towMake, an 8-minute tow at four streams and ponds. In lakes, =large rivers, estu- levels in the water .column (2 minutes at each aries and coastal waters, filter 1.5 m3 (horizon- level:..just above the bottom, 1/3 total depth, tal tow)lo 5 m3 (oblique tow) of water through 2/3 total depth, and just below the surface) to nets for adequate representation of species pres- estimate zooplankton abundance. ent. Horizontal towTake samples for estimating 2.3.3 Sample, preservation zooplankton distribution and abundance within a particular layer. of water with a messenger- For identification and enumeration, preserve operated_ net equipped with a flow- through grab samples in a final concentration of 5 per- measuring, vane (such as- the Clarke-Bumpus cent neutral (add sodium tetraborate to obtain a sampler). Each tow lasts from 5 to 8 minutes. pH of 7.0 to 7.3) formalin. Adding either 70, Vertical towLower a weighted net to the percent ethanol or percent neutral formalin, desired depth, record the amount of line ex- each with 5 percent glycem (glycerol) added, to tended, and retrieve ..at a rate of 0.5 to 1.0 preserve the concentrated net samples. Formalin meters per second. The volume of water strained is usually used for p serving samples obtained can be ,estimated. Duplicate vertical tows are from coastal waters. Idetritus-laden samples, suggested at each station. add 0.04 percent Rose Bengal stain to help To sample most sizes of zooplankters, two differentiate zooplankter from plant material. nets of different mesh size can be attached a For chemical analysisaken, in part, from short distance apart on the same line. Recommended Procedures for Measuring the Productivity of Planktontanding Stock and Net casting Related Oceanic. Properties,ational Academy of Sciences, Washington, D. 1960), the con- Zdoplankton can also be sampled from shore centrated sample is placed in a fine-meshed by casting a weighted net as fat as possible, (bolting silk or nylon) bag, drained of excess allowing the net to reach depth, and hauling to water, placed in a plastic bag, and frozen. for shore at the rate of 0.5 to 1.0 meters per second. laboratory processing. If the sample is taken Take several samples to obtain a qualitative from an estuarine or coastal station, the nylon estimate of relative abundance and species bag is dipped several times in-distilled water to present. remove the chloride from interstitial seawater Suggested net sizes are: No. 6 (0.239 mm which can interfere with carbon analysis. aperture) for adult copepods in estuarine and coastal waters; No. 10 (0.158 mm) for cope- 3.0 "SAMPLE PREPARATION podites in saline water or microcrustacea in fresh water; and No. 20 (0.076 mm) for rotifers and 3.1' Phytoplankton nauplii. The No. 20 net clogs easily' with phy- As the phytoplankton density decreases, the, toplankton because of its small aperture size. amount of concentration must be increased and, Rinse messenger-operated samplers with clean,.accordingly, larger sample volumes are required. S 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,11 liter is an adequate routine sample volume, The following three methods may be used for concentrating preserved phytoplankton, but sedimentation is preferred. 3.1.1Sedimentation 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 ofdepth. Aftersettling,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, phyto-. plankton. 3.1.2Centrifugatibn During centrifugation," some of the more fragile forms may be destroyed or. flagella may become detached. In using plankton centrifuges, many of thecells may be, lost; modern continuous-flow centrifuges avoid this. Figur1.Plexiglas plankton settling tube with side drain and detachable cup. Not 3.1.3 Filtration drawn to scale. To concentrate samples by filtration, pass through .a membrane. filter. A specialfilter Take care to recover organisms (especially the apparatus and a vacuum source are required. Cladocera) that cling to the surface of the water Samples containing large amounts of suspended in the settling tube. material (other than phytoplankton) are difficult to enumerate by this method, because 4.0 SAMPLE ANALYSIS the suspended matter tends to crush' the phyto- 4.1Phytoplankton plankters or obscure them from view. The i=) vacuum should not exceed 0.5 atmospheres. 4.s1.1Qualitatize analysis of phytoplankton .,,,Concentration by filtration is particularly useful The optical equipment needed includes .a good for samples low in plan .and_silt content_ quality compound binocular microscope with a mechanical stage. For high magnification, a sub- 3.2i1Zooplankton- stage condenser isequired. The Ocular lens The zooplankton in grab samples are con-should be 8X to 1 .Binocular e6pies are centrated prior' to counting by allowing them to generally preferreover a monocular e ce settle for 24 hours in laboratory cylinders of -because of reduced fatigue. Four turret-mounte appropriate sizeor in sOcially constructedobjective lenses should be provided with mag- settling tubes (Figure 1). nifications of approximately 10, 20, 45, and ;., ;. 6 -' 0 PLANKTON COUNTING 100X When combined with the oculars, the tween "live" cells, i.e., those that contain any following chaoeteristicsareapproximately part of a protoplast, and empty frustules or c.QrreCt. shells. The availability of taxonomic bench refer-_ ences and the skill of the biolOgist will govern Maximum working the sophistication of identification efforts. No ObjectiveOcular Subject distance betweenDepth of single reference is pletely .adequate for all lens lens,magnification objective and focus, A I cover slip, mm phytoplankton. So e general references that 10X10X 100X 7 '8 shoUld be availableare, listed below. Those 20X.10X 200X 1.3 2 marked with, an asterisk are considered essential.. 45X'-1-0X 450X 0.5-0.7 1 American Public Health Association, 1971. Standard methods for the examination of water and wastewater. 13th, edition. 100X 10X 1000X 0.2 0.4 Washington, D.C. Bourrelly, P. 1966-1968. Les algues d'eau douce. 1966. Tome I-111, Boubee & Cie, Patis. , An initial examination is needed because most Fott, B. 1959. Algenkunde. Gustav Fischer, Jena. (2nd revised 'phytoplankton samples will ,contain a diverse edition, 1970.) Prescott, G. W. 1954. How to know, the fresh-water algae. W. C. assemblage of organisms. Carry out the identi- Brown Company, Dubuque. (2nd edition, 19640 fication, to species whenever possible. Because *Prescott, G. W. 1962. Algae of the Western Great Lakes Area. the size range. of the individual organisms may (2nd edition), W. C. Brown, Dubuque. - extend over several orders of. magnitude,, no *Smith, G. M. 1950. The freshwater algae of the United States. single magnification is completely satisfactory (2nd edition), McGraw-Hill Book Co., Newyork, 4 for identification. For the initial examination, Ward, H. B., and G. C. Whipple. 1965, Fresh- w, biology. 2nd editiontion edited by W. T.dmonson. fohn WilekInd Sons, New place one or two drops of a concentrate on a York. glass slide and cover withNo. 1 or No. 1-1/2 *Weber, C. I. 1966. A guide to the common`diatoms at water cover slip. Use the 10X objective to examine the 'pollution surveillance system stations. ,USDI, FWPCA, Cin- entire area under the cover slip and record all cinnati. identifiable organisms. Then examine with the' West, G. S., and F. E. 'Fritsch: 1927M treatise on the British freshwater algae. Cambridge Un.v. Hess. (Reprinted 1967; J. 20 and 45X objectives. Some very small or- Cramer, Lehre; Wheldon esley, Ltd.; and Stecherthafner, ganisms may require the use of the 100X Inc., New York.) objecti+e (oil immersion) for identification. The initial 'examination helPs to obtain an estimate Specialized ,references that may' be required of population density and may indicate the need'for exact *identification. within certain for subsequent dilution or concentration of thenomic groups include: sample.,to recognize characteristics of small N3rant, K., and C. Apstein. 1964. Nordisches /Zlankton. A. Asher forms hot obvious during, the routine counting & Co., Amsterdam. (Reprint of the 1908 publication published procedure-T--and to decide if more than one type by Verlag von Lipsius & Tischer, Kiel and Leipzig.) Cleve-Euler, A. 1968. Die diatomeen von Schweden and Finn- of counting procedure must be used. land, I-V. Bibliotheca Phycologica, Band 5; J. Cramer, Lehre, When identifying phytoplankton, it is useful Germany. 4 to ermine fresh, unpreserved samples. Pres- Cupp, E. 1943. Marine plankton lliatoms of the west coast of may cause some forms to become, dis- North America. Bull. Scripps. Inst. `Oceanogr., Univi Calif., torted, e flagella, or be lost together. These 5:1-238. - can be determined by a comparison between Curl, H 1959. The "phytoplankton of Apalachee Bray and the Northwestern Gulf of Mexico. Univ. Texas Inst. Marine Sci., fresh and preserved samples. Vol. 6, 277-320. Ai the sample is examined under the micro- *Drouet, F. 1968. Revision of the classification,of the Oscilla- scope identify the phytoplankton and tally toriaceae. Acad: Natural Sci., Philadelphia. under the following categories: coccoid blue- *Drouet, F., and W. A. Daily. 1956. Revision of the cocdoid green, filamentous blue-green, coccoid green; Myxophyceae. Butler Univ. Bot. Stud. XII., Indianapolis. filamentous green, green flagellates, other pig- Fott, B. 1969., Studies in phycology. E. Schweizerbaresche, mented flagellates, centric diatoms, and pennate Verlagsbuchhandlung (Nagele u..0beriniller), Ger- diatoms.Intallying diatoms, distinguish be- many. , BIOLOGICAL METHODS Fritsch, F.1E. 1956. The, structure and reproduction of the directly. In those samples 'where algal concen- algae. Volumes I and II. Cainbridge UniverSity Press. trations are extreme, or where silt or detritus G6tler, L. 132. Cyanophyceae. 1n: Rabehnorst's Kryptoga- may interfere, carefully dilute a 10-m1 portion ine n-F lora, 1 4 : 1-1 096. Akademische Verlagsgesellschaft m.b.H., Leipzig. (Available from Johnson Reprint Corp., New of the sam4ble" 5 to 10 times with distilled water. York.) I In samples with very low populations, it may be Glezer, Z. I. 1966. Cryptogamic plants of the U.S.S.R., volume necessary to concentrate organismsto minimize VII: Silioflagellatophyceae. Moscow. (English Transl. Jerusa- lem, 1970) (Available from A. Asher & Co.; Amsterdam.) statistical counting errors. The analyst -should Oran, H: H., and E. C. Angst.. 1930. Plankton diatoms of Puget recognize, however, that manipulations involved ,Sound. I4niv. Washington, Seattle. . in dilution and concentration may introduce iendeY, N: I. 4964. An introductory account of the smaller errO,5. algae of British coastal waters. Part V: Baccilariophyceae (Dia- 4Mong the various taxa are forms tat live as: toms). Fisheries Invest. (London), Series IV. . , solitary cell,' as components of naturgroups Huber-Pestalozzi, G., and F. Hustedt. 1942. Die Kieselalgen. In; -'A. Thienemann (ed.), Das Phytoplankton des Susswassers, Die or aggregates (colonies), or as both. Binnengewasser, Band XVI,, Teil II, Halfte II. E. Schweizer- every cell, whether solitary or in a group; can be baresche Verlagsbuch-handlung, Stuttgart.(Stechert, New individually tallied, this procedure is difficult, York, reprinted 1962.) . time consuming, and seldom worth the effort. *Hustedt, F. 1930. Die Kieselalgen. In: L. Rabenhorst (ed.), Kryptogamen-Flora von Deutschland, Osterreich, and der The unit or clump count is easier and faster and Schweiz. Band Vii. Akademische Verlagsgesellschaft m.b.h., isthe system used commonly withinthis Leipzig. (Johnson Reprint Co., New York.) Agency. In this ocedure, all .unicellular or Hustedt, F. 1930. Bacillariophyta. In: A Pascher (ed.); Die colonial (multi-cellular) organisms are tallied as Suswasser-Flora Mitteliuropas, Heft 10. Gustav Fischer, Jena. (University Microfilms, Ann Arbor, Xerox.) dingle units and have equal numerical weight on Hustedt,I F. 1955. Marine littoral diatoms of Beaufert, North the bench sheet. Carolina. Duke Univ. Mar. Sta. Bull. No. 6. Duke Univ."Press, The apparatusandndthniques used in"' Durham, N. C., 67 pp. counting phytoplankton are d scribed here. lienee-Mixie, F. 1938. Fiore Desmidiale de la region de Mon- treal. Laprairie, Canada. *Patrick, R., and C. W. Reimer. 1966. The diatoms of the United . States. VoL I. Academy of NturatSciences, Philadelphia. Sedgwick-Raftei. (S-R) counting Chamber Tiffany, L. H., and M. E. Britton. 1952. The algae of Illinois. Reprinted in 1971 by Hafner Publishing Co., New York. Tilden, J. 1910. Minnesota algae, Vbl. I. The Myxophyceae of The S-R cell is 50 mm long by .20 mm wide by North America and adjacent regions including Central America, 1 mm deep; thus, the total area of the bottom of ) Greenland, Bermuda, the West Indies and Hawaii. Univ. the cell is 1000 mm2 and 'the total volume is 'Minnesota. (First and unique volume) (Reprinted, 1969, in Bibliotheca Phycologica, 4, J;Cramer, Lehre, Germany.) 1000 mm3 or one mk Check the volume of each counting chamber with a vernier caliper an,d micrometer..Because the depth of the' chamber 4.1.2 Quantitative analysis of phyt plankton normally precludes the use' of the 45X or 100X `To calibrate the microscope, the ocular must objectives, the 20X objective is generally used. HowevF, special long-working-distance, higher- be' equipped with a Whipple grid-type micro- power obleitives can be obtained. meter. The .exact magnification with any set of Far the procedure, see Standard Methods, oculars varies, and therefore, each combination 13th Edition. Place a 24 by 60 mm, No. 1 cover- ofoculars,and objectives must be calibrated by glass diagonally across the cell, and witha large- matching the ocular micrometer against a stage bore pipet or eyedropper, quickly 'transfer a 1-ml micrometer. Detail§ of the procedure are given a,liquot of well-mixed sample into the open corner of the Chamber. The sample should be di- in Standard Methods, 13th Edition rected diagondlly across the bottom of the cell. When Counting and identifyingphyto- Usually, the cover slip will rotate into place as ,plankton, analysts will find that samples from the cell is filled. Allow the S-R cell to stand for most na-tural, waters seldom need dilution or at least 15 minutes to permit settling. Because concentration and that they can be enumerated some ,organisms, notably blue-green algae, may 8 PLANKTON COUNTING 0,9 float, examine the underside of the cover slip and Palmer-Maloney (P-M) Nannoplankton Cell adfl these organisms to the total count. Lower The P-M cell Was especially designed for the objective lens carefully into position with enumerating nannoplankton with a high-dry the coarse focus adjustment to ensure that the objective-(45X). It has a circular chamber 17.9 cover slip will not be broken. Fine focus should mm in diameter and 0.4 mm deep, with a always be upzfrom the cover slip. volume of 0.1 ml. Although useful for exam- When making the strip count, examine two to ining samples containing a high perceniage of four "strips" the length of the cell, depending nannoplankton, more counts may be req*edi to upon the Tensity of organisms. Enumerate- all obtain a valid estimate of the larger, but less forms that are totally or partially covered by the numerous, organisms present. Do not use this image of the Whipple grid. cell for routine counting unless the samplesha:ve When making thefield count, examine a high counts. minlintun of 10 random Whipple, fields in a0 Pipet an aliquOt of well.:mixed sample into least" two identically prepared S-R cells. Be sure one of the Q X 5 mm channels on either side of to adopt a; consistent system, of counting organ- the circular chamber with the coveOlip in place. isms that lie only Partially .within the grid .or'After 10 minutes, examine the sample under the that touch` one of the edges.. high-dry objective and count at least'20 Whipple To calculate; the concentration of organisms fields. with the S-R cell, for the strip count: To calculate the concentration of organisms: ,z >c woo mm3 X 1000 mm3 No. per ml = per m1=7LXDXWXS AXDXF a where: where: C =number of organisms,counted (tally) C number of organisms counted (tally) A =area of a field (Whipple grid image), mm2, L =length of each strip (S-R cell length), mm D. =depth of a field (P-M,cell depth), mm D =depth of a strip (S-R cell depth), mm F =number of fields counted W =width of a -iffiti(Whipple grid 'image width), nun Bacterial Counting Cells and Hemocytometers S =number of strips coted The counting cells in this group are precisely- 'machined glasnlides with a finely ruled grid on To calculate, the concenhqrtion of-organisms a counting, plate and specially-fitted ground with the field count: cover slip. The counting plate proper is sepa- ( rated from the cover slip mounts by parallel C X 1000 mm3 trenches on opposite sides. The grid is ruled such No. per ml AXDXF that squares as small as 1/20 mm (50 to a side are formed within a larger 1-mm square. With the cover slip, in place, the depth-in Petroff- where: Hausser cell, is1/.50 mm' (20p) and inthe C =actual count of organisms.(tally) hemocytometer 1/10 mm (100p). An optical A =area of a field (Whipple grid image area), micrometer is not used. mm2 With a pipet or-medicine dropper, introduce a D =depth of a field (S-R cell depth), mm sample to the cell ant at high magnification . F = umber of fields counted identify and count all, the forms that fall with' the gridded area of the cell. Mu tiply or divide the number of cells per To calculate the number of organisms millili er by a correction factor for dilution milliliter, multiply all the or anism§ found in the (inclu ng that resulting from the preservative) gridded area of the cell by the appropriate or for concentration. factor. For example, the multiplication factor BIOLOGICAL MiTtIODS for the-Petroff-Hausser.bacterial counting cell is based on the volume over the entire grid. The Inverted Microscope dimensions areIfilm X I mm X 1 /50 mm,. This instrument differs from the conventional which gives a volume of 1/50 mm3 and-a factor microscope in that the objeCtives are mounted_ of 50,000. , Carefully follow the manufacturer's instruc- below the stage and the illumination comes from tions that come withthe chamber 'Vvhen above. This design allows cylindrical counting purchased. Do not att%mpt routine countaruntil chambers (which may also be sedimentation experienced in its use and the statistical validity tubes) with thin clear glas4ottoms to be placed of I the results are satisfactory. The primary on the stage and sedimeTted plankton to be disadvantage of this type of counting cell is theexamined from below, and it permits the use of . extremely limited capacity, which results. in 4 shortfocus,high'-wgnificationobjeetives, -large multiplication, factor. Densities as high as including oil immersion. A. wide range of con- 50,000 cells/ml are seldom found in natural centrations is automatically obtained by merely' waters except during bloomi. Such populations altering the height of the chamber. Chambers \Inay.be found in sewage stabilization ponds or in can be easily and inexpensively made: use tubu- laboratoiy cultures. lar Plexiglas for large capacity chambers, -and For statistical purposes, a normal sample must -flat, plastic plates of various thicknesses, which . be either concentrated or a large numberof have been carefully bored out to the deSired °mounts Per sample should be examined. dimension, for smaller Chambers.;it then dement a No. 1 or No. 1-'1/2 cover slip to form the. cell Membrane Filter. bottom. Precision-made,all-glasscounting A", specialfiltration appar.atus and vacuum chambers in a wide variety of ym nsions are source are required, and a 1-inch, 0.4511 mem-.also available. The counting ue differs brane filter is used. little Aom the S-R procedure, aneither the Pass a known volume of; the water sample strip or- separate field-counts can be used. The through he membrane filter under a vacuum of Whipple eyepiece micron is also used. atmospheres. (Note: in coastal and marine Transfer a: sample into the desired, counting 'waters, rinse with distilled water to remove salt.) chamber (p4ur with the large chambers, or pipet ;Allow the filter to dry at room temperature fer,)with 2-ml or smaller, chambers), fill to the point 5 minutes, and place it on top of two drops of of Overflow, and apply a glass cover slip. Set tie immersion oil on a microscope.slide. Place two chamber aside and keep at room temperature drops of oil on top of the filter and allow it to until ?sedimentation is complete. On the average, dry clear (approximately 48 hours) at room allow 4 hours per 10 mm of height. After a suit-' temperature, cover withacoverslip,and able period .of settling, place the chamber on the enumerate the organisms. The occurrence of , microscope stage and examine with the use of each species in 30 random fields is recorded. the 20X, .45X, or 100X oil immersion lens. Experience 'is 'requiredtodetermine the Count at least two strips perpendicular to each proper amount of,,water to be filtered. Signifi- other over the bottom of the climber ankl aver- cant amounts of suspended matter may obscure age\sthevalues.Alternatively, randomfield or crush the organisms. counts can be made; the number depends on the Calculate the oWginal concentration in the density_ of organisms found. A.S% a general rule, sample as a function of' a conversion factor count a minirmfm of 100 of the most abundant obtained from a prepared table, the number ofspecies. At higher magnification, count more' quadrates or fields per filter; the amount of_fields than under lower power. sample filtered,, and A the dilution. factor. (See When,a 25.2 mffi diameter countingchamber Standard Methods, Inn Edition.) ," is used (the most convenient size), the conversion 10' I PLANKTON COUNTING of counte-td numbers per ml is quite simple: method, however,. toefturequantitative c x 1000 mm3 removal of cells smaller than 10 microns in No. per ml (strip count)LXWXDXS diameter. Thoroughly mix the plankton concentrate' in a where: vial with a disposable pipet, anclideliver several C =number of organisms counted (tally) drops to a No. 1, circular 18-mm coverglass. Dry L =length of a strip, mm the samples on a hotplate at 95°C..(Caution: W= width' of astrip(Whipple gridimage,°overheating may cause, splattering and cross- width), mm contamination of samples.) When gry, examine D =depth of chamber, mm , the coverglasses to determine if there is suffi- S = 17mber of strips counted cient material for a diatom count. If not, repeat C X 1000 mm3 No. per ml (field count) = the previous steps one or two more times, A X D"X F vr- depen,ding upon the density of the seclimented sample. Then heat the samples dn a-heavy-duty -where: ( C = .number of organisms counted (tally) hotplate 30 minutes. at approximately 570°C to A =area of a field (Whipple grid image area), drive 'off alt organic matter. Remove grains of mm2 sand or other large objects Qn the cover glass D=depth of chamber; mm with a dissection needle. The oil immersion,. F=number of fields counted objective has,a very small workint distance, and the slide may be unusable if this is not done. Diatom Analysis' Label the frosted end of a 25-,- X 75.rnm Study objectives often require specific identi- microscope slide with the sample identification. ficatidn'of diatoms and information about the Place the labelled slide on a moderately warm relative abundance of each species. Since the hotplate (157°C), put a drop of Hyrax , or taxonomy of this group is based on frustule AT/dor 5442 (melt anduse at about 138 °C) characteristics, low-power magnificationis 4'mounting medium (Index ofRefraction seldomsufficient, and permanent diatom 1.66-1.82) at the center, and heat the slide until mounts are prepared and examined under oil the solvent (xy,lene or toluene) has evaporated immersion. (the solvent is gone when the Hyrax becomes To concentratethe diatoms, centrifuge 100 'hard and brittle upon cooling). ml of thoroughly mixed, sample for 20 minutes While the coverglass and slide are stillhot, at WOO X g and -decant the supernatant with a grasp the coverglass with a...tweezer, inveft, and suction tube. Pour the concentrated sample into Place On the drop of Hyrax on a slide. It may be a disposable vial, and allow to stand at least 24 necessary to iedd Hyrai at the margin of the hours before eurther. processing. Remove the coverglass. Mlle additional bubbles of. solveht supernatant water from the vial with a suction vapor may appear under the coverglass when it is tube. If the water contains more than 1 gm of.placed on the slide. When the bubbling' ceases, dissolvedsolidsper.liter '(as in the case of remove the slide from the hotplate and place on brackish water or marine samples), salt "crystals a firm,flat- surface. Immediately apply slight form' when the sample dries and obsCure the pressure to the coverglass,with a pencil eraser (or diatoms 'onthe' finished slides.In this case, similar object), anSI maintain until the Hyrax reduce the concentration Of salts by refilling th cools and hardens (about/ 5 seconds)'. Spray a vial with distilled water, resuspending the.plank- protective coating of clear acquer on the frosted ton, and allowing the vial to stand 24 hours end of the slide, and scrape the excess Hyrax before repoving the supernatant liquid. Repe from around the coverglass.. the diluti6n several times if necessary. Identify and countthe., diatoms athigh If the plankton counts are less than 1000 per magnification under oil. Exarhine random lateral ml,concentratethe diatoms from alarger strips th 'width of the Whipple grid, and iden- volume of sample (Ito 5. liters) by _ allowing tify)and ount all diatoms within the borders of them to settle out. Exercise caution in using this the/ grid until 250 cells" (500 halves) are tallied. BIOLOGICAL METHODS 6 Ignoke small cell' fragments. If the slide has very Edmondson, W. T. (ed.). Ward, H. B. and G. C. Whipple. 1959: . few diatoms, limit:the analysis to the number of Fresh-water biology. John Wiley and Sons, New York, 1248 tells encountered in 45- nitnutes of scanning. Faber, D. J. and E. J. Jermolajev. 1966. A new copepod genus in *When the, count is completed, total the tallies the _plankton of the Great Lakes. Limnol. Oceanogr. 11(2):. Ed calcUlate the percentages of the indiVidual 3.01 -303. species. Ferguson, E., Jr. 1967. New ostracods from the Playa lakes of eastern New Mexico and western Texas. Trans. Amer. Microsc. 4.2 Zooplankton Soc. 86(3):244-250. Hyman, L.IL1951. TheInvertebrates: Acanthocephala, 4.2.1' Quglitative analysis of zooplankton Asch e 1m in thes,an dEctoprocta. The pieudocoelomate Bilateria. Vol. III. McGraw-Hill, New York,,572 pp. In the initial examination, remove 'excess Light,. S. F. 1938. New subgenera and species of.diaptbmid preservative from the sample with the use of an copepods from the inland waters of California and Nevada: aspirator bulb attached to' a sm011 pkee of glass Univ. Calif. Publ. in Zool. 43(3): 67 -78. UniV. Calif. Press, tubing, whosestrifice is _covered with a piece of Berkeley. Marsh, C. C. 1933. Synopsis of the calanoid.crustaceans, exclu- No. 20 mesh, netting. Swirl the sample, and with sive 'of the Diaptomidae, fotind in fresh and brackish waters, alarge-bore pipet, remove a- portion of -the'chiefly of North America. No. 2959, Proc: U. S. Nat. Mus. 82 suspension and place 2 ml into each section of a (Art. 18): 1-58. ' four-compartment g,Idss culture dish (100 X 15, Pennak, R. W. 1953. Fresh-water invertebrates of the United' mm). Examine, atotalof -8 ml for adult States. The Roland Press Co., New York. 369 pp. Rubel.; E. 1968. Description of a salt marsh copepod cyclops Copepoda, Cladocera; and other large forms (Apocyclops) spartinus' n. sp. and a comparison with closely with the use of ,a binocular dissecting micro- related species. Trans. Amer. Microsc. Soc. 87(3):368-375. scope at a magnification of 20 to 40X. Count Wilson,. M. S. 1956. North American Harpacticoid copepods. and identify rotifers at a higher magnification 1. Comments on the known fresh- voier species of the', (100X). All animals should be identiflea to _Canthocamptidae. specles if possible. For qualitative analysis. of 2. Canthocamptus oregonensisn.sp.from 'Oregon and relative frequency, the follomingclassification is California. Trans. Amer. Mimi:6c. Soc. 75 (3): '290 -307. Wilson, M. S. 1958. The copepod genus Halicyclops in North suggested: America, with a description of a new species from Lake Pont- chartrain, Louisiana, and the Texas coast. Tulane Studies ZooL 6(4): 176-189. Species in Relative Zimmer, C. 1936. California Crustacea of the order cumacea. fields, % frequency No. 2992. Proc.. U. S. Natl. Mus. 83:423-439. 60- 100 abundant 3O 60 very common 4.2.2 Quantitative analysis ofj)oplankton 5. --30 common 1- 5 occasional Pipet Method <1 rare Remove excess liquid using a screened (No.0 The following taxonomic bench references are mesh net) suction device until a 125: to 250- 1 recommended:. sample volume remains:Pour the sampinnto a Calman, W. T. 1912. The Crustacea of the order Cumacea in tie conical c9ntainer graduated in milliliters, an collection of theUnitedStatesNational Museum.. No. , allow the zooplankton to settle for 5 minut s. 18 6 -Proc. U. S. Natl. Mus. 41: 603-676. es. Read thesettled volume of zboplankt Cbien, S. M. 1970. Alonella fitzpatricki sp. n. and A. leei sp. n:, multiply the settled volume by a factor of f1 e 8,Cladocera from 'Mississippi, Trans. Amer. Microsc. Soc. 4): 5321,538. to obtain the totalclilltet1 volume; and ad Conseil Permanent International Pour L'Exploration De La Mer. enough water to oftain this volume. Insert a . 1970. Fiches D'Iderilification du Zooplankton. Sheets No.'s 1-ml Stempel pipet intothe 'water-plankton 1-133. mixture, and stir rapidly witli the 'pipet. While Davis, C. 1949. The pelagic Copepoda of thenortheastern the mixture is stilt agitated, withdraw a 1-ml Pacific Ocean. Univ. Wash. Publ. in Biol. 14:1-188. Univ. Wash. `.Press, Seattle. subsample from the center of the water mass. Davis, C. 1955. The marine and freshwater Plankton, Mich. State Transfer the'subsprriple to a ogridded culture-dish Univ-Press, East Lansing. (110 X 15mm) with 5-mm squares. Rinse the ' 12 PLANKTON BIOMASS, pipet with distilled water into a cultureAish to When calculating the nunibe -.-Of Plankton, remove any adherent, organisms. Enumerate determine the volume of the counting chamber (about 200. zdeplankiers) and identify under a from its inside dimensiont. Convert the tallies to dissecting microscope. organisms per liter With the use of the following To calculate the number of planktOn with an relationships: =petered collecting device: T X C Rotifers per liter .1' X V, Total no. DV= X TN SV )1, 7 TX C Microcrustami per liter = SX V where: To calculate the number of plankton with T =total-tally . metered collecting device:' C =total volume Of sample concentrate, ml , . P volume of 10stripsinthe Counting chamber, ml V =- volume of netted or grab sample, liters TN XDV SV S =volume of counting chamber, ntI No. per m3 of water = Q 5.0 BIOMASS DETERMINATION- where: Because natural planktop populations are DV = total diluted voliime, mi cornposed of many 'types. of orm (i.e.', plant, animal, and bacterial), it is difficult to SV = total subsample volume, ml 9 obtain, quantitatiVe values for 'each of the .cOrn- TN = total no. iooplankteits in sainple po nen tpopulations.Currently-Used indices Q = quantity of water strained, m3 include dry and ash-free weight, cell volurne, cell surface area, total carbon, . total, nitrogen; and.' Counting Clia?riber. t chlorophyll content.? The dry and ash-free Bring the entire conCettrate - (di an appro- weight methods yield data that.include the par- ., priate aliquOt) to a volume of 8 mix well, ticulate inorganic materials as Well as the plank- and transfer to a counting chamber 80'X 50 X '2 ton. Cell volume and cell surface area determi- mm (8-ml. c'apacit'y). To fill, use the technique nations can be made on individual components:. previously described for the Sedgwick-Rafter of the population, ,and -thus yield data on the. plant, the animal, or th6:.bacteriab volume; Or cell. The proper degree of sample concentration Surfacearea,or biith: Chlorophyll determi- can be_determined only by experience: nations yield data on the phytoplankton. Using a compound microscope-equipped with an ocular Whipple grid, enumerate and identify'',5:1 Dry and Ash-Free Weight the rotifers (to species if posible) in 10 strips s scanned at a magnification of 100X (one-fifth of To reduce the amount of contamination. by dissolVed solids, wash the saMple with... several the chamber volume). Enurngrate the aupliivolumes of distilled 'Water by Centrifugation or also during the ,.rotifer count: Count the adult settling. After -washingconcentrate the Sample microcrustaceaunder 'abinocular dissecting-by centrifugation or settling. If possible, take, microscope at a magnification of 20 to 40X by sufficient, sample to, provide several aliquots each scannAg the entire chaMber. Species identi- having at least 10 mg dry weight.Process at ficati of. rotifers and microcrustacea often least two replicate aliquots for each sample. requiredissection and e$amination under a (Generally,. 10 mg :chi weight is equivalent- to Compound microscope (see.Pennak, 1953). 1.00 mg wet weight.) 13 A BIOLOGICAL, M ETIIODS 5.1.1 1)ry weight the concentrate and store frozen in a desiccator. Place the aliquot of concentrated sample in a Keep the stored samples' in the dark . to 'avoid tared porcelain crucible, and dry to a constant photochemical breakdOwn of the chlorophyll. weight atI 05°C (24 hours is usual/ sufficient): Place the sample in a tissue grinder, cover with Subtract the weight of enicible to obtain the dry 2 to 3 milliliters of 90 percent aqueous acetone weight. (use reagent grade acetone), add a small amount (0.2 ml) of saturated aqueous solution of magne- 5.1.2 Ash-free weight sium carbonate and macerate. . Transfer the sample to a screw-capped cen- After the dry weight is' detdrinined, place the trifuge tube, add sufficient 90 percent aqueous crucible in a muffle furnace at 500 °C for I hour. acetone to bring the volume to 5 ml'; and steep Cool, rewet the ash with distilled water, and at 4 °C for 24 hours in the dark. Use the solvent bring' to constant weight atI 05°C. The ash is sparingly, avoiding unnecessary pigment dilu- wetted to reintroduce the water of hydration of tibn. midway during the extraction the clay and other minerals that; -though not period and again before clarifying. driven off atI 05°C, is lost at 500°C. This water To clarify the extract, centrifuge 20 minutes loss often amounts to 10_percent of the weight at 500 g. Decant the supernatant into a clean, lost during ignitiOn and, if not corrected for, will calibratedvessel(5-m1,- screw-capped,cali- be interpreted as organic matter. Subtract the brated centrifuge tube) and determine the vol- weight, of crucible and ash from thkcyweight umMinimize evaporation by 'keeping the tube to obtain ash-free weight. capd. 5.2 Chlorophyll hree procedures for analysis and concen- tration calculations are described. All algae contain chlorophyll a, and measuring this -pigment can yield some insight into the Tricliromatic Method relative amount of algal standing crop. Certain Determine the optical density (OD) of the algae also contain chlorophyll b and c. Since the extract at 750, 663, 645, and 630 nanometers chlorophyll concentration varies with species and (nm) using ii- 90 percent aqueous acetone blank. with environmental and nutritional factors that Dilute the extract or shorten the light path if do not necessarily, affectthe standing crop, necessary, to bring the OD6 6 3 between 0. :i0 and biomass estimates based 9n chlorophyll-measure- 0.50. The 750 nm readingois used to correct for' ments are relatively imprecise. Chlorophyll can turbidity.Spectrophotometers ,,hang a reso- be measured in vivo .fluorometrically or -in ace- lution of 1.nm or less are prefei(red. Stopper the . toneextracts(in, vitro)by fluorometryor cuvettes tominimize evaporation during the spectrophotometry. time the readings are being-made. Thechlorophyll concentrations in the extract 5.2.1 In vitro measurement . \ are deermined by inserting the corrected 1-cm The .algae differ ,con'siderably, in the eale_ f 1 OD's. inthe following" equations. (UNESCO. pigment extraction. The diatoms extract, eaSil . whereas the coccoid greens ,extract withr'',.diffi- culty. Complete extractionof pigments from all Ca = 11.641)663 2.16D645 0.10D630 taxonomic groups, therefore, requires disruption cb =-3.94D663+ 20,97D645 3.66D630 of the cells with a tissue irider or blender, or Cc = -5.53D6 63 14.81D645+ 54.22D63O by freeiing or dryingGenerally, pigment is more difficUlt to extract from old cells than from young cells. )where Ca, Cb, Ccre the concentrations, in Concentrate -the algae with 'a laboiltory cen- milligrams per liter, of chlorophyll a, b, and c, trifuge,. dr collect on a membrane filter (0.45-ixrespectiOly, in the extract; and D663, D645 pOioSity.)or a glass fiber filter (0.45,-// effective and D630 are the 1-crii OD's at the respective pore size).- Jf the. analysis will be .delayed,- dry wavelengths:after subtracting the 750-nm blank. 14 PLANKTON PIGMENTS ' The concentration of piguNnt in the phyto- To determine the chlorophyll a,zero the plankton grab sample is expinsed as mg/m3 or fluorometer with a distilled water blank before pg/m3 of pg/liter and is calculated as follows: taking the first sample reading at each sensitivity level. , Co X volume of extract (liters) Mix the phytoplankion sample thololighly to mg chlorophyll a/m3= volume of grab sample (m3) ensure a homogenous suspensioiCof algal cells. Pour an aliquot of- the well-mixed sample into a Fluorometric (for chlorophyll a) cuvette, and read the fluorescence. If the reading (scale deflection) is over 90, units, use allower The fluorometric method is much more sensi sensitivity setting, e.g., 30X > 10X > 3X > 1 X. tive than the photometric method and permits Conversely, if the reading is less than 15 units, accurate ,determination of much , lower con- in-crease thesensitivity setting. If the samples fail centrations of pigment and the,nse of smaller t6 fall' in Ange, dilute accordingly. Record the sample volumes. Optimum sensitivity is:obtained fluorescent units based on a common sensitivity at excitation and _emission .wavelengths of 43p factor, e.g., a reading 50 at 1X equals 1500 at and 663 nal, respectively, using a R-136 'photo- multiplier tube. Pluorometers employing filters 30X. should beequipped with Corning CS-5-60 excitation and CS-2-64 emission fitters, or their Pheophytin Eorrection ,equivalents. Calibrate the fluorometer with a chlorophyll solution of known concentration. `Pheophytin is a natural degradation product Prepare a chlorophyll extract and determineof chlorophyll and often occurs in significant a, theconcentration of chlorophyll a by thequantities inphytoplankton. Pheophytin spectrophotometric method as previously, de- although physiologically inactive, has an abrrp- scribed. tion peak in the same region of the visible Prepareserialdilutions of the extractto spectrum as chlorophyll a and can be a source of provide' concentrations of approxi*tely 0.002, error in chlorophyll determinations. In, nature, 0.006, 0.02 and 0.06 mg chlorophyll a per liter chlorophyll is converted to pheophytin upon the of extract, so that a minimum of two readings loss of magnesium from the_porphyrin ring. This are obtained in each sensitivity rangeof the conversion ban be accomplished in the labors fluorometer (1/3 and 2/3 of full scale). With the tory by adding acid to the pigment extract. The use of these values, derivefactors to convert the amount of pheophytin a in the/tract can be fluorometer readings in each sensitivity range to determined by reading the OD6'63 before and milligrams of chlorophyll a per liter of extract. after acidification. Acidification of a solution of pure chlorophyll a results in a 40 percent re- Conc. chlorophyll a (mg/I) duction in the OD6 6 3, yielding a "before:after" Fs fluorometer reading OD- ratio (663b/663a) of l.70. ,Samples with 663b/663a ratios of 1.70 are considered free of where Fs is the fluorometric conversion factor pheophytin d, and contain algal' populations and s is the sensitivity, range (door). consisting mostly of intact, nondpcaying organ- isms. 5.2.2 hi vivo measurement Conversely, samples containing pheophytin" a Using fluorescence to determine chlorophyll a but not chlorophyll a show no reduction in in vivo is much less cumbersome than methods OD6 6 3upon acidification, and have a -involving extraction; however, itis reportedly 663b/663a ratio of 1.0. Samples containing both considerably less efficient than the extractionpigments will haire ratios between 1.0 and 1.7. method and yields about one-tenth as much To determine the concentration of pheophy- fluorescence, per unit weight as the same amount tin a, prepare the extract as previously described insolution. The fluorometer should be' cali-and determine the OD663. Add one drop of brated with a chlorophyll extract that has been I NHC1 to the cuvette, mix well, and reread the analyzed with a spec.trofluorometer. OD?5 13 and OD6 6 3after 30 seconds. 15 BIOLOGICAL METHODS Calculate the chlorophylla and pheophytina microscope. ,Assume the cells to be spherical as follows: cylindrical, rectangular, etc., and from the linear dimensions, compute the average surface area Chlorophyll a (mdm3) = 26.7 (663b .663a) X E ) per species. Multiply by the number of VXL organisms permilliliter(Welch,1948,lists mathematical formulas for computing surface Phcophytin a (mg/m3). 26.7 (1.7 X 663a- 663b) X E VXL area). where 663b is the 1-cm corrected OD663 before 6.0 PHYTOPLANKTON PRODUCTIVITY acidification; 663a is the OD693 after acidifi- cation; E the volume of acetone used for tip Phytoplanktonproductivity measurements extraction (ml); V the volume of water filtered indicate the rate of uptake of inorgartic carbon (liters); and L the path length of the cuvette by phytoplankton during photosyntheilsand are (cm). useful in determining the effects of pollutants and nutrients,on the aquatic community. 5.3 Cell Vojume Several -different methods have' been used to 5.3.1 Microscopic (algae and bacteria) ., measure phytoplankton productivity.Diurnal curve- techniques, involving pH and dissolved Concentrate an aliquot of sample by settling oxygen measurements, have been used in natural, or centrifugation, and examine -wet at a 1000X 'aquatic communities byp number of investi- magnification with a microscope equipped with gators.Weptlake, Owens, apd Tailing (1969), a calibrated ocular micrometer.' Higher magnifi- pre?nt an excellent discussfdrc;c4nceming. the cation may be necessary for small algae and the limitations, advantages; and disadvantage-s of bacteria. Make optical measurements anddiurnal curve techniques as applied to non- dehtlne thP volume of 20 representativeisolatednatural communities. The oxygen indivi uars of each major species. Determine the method of Gaarder and Gran (1927) and the average volume (cubic microns), and multiply by carbon-14 methpd of Sfeeman-Neilson (1952) number of organisms per milliliter. are techniques for measuring in situ phyto- plankton productivity. Tailing and Fogg (1959) ' 5.3.2 Displacement (zooplan Icton) discussed th)e relationship between the oxygen Separate sample from preservative by pouring and carb6n214 methods, and the limitations of through a piece of No. 20 mesh nylon bolting both methods. A number of physiological cloth placed in the bottom of a small glass factors must be considered in hie interpretation funnel. To hasten eva oration, wash sample with of the carbOn-14 method ttor measurement of a small amount of 50 ercent ethanol to remove phytoplankton productivity. Specialized appli- excess interstitial fluid d place* on a piece of cations of the' carbon-14 method include bias filter or blotting paper. Place drained plank- assay of nutrient limiting factors and measure- ton in a 25-,50-,or. '100-ml( pending on ment of the potential for algal growth. sample size) graduated cylinder, and add a The carbon-14 methods' and the oxygen known volume of water' from a burette. Read method have the widest use,,and the following the water level in the graduated cylinder. The procedures are presented for the in situ field, difference between the volume of the zooplank- measurement of inorganic carbon uptake by ton 151us the ad,'_d water and the volume of the theselniethods. water alone isthe displacement volume and, ( therefore, the volume of the total amount of 6.1Oxygen Method zooplankton in the sample. General directionfrfor the oxygen method are_ 5.4Cell Surface Area of Phytoplankton found in: Standard Methods for the Exami- Measure the dimensions of several represen- nation of Water and Wastewater, 13th Edition, tative individuals of each major species with a pp. 738-739 and 750451. . 16 , 11 y / PLANKTON PRODUCTIVITY .., Specific modifications and additionsfor Apparatlis A fuming chamber is not re- apparatus, procedures, and calculations are: quired. Use the methods of Strickland. and Apparatus Rinse the acid-cleaned sample1 Parsons (1968) to prepare ampoules containinga bottles with the water being tested prior to use. carbonate solution of the activity desired. Procedure Obtain a profile of the input .of Prodedure -'The carbon-14 concentration in solarradiationforthe photoperiod with a thaltered sample should yield the number of pyrheliometer.Incubate the samples at least 2 cou isrequiredforstatisticalsignificance; hours, but never longer than to that point where Strickland and Parsons suggest a minimum of oxygen-gas bubbles are formed inthe clear 1,000 counts per minute. Obtain, a profile of the bottles or dissolved oxygen is depleted in the input of solar radiation for the photoperiod with dark bottles. a pyroheliometer. 'Incubate up to4 hours; if Calculations Using solar radiation profile measurements are required for the entire photo- and photosynthetic rate during the incubation period, overlap 4-hour periolis,from 'dawn until period, adjust the datato represent phyto-dusk (e.g.; 0600-1000, 0800-1200, , plankton -S productivityfor the entire photo- 1400-1800; 1600-2000). A" 4-hour incubation period. period may be sufficient: however, provided A energy input is used as the basis for integrating the incubation periokl into the entire ph 6.2 ,Carbon1.4 Method period. To diy ,and store the filters, place' he General difections for the carbon-14 method membranes in a desiccator for' 12 hours following I are found iStandard Methods for the Exami- filtratio(n. Fuming with Hq is ndt r ired, and nation of er and Wastewater,g13th Edition, dried filters may be stored indefinite pp. 739-741 d 751-752. Calculations Using solar radiation profile and Specific modifications and additionsfor photosynthetic ratesduringtheincubation apparatus., procedures, and calculations are listed period, adjust data to represent phytoplankton productivity for the entire photoperiod. . below: 7.0 REFERENCES 7.1Sample Collection and Preservation 7.1.1G,eneral considerations Hutchinson, G. L.1957. A treatise on limnology, Vol. 1. Geography, Physics, anj1Chemistry.John Wiley and ons, Inc., New York. Ainson, C. 'E.1967. A treatise on limnology, Vol. 2, Introduction to lake biology and thelimnoplankton. John Wiley and Sons, ne., New York. . Reid, G. K. 1961.Ecology of inland waters and estuaries. Reinhold Publishing Co,, New York. Ruttner, F. 1953.Fundamentals of limnology (trans). by D. G. Frey and ,F. E. J. Fry), Universityof Toronto press, Toronto, Canada. 7.1.2Pifytofilankton Ingram, W. M., and C. M. Palmer. 1952.5implified proceduresfor collecting, examining, and recording plankton. JAWWA. 44:617. Anckey, J. B. 1938. The manipulation and counting of river planktonand changes in some organisms due to formalinpreservation. / Pub. Health Rep. 53:2080. -- Weber, C. 1. 1968. The preservation of phytoplanktori grab samples.Trans. Amer. Microscop. Soc. 87:70. Welch, P. S. 1948. Limnological methods. Blakiston Co., Philadelphia. 7.1.3 Zooplaamn Arnon, W., et al. 1965. Towing eliaracteristics of plankton sampling gear.Limnol. OcciinOgr. 10(3):333-340. 1955. Physical and biological processes determining the distribution of zooplanktonin a tidal estuary Biological Bull. Barlow, f 109(2):211-225, Barnes, H., and D. J. Tranter. 1964. A statistical examination of theCatches, numbers, and biomass taken by -three c mmonly used plankton nets. Aust. J. Mar. Freshwater Res. 16(3):293-306. 7 r4E. BIOLOGICAL MED:1(MS .4 Gayly, I. A. E. 1962. Ecological studies on,New Zealand lacustrine zooplankton with special reference to Boeckella propinqua Sal( (Corlepoda: Calanoida). Aust. J. Mar. FreshwaterRes. 13(2):143.197. Brooks, J. L. 1957. The systematiosof North America Daphita. Yale Univ. P2'ess,New Haven. Culver, D. A., and G. J. Brunskill. 1969. Triyetteville Green Lake, New York. V.Studies of primary production and zooplankton in a meromictic marl lake. Limnot Oceanogr. 14(6):862-873. Curl, H., Jr. 1962. Analysis of carbon in marine plankton organisms. J.Mar. Ros. 2'0(3):181-188. Dovel, W. L. 1964. An approach to sampling estuarine macroplankton. ChesapeakeSci. 5(1-2): 77-90, Faber, K. J. 1966. Free-swimNing copepod naupW of Narragansett Ba'y, Witha key to their identification. I. Fish. Res. Bd. Canada, 23(2):1$9-205. Faber, K. J. 1966. Seasonardtcurrence and abundance of free-swimmingcopepod,nauplii in Narragansett Bay. J. Fish. Res. Bd.Canada, -23(3):415-422. Frolander: Hi F. 1'957. A plankton volume indicator. J. Cons. Perm. int.explor. Mer. 22(3):278-283. Frolander, H. P. 1968. Statistical variation in zooplankton numbersfrom subsampling with a Stempel pipetle:..TWPCF, 40(2), Pt. 2:R 82-R 88. Galbraith, M. G., Jr. 1967 Size-selective predationon Daphnia by rainbow trout and yellow perch.(Trans. Amer. Fish. Soc. 96(1):1-10. Hall: D. J.(1964: An 'experimental approach to the' dynamics (ii:fa natural population of baphnia galeata mendotae. Ecology, 45(1):94-11:2. Hazelwood, D. H., and R. A'. Parker. 1961. Population dynamics ofsome freshwater zooplankton. Ecology, 42(2):266-274. .., Herman, S. S., J. A. Mihursky, and A. J. McErlean.1968. Zooplankton and environmental characteristics of the Patuxent River Estuary. Chesapeake Sci. 9(2):67-82. 4r. . JohnsOn, W. E. 1964. Quantitativeaspects of the pelagic 'e'ntomostracan zooplankton of a multibasin lake systemover a 6-year period. Verh. Internat. V.erein. Limnol. 15:727-734. . Jossi, J. W. 1970. Annotated bibliography of zooplankton sampling devices.U. S. Fish: Wildl. Serv., Special Scientific Report. Fisheries. No. 609. Likens, G. E., and J. J. Gilbert. 1970. Notes on quantitative sampling of naturalpopulations of planktonic rotifers. Limnol. Oceanogr. 15(5):816-820. .. McGowan, J. A.rhnd V. J. Fraundorf. 1966. The relationship between size ofnet used and estimates of zooplankton diversity. Limnol. Oceanogr. 11(4):456-469. Nall2ral Academy of, Sci. L969. Recommendedprocedures-for measuring the productivity of plankton standing stock and related .-- Oceanic properties. Washington, D. C., 59 pp. Paquette, R.G., and H. F. Frolander. 1967. Improvements in the Clarke-Bumpusplankton sampler. J. Cons. Perm. int, explor. Mer. 22(3)284-288. i Paquette, R. G., E. L. Scott, and P. N. Sund. 1961. An enlarged Clarke-Biimpus sampler.Limnol. Oceanogr.6(2):230-233. Pennalc, R. W. 1957. Species composition of limnetic zooplankton communities. Limnol.Oceanogr. 2(3):222-232. Smith, M. W. 1961. 'A limnological reconnaissance of a Nova Scotian brown-water lake: J. Fish.Res. Bd. nada, 18(35:463-478. Smith, P. E., R. C. Counts, and R. 1..Olutter. 1968. Changes in filtering efficiencOofplankton nets due 6-clogging under tow. J. Cons. Perm. int. explor. Mer. 32(21; 2V-* ' ..i. Smyly, W: J. .1). -1968 Some oeserviitiOris on the effect' of sampling fechniqueunder ,different condi s on numbis of some fresh-water planktonic Entornostrica.ancIROlifesa caught by a Winer-V*1e.). Nat. Hist. 2:50-575. Stross, R. G., J. C. Neess, and A. D. Haslek 196L Turnover time and productionof plank tonic crustacea in limed 'and reference portion 'of a bog lake-Ecology, 42(2):237-245. . Tranter, D. J., J. D. Kerr, and A. C. Heron. 1968. Effects of haulingSpeed on zooplankton catches. Aust. J. Mar. Freshwater Res. 19(1)i65-75. . Ward, J. 1955. A description of a new zooplankton counter. Quart. L Microscopical Sci. 96:371-373. , Yentsch, C. S., and A.C. liuxbury. 1956. Some factols affecting the calibration numberof the Clarke-Bumpus quantitative plankton sampler. Limnol. Oceanogr: 1(4):268-273. / Yentsch, C. S., and F. J. Hcbard. 1957. A gauge for determining plankton volUme bythe mercury immersion method. J. Cons. Perm. int. explor. Mer. 32(2):184-19 .. 1 I , 7.2 Sample pieparati n and analysis/ q r 7.2.1Sample alysisphytoplanktonl Haste, G. R., and G. A. FryxelL 1970. Diatoms: cleaning and mounting for light andelectron microscopy. Trans.Amer. Microscop. Soc., 89(4):469-474. 18 4=..) ' PLANKTON REFERENCES 6 Holmes, R. W. 1962. The preparationof matine phytoplankton for microscopic eicamination andc*meration on molecular filters. U. S.1Fish and Wildlife Serv., Special Scientific Report. Fisheries No. 433, 1-6. ' Jackson, H W., and L. G*."Williams. 1962. Calibration and use of certain plankton counting equipinent.Trans. Amer. Microscop. Soc. 81:96. - ofiiversplaaton and changes in some organisms due to formalin preservation., Lackey, J. B. 1938. The manipulation and counting ._----- PubL Health Repls. 53(47):2080-93. Levinson, S. A., R. P. MacFat. 1956. Clinical laboratory diagnosis. Lea and Fcbige 7.2.2 Biomass.de terminatiOn . Chlorophyll Lorenzen, C. J. 1966. A method for the continuous measurement of in vivo chlorophylrZoncentration.Deep Sea Res.13:223-227. Lorenzen, C.J.1967. Determination of chIcirophyll and pheopigments: spectrophotornetricequations. Limnol. Oceanogr. 12(2): 343-346. Moss, B. 1967. A spectrophotometric method for the estimation of percentagedegradation of chlorophylls to pheo-pigments in extracts of algae. Limnol. Oceanogr. 12(2):335-340. Strickland, J. D. H., and T. R. Parsons. 1968. A practical handbook of seawater analysis.Fisheries Res. Board of Canada, Bulletin No. 167, 311 pp. methodology. 1. Determi- United.Nations Educational, Scientific, and Cultural Organization. 1966. Monographs on oceanographic nation of photosynthetic pigments in sea water. UNESCO, Paris. 69 pp. Yentsch, C. S., and D. W. MenzeL 1963. A method for the determination ofphytoplankton chlorophyll and phacophytin by fluorescence. Deep ca Res. 10:221-231. Cell Surface Area Mackenthun, K. M. 1969. The practice of water pollution biology. U.S. Dept.Interior, FWPCA. 281 pp. Mullin, M. M., P. R. Sloan, and R. W. Eppley.1966.Relationship between carbon content, cell volume, and area in phytoplankton. Limn$1. Oceanogr., 11(2):307-311. Welch, P. S.11948. Limnological methods. Blakiston Co., Philadelphia. 344 pp. 7.3 Phytoplankton productivity American Public Health Association. 1970. Standard Methods for the Examinationof Water and Wastewater, 13th Edition, ,PHA, Washington, D. C. Beyers, R. J., J. L. Larimer, H. T. Odum, R. A. Parker, and N. E.Armstropg..1963. Directions for the deteimination of changes in carbon dioxide concentration fromchanges,in pH. Publ. Inst. Mar. Sci.Univ. Texas, 9:454-489. Beyers, R. J., and H. T. Odum. 1959. The use of carbon dioxide 'to constructpH curves for the measurement of productivity. LimnoL Oceanogr. 4(4):499-50 . Bransome, Edwin D., Jr. (ed.) 1970. The current status of liquidscintillation counting. Grune and Stratton, Inc., New York. 394 pp. Chase, G: D., and J. L. Rabinowitz. 190. Principles of radioisotope methodology.3rd edition. Burgess Publ. Co., Minneapolis. 633 pp. Edwards, R. W., and M. Owens. 1962. The effects of plants on river conditionsIV. The oxygen balance of a chalk stream. J. Ecol: 50:207-220. Fee, E. J. 1969. Numerical modelfor-the:estimation of photosynthetic production, integrated over time and depth innatural watirs. LimnoL Oceanogr. ,14(6):906 -911. Gaarder, T., and H. H. Gran. 1927. Investigations of the production of plankton in theOslo Fjord. Rapp. et Proc Verb., Cons. Internat.'. Explor. Mer. 42:1-48. 19 BIOLOGICAL METHODS Goldman, C. R. and R. C. Carter. 1965. An investigation by rapid Carbon-14 bioassay offactors affecting the cultural eutrophication of Lake Tahoe California- Nevada. J. WPCF, 37(7):1044.1059. . Goldman,,Ci R. 1969. Measurements (in situ) on isolated samples of natural communities,bioassay technique for nutrient limiting factors. manual on methods for measuring priMary production in aquatic environments (R. A.Vollenweider, ed.) 1BP Handbook, No 12. F. A. Davis, Philadelphia. pp. 79-81., Goldman, C. R. 1963. Measurement of primary productivity and limiting factors in freshwater withC-14. ln: Proc. cord% on primary prOductivity measurement, marine and freshwater (M. S. Doty, ed.) Univ.- of Hawn*Aug.-Sept. 1961. U. S. Atomic Energy Commission, Div. Tech. Inf. T.1.D. 7633, 103-113. Goldman, C. R. 1968. The use of absolute activity for eliminatingserious errors iii the measuremetWof primary productivity C-14.t J. Cons. Int. Explor. Mer. 32:172-179. Ankins, D. 1965. Determination of primary productivity of turbidwaters with' carbon-14. J. WPCF, 37:1281-1288. lifts, H. R., 4nd B. 15. Scott. 1961. The determination of zero-thickness activityin geiger counting of C14 solutions used in marine productivity studies. Limnol. Oceanogr. 6:116-123. ) .t,litts, H. R. 1963. 'the standardization and comparison of measurements ofprimary production by the carbon-14 technique. In: Proc. Conf. on Primary Productivity Measurement, Marine and Freshwater (M.S. Doty,ed.) Univ. of Hawaii, Aug.-Sept. 1961. U. S. Atomic .Energy Commission, Div. Tech. Inf. T.1.D. 7633, 103-113. Joint Industry /Government Task Forte of Eutrophication. 1969. Provisional algalassay procedure. pp.16.29. McAllister, C. D. 1961. Decontaminalion of filters in the C14 methoii, of measuringmarine photosynthesis. Limnol. Oceanogr. 6(3):447-450. Odum, H. T. 1956. Primary production in flowing water. Limnol. Oceanogr. 1(2):102-117. Odum, H. T. 1957. Primary production measurements in eleven Florida springs anda marine turtle grass -community. Lin' ol. Occanogr. 2(2):85 -97. Odum, H. T., and C M. Hoskin, 1958. Comparative studs on the metaboliSm of marinewaters. Publ. Inst. Mar. Sci., Univ. of Texas, 5:16-46. Owens, M., and R. W. Edwards. 1963. Some oxygen studies In the River Lark. Proc. Soc. for Water Treatmentand Examination, 12:126-145. Park, K., D. W. Hood, and H. T. Odum. 1958. Diurnal pH variation irr Texas bays and its applicationto primary production estimation. Publ. Inst. Mar. Sci., Univ. Texas, 5:47-64. . Rodhe, W., R. A. Vollenweiderand A. Nauwerck. 1958. The primary production and standingcrop of phytoplankton. ln: Perspectives in Make Biology (A. A. Buzzati-Traverso, ed.), Univ. of California Press.pp. 299-322. Saijo, Y., and S: lchimura. 1963. A review of recent development of techniques measuring primary production.ln: Proc. conf. on primary productivity measuremen, marineand freshwater SS. Doty, ed.) Univ. Hawaii, Aug.-Sept. 1961. U. S, AtomicEnergy Commission; Iiiv. Tech. Inf. T.I.D.633, 91-96. Steeman-Neilson, E. 1952. The USe f radioactive carbon (C-14) for measuring organic production in thesea. J. Cons. Int. Explor. Mer. 18:117-140. Strickland, 7.1). II., and T.R. Pars is. 1968. A practical handbook of seawater analysis. Fish. Res. Bd. Canada, Bull. No. 167, 311 pp. Tailing, J9f., and G. E. Fogg. 59. Measurements (in situ) on isolated sample's on natural communities, possible limitations and artificial modifications. ln: Aanual of methods for measuring primary production in arniatic environments (R. A. Vollenweider, ed.) 1BP Handbook, No. 12,4F.. Davis, Philadelphia. pp. 73-78. Thomas, W. H. 1963. Phisiologic I gctors affecting the interpretation. of phytoplankton productionmeasurements. ln: Proc. conf. on primary productivity measureme t, marine and freshwater (M. S. Doty, ed.) .Univ. Hawaii, Aug.-Sept. 1961.U. S. Atomic Energy Commission, Div. Tech. Inf. T.1.7633, 147-162. Verduin, J. 1952. Photosynthesis d growth rates of two diatom communities in western Lake Erie. Ecology, 33(2):163168. Westlake, D. F., M. Owens, and J.. Tailing. 1969. Measurements on non-isolated natural communities. ln: A manual on methods for measuring primary production in aquatic environments (R. A. Vollenweider, ed.) 1BP Handbook, No.12. F. A. Davis, Philadelphia. pp. 90-100. r PERIM!) a M' PERIPHYTON Page 1.0 INTRODUCTION 1 2.0 SAMPLE COLLECTION AND PRESERVATION 2 2.1 Qualitative Sampling 2 2.2' Quantitativef Sampling 2 3.0 SAMPLE PREPARATION AND ANALYSIS 3 3.1 Sample Preparation 3 3.2 Sample Analysis 3 11/4 4.0 BIBLIOGRAPHY 5 PERIPHYTON lc 1.0 INTRODUCTION: (1908 and 1909) made wide use of components in this community. in the development of the Periphyton is an assemblage of a wide variety saprobic. system of water quality classification. of organisms' that grow on underwater substrates This system has been continued and developed and includes but is not limited to, bacteria, in Middle and Eastern Europe (Srameck-Husek, yeasts and molds, algae, protozoa, and forms 1946; Butcher, 1932; 1940,...,1146; Sladeckoya, that may, develop large colonies such as sponges 1962; Sladecek and Sladeckoiia, 1964; Fjerding- and corals. Al organisms within the community stad, 1950, 1964, 1965). are not necessarily attached butsome may bur- of The study of periphyton was introduced in row pr, live within the. cgmmutlity structure the United States in the 020's and expanded in- the attached forms. the1930's. The use of the community'has \Literallytranslated, periphyton means gown steadily and rapidly in waterqualit}I in- ` /around plants," such as organisms overgrowing vestigatias (Blum, 1956; Cooke, 1956;Patrick, pond weeds, but through widespread, usage, the 1957; Cairns, et al., 1968). . terrp has become associated with communities The periphyton and plankton are theprincipal of microorganisms growing on substrates of any primary producers in waterways they convert nature. Aufwuchs (Seligo, 1905), the German nutrients to . organic living mterials and store noun for this community, does not have an light energy through the pr6cesses of photo- equivalent English translation, but essentially synthesis. In extensive deep waters, the plankton means growing on and aroundthings. Other are probably the predominantprimary pio-, terms thatareessentially synonymous with ducers. In shallow lakes, ponds, and rivers, the periphyton or describe important or predomi- periphyton are the predominant primary pro- nant components of the periphytic community ducers.. are: nereiden, bewuchs, bison,belag, besatz, Periphyton is the basis of the trickling filter attached,sessile,sessile-attached,sedentary, system form of secondary sewage treatment. It seeded-on, attachedmaterials,slimes,slime is the film of growths covering tOe substrate in growths, and coatings. Some of these terms are the filter that consumes nutrient, micro-solids, rarely encountered in the literature. Terminology and bacteria from the primary treated sewage based on the nature of the substrate is as passing through the filter.s these growths ac- follows: cumulate,they 'eventually sl ugh from the sub- strate, pass 'through theflit ,and are captured , Substrate Adjective in the filial clarifier; thus, they change chemical' various epiholitic, nereiditic, sessile and biological materials to a solid that can plants epiphytic be removedwiththephysicalprocess of animals epizooic,_ settling: Excellent dstudies and reportson this7 wood epidendritic, epixylonic prOdess have been published by WisntecsAki rock epilithic (1948), Cooke (1959), and Holtje (1943)' Mosta b ove-listedLatin-root allrectives are The periphyton community is an ekcellen,t derivatives of nouns such asepiholKepiphyton, indixpator of water quality. Changes may range epizoa, etc. (After. Sraineck-Huse,1946a from subtle alteration of species composition to.: Sladeckova, 1962). extremely dramatic results, such as whe,1'ili°07°- addition of organic wastes to 'waters supp sting Periphyton, was recognized as an important a. community of predominately diatomgirowths component of fquatic communities before the result in their replacement by extensive shine beginning of the 20th century, and the study of colonies: composed predominately of bacteria periphyton was initiated in Europe in the early such as Sphaerotilus or Leptomitus and vorticeli 1900's. Kolkwitz and Marsson in two articles..lid protopans. ti""' P 4 'BIOLOGICAL. METHODS I Excessive growth stimulateik; by increased fittoralareas in lakes and shallow or riffle areas nutrients can resultin( large,filamentous in flowing water where the'greatest number and streamers that are esthetically and variety of organisms are found. Combine the interfere with suety water uses as swimming, scrapings to a volume of 5 to 10 ml fora tuf- wading, fishing, and boating, and Can also affect ficient sample. In lakes and streamswhere tong the quality of the. overlying, water. Photo- strands of filar lent algae occur, weigh. - synthesis and respiration can affect alkalinity sample. (U. S. FWPCA,4967) and dissolved oxygen con- After scraping has been completed, store the centrations (O'Connell and Thomas, 1965). of material in bottles containing 5 percent forma- lakes and loreams. Metabolicbyliroducts lin. If the material is for chloroph ll analysis, do releasedtothe overlying water may impart not preserve. Store at 4°C in theark in 100 ml tastes and odors to drinking waters drawn from of 90 percent aqueous acetone.se bottle caps thetostrtramorlake,awidesprekl probleM with a cone-shaped polyethylene seal to prevent throughout the United States (Lackey, 1950; evaporation. Silvey, 1966; Safferman, et al.,1967).- Large clumps of growth may break from the site iof 2.2, Quantitative Sampling attachment and eventually settle to form accu- ,) mulations of decomposing, organic, sludge-like The'standard (plain, 25 X 75 mm) glass micro- materials. scope slide is the most suitable artificial sub- Periphyton have proven useful in reconnais- strate for quantitative sampling. If less fragile sance surveys, water quality monitoring Studies, material is preferred, strips of Plexiglas may be short-term investigations, research and develop- used in place of glasS slides. ment, and enforcement studies. The investiga- Devices for, cSing the substratescan be tion, objectives dictate the nature, approach, and modified to suit a paOcular situation, keeping methodology- of sampling the periphy ton com- in mind that the depth of exposure must becon- munity. Factors to be considered are the time sistant for all sampling sites. In large rivers or and duration of the study and the characteristics lakes,afloatingsampler (API-IA, - 1971)is of the waterway. advantageous when turbidities are high and line Sladeckova (1962)published, anextensive substrates must be exposed near the surface.Vn review of methodology used in investigating this small, shallow streams or littoral areas of lakes community. where turbidity is not a critical factor, substrates may be exposed in several ways. TwQ possible 2.0 SAMPLE COLLECTION AND PRESER- methods are:(a)attich- fire- substrates with VATION PLASTIC TAK adhesive to bricks or flat rocks in the stream bed, or (b) anchor Plexiglas racks 2.1Qualitative Sampling to the bottom to hold the substrates. Inareas where siltation is a problem, hold the substrates Time limitations often prohibit the use of in a vertical position to avoicra covering of silt. artificial substrate samplers for quantitative col- If desired, another set of horizontally-exposed lection, and thus necessitate qualitative sampling substrates could be used to demonstratethe from naturalsubstrates.Periphytonusually effeCts of siltationon the periphyton com- appear as brown, brownish-gfeen,or. green munity. growths on the substrate. In standing or flowing The number of substrates to be exposed at water, periphyton may be qualitatively collected each sampling site depends on the type and by `scraping the surfaces of several different number of analyses to be performed. Because of rocks and logs With a pocket knife or some other unexpected fluctuations in water levels,cur- sharp object. This manner of collecting may also rents, wave action, and the threat of vandalism, be used as a quantitative method if accurate-duplicate samplers should be used. A minimum measurements are made of the sampled areas. of four replicate substrates should be taken for When sampling this way, limit collections to each type of analysis. 2r -. - ..',PERIPHYTON The length of exposure depends upon many factors,incltiding the survey time schedule, Donner, J. 1966. Rotifers. Butler and Tanner, Ltd., London. gioWth "patterns, which are *seasonal, d pre- Edmundson, W. T. 12,59. FreshWater biology. .1e3hn Wiley" ant vailing hydrologic conditions. On the assump- Sons, New Yprk. tion that periphYton growth rate -on c can sub- PennakR. 1953. - Freshwater invertebrates of the United strates ptoceeds exponentially for 1 o 2 weekS States.11-cmald Press, New York.` and then gradually declines, the optimum eX- posure period is 2 to 4 weeks. . Microcrustacea Edmondson, W. T. (see above). 3.0 SAMPLtPREPARATION AND ANALYSIS Pennak, R. W. (see above). 3.1 Samplereparation. 3.2.2 Counts and enumeration Sample preparation varies according to the method of analysis; see the 13th edition of Sedgwick-Rafter Method Stan dardMethods, Section 602-3 ,(APHA, Shake vigorously to mix the sample, transfer 1971). ml to a Sedgwick-Raftei cell, and make strip 3.2 Sample Analysis counts,, as described int, the Plankton Sectiont except 'that a cell count is made of all organisms. 3.2.1 Identification If the material is too concentrated for a direct -In addition to the taxonomireferences listed coupdilute a 1-ml aliquot with 4/n1 of dis- in the Plankton Section, the following bench.tilledater; further dilution "iriay-bfe nec9ssary. Even after vigorous shaking, the scrapings may references are essential for day-to-day periphy- contain large clumps of cells. These clumps can ton identification. result in ,an uneven distribution of material in the counting chamber that could'seriously affect Algae' the accuracy of the.count. Should this condition Desikachary, T. W. 1956, Cyanophyta. Indian eounc. Agric. Res.','New Delhi: occur, stir 50 ml of the sample (or a proper Fairdi, M. 146.11FA monograph of the fresh water species of dilution) in a blender for .1 Minute and reeX Cladophbra and Rhizoclonium. Ph.D. Thesis, Univ. Kansas "amine.Repeatifnecessary.Caution: Some (available in Xerox from University Microfilms, Ann Arbor). colonial 'organisms cannOtbe identified in a frag- Islan, A. K., and M. Nurul. 1963. Revision of the genus Stigeo- mented condition. Therefore, the sample must clonium. Nova Hedwigia, Suppl.10. J. Cramer, Weinheim Germany. be examined before being blended. Ranantlian, K. Tt.1964. Ulotrichales. Indian Couhe. Agric. The quantitative dete'rmination of orgapisms New Delhi. on a substrate can then be expressed as: Ftandlowa, M. S. 1959. Zygnemaceae. Indian Co,unc. Agric. Res., New Delhi. C X 1000 MM3 X V X. DF Tiffany, L. H. 1937. Oedogoniales,Oedogoniaceae4P..In: North No. cells/mm2 LXWXDXSXA American Flora, 1(1):1-102. N. Y. Bof. Garden, Hafner Publ. New York. ;Oere. = number of cells counted (tally) Fungi C Cooke, W. Bridge. 1963. A laboratory guide to fungi in polluted V = sample volume, ml waters, sewage, and sewage treatment systems. USDHEW, DF .= dilution factor USPHS, DWSPC, Cincinnati. = length of a stri,mm Protozoa = width of arip (Whipple grid image, Bick, H. 1967-69. 'An illustrated guide tociliated protozo width), mm N (used as biological indicators in freshwater ecology). Parts1-9. D = depth of a strip (S-R cell depth), m \ World Hlth. Organ., Geneva,, Switzerland. S = number of strips counted Ku o, R. R. 1963. Protozoology. Charles Thomas, Publ, Spring- fie, Ill.. A =. area of substrate scraped, mm 3 BIOLOGICAL METHODS . . Diatom Species Proportionaittount ,7 Displacement.Use :displacement' forlarge Before 'preparing the diatom slides, use an growths of periphyton when excess Water can be oxidizing agent to digest the gelatinous stalksereadily removed. Once the excess water is re: _ and other extracellular organic materials'causing moved, proceed as per Plankton Section; how- cellclumping. Before the oxidant is added, ever,. do not pour sample throng ia No. 20 . however, centrifuge or settle the sample to- re- mesh, nylon bolting cloth. move the formalin. v . '1# If centrifugationispreferred, transfer the Chlorophyll ", .sample to -a conical tube and centrifuge .10 The chlorophyll content of the periphytbn is minutes at 1000 X G. Decant the formalin, re- used to estimate the algal biomass and as an suspend the sample in 10 ml of distilled water, and recentrifuge. Decant, take up the sample in ,indicatonrof the nutrient content (Or trophic a ml of 5 percent potassium (or ammonium),status) or toxicity of the water and the taxo- persulfate, and transfer, back to the (rinsed) nomic composition of the community.' Periphy- ton *owing in surface water relatively free of. sample vial. ,` - organic pollution consists largely of algae, which If the settling method is preferred, follow the 4contain apProximately. 1to 2 percent chloro- instructions given in the Plankton Section for phyll-a-by dry, weight. If dissolved or particulate removing salt from the diatom cone ntrate, but organic matter is present in high concentrations, add persulfate or hydrogen perox' e instead of large distilled water. After the formalin is replaced by populations. of filamentousbacteria, the oxidant, heat the sample to ,95°C for 30 stalkedprotozoa, and other nonchloropbyll bearing microorganisms develop and the percent- minutes (do not bpil). Cool, remove the oxidant age of chlorophyll a is then reduced. If the by 'centrifugation or settling, and take up the biomasschlorophyll a relatio'nship is expressed diatoms in 2 to 3 ml of distilled water. Proceed = with the preparation of the permanent diatom,as a ratio (the autotrophic index),,,ralues greater ', mount as described in the Plankton Section.. than 100 may result from organic pollution Label the slide with the 'station location and (Weber and McFarland,. 1969; Weber, 1973). Ash -flee Wgt (mg/m2) inclusive sample dates. Carry out the diatom Autotrophic Index = strip count as described in the Plankton Section, Chlorophyll a (mg/m2) _except that separated, individual valves (half cell walls) are as such, and the tally is divided To obtain information! on tilt physiological by two to obtain cell numbers. condition/ (or health) of the algal periphyton, measure the amount of phefophytin a, 'a physio- 3.2.3 Biomass logically inactive,degradgron product-of chloro- phyll a. This degradatiOn product has absorp- Cell-Volume tion peak at nearly the same wavelength chlo- See the'Plankton Section. rophyll a and, under severe environmental condi- tions, may be responiible for most if riot all of Dry arid Ash4ree Weight th- 3inbe acetone extract. The presnce See the Plankton Section. of rejativellarge ?,thoun&of-pheoplwtin a is an abnormal ondition indicating water quality centrifugation, Sedimentation and Displacemi t degra ati bn. (See-the Plankton Section.) iCentrifugation.Place sample in graduated, ract chlorophyll, grind and steep t centrifuge tube and centrifuge for 20 minutes at p riphy in in 90 percent 'aqueous acetone (s 1000 X G. Relate the volume in Milliliters to t on Section). Because of the normal sea-, area sampled. sonal succession of the algae, the taxonomic Sedimentation.Placesample. in graduat composition and the efficiency of extraction by Cylinder and allowmpie to settle at least steeping change continually during the year.' hourRelate the v lume in milliliters to the Although, mechanical or other cell disruption .area)sampled. . may not increase the recovery of pigment from 1 4 a 1 PalPHYTON 'every sample,' routine grinding will significantly,.trembly sensitive to photodecompositiOn and. increase (10' percent or moie) the average re- lose 1more than 50 percent of their',, optical covery of chlorophyll 'from' samples c011etted activityif eiposed to direct sunlight for only 5. over a period of several. months. Whet le. glass-'mingles. Therefore, samples placed in acetone in slides are used as substrates, place the,individualtithe field .must be, prolated fro smore than slides bearing the periphy ton directly iii separate Monttary exposure to dire su small bottles (containing 100 ,;m1) of .acetone sboul%,b,,e,;,. placed. im 'edia in the' dar when removed from the.s4mpler.(,Similarly, Sai'nples not placed in acetonein thefief 7,yi -.should be iced until processed. if - samples a e periphyton removed from other. artifical not to .ke processed on the day, "collected, ho or natural substrates in the fieldimmediately in ever, they should be frozen-and held at'.220°C. 90 perc'ent aqueous acetone, (Samples should be Vor the chlorophyll , see.the Plankton maCerated, however, when.retutned to the -lab.),4Section.. ! Acetonesolutions' of chlorophyllare1611(1 4.0 BIBEIOGRATilY . f' American Public He4Itli, Associitiod. 1971-. Standard metliods,for the examinationof water undwastewater, 13th ed., APHA, New York. . Blum, J. L. 1954.`The deologytif river algae. Bot. Rev. 22(5):.01'. Butcher, R. W. 1932. Studies in the ecology of rivers. II. tho microflbra of rivers with special reference to the algae on the rr bed. 2 Ann. pot. 46113-461.,` : .!'' 1 Butcher, R. W 1940. Studies in the ecology of rivers. IV. Obtervations on the growth anddistribution of sessile algae in the River HtliV, . ., ...... Yorkshirej.'ology, 28:210-223. , . ,! °:.' - Butcher, R. W. 146/.§tudies in the ecology of ,rivers, VIL.TheelgaV.oforganically.enticb41waters. J. Ecology, 35:1 86ii 91.. Butcher, R.. 1959, BiologiCaiassessment of fiver pollution, 'Proceedrngs.Linnean Society, 170:159-165;.Abstracyln: J. Sci. Food., .. 4,' . , ._ Agri. 10:("1 1):104. - .o . 0 - . ( ,k.. Cairns, J., Jr., D. W. Albough, E.- BuSey, ,a,hd M.. b. ChalAy: 1968. The sequential comparison index . A simplified, method for nonbiologists to estifnate relative diffe(ences in biologiealdiversity ftc stream pollution studies.IWPCF,40(9):16074613. ' Cooke, W. B. 1956, Colonization of artificial bare areas by microorganisms: Bot. Rev. 22(9):6136) Cooke, W.. B. 1959: fungi in pollUted water and sewage. 'IV:The occurrence of fungi in a trickling Filter-type sewage treatment plant.. In Proceeding, 13th Industrial Waste'Conferen6e, Purdue University, Series No. 96, 43(3):26-45. Cummins, K. W., C. A. Tyron, Jr.,. and R. t../Aartman (Editors): 1966-. Organism-substratt relationships in streams. Spec. Publy No. 4,- 'Pyrnatumnelab. of Ecol:,Univ. PittsbUrgh 141 pp. , Fjerdingstailo. E. 1950. The micrOflora' of the RiverMolleaa with special reference to the relation of-the algae to pollution. Folia LimnolScand, No. 5, Kobenhaven. 123 Fjerdingitad, E. 1964: Pollution of stream estimated 1)9, bentital phytomicroorganisms. i. Astprobic system blsed on communities dr go and ecoroOcal faCtor,s, 1-1)/frirobiol. 49(1):63 -131. . Fjerdingstad,E. 1965. TaxonbrO:and saprobic valency of "be.riShic plrytornicroorgaqismi. Hydrobiol. 50M:475 -604. . Hawkes, H. A. 1963. Effects of-domestic and industrial clischrge on the ecology Of riffles in midland streams. Intern. J. AirW;ter Poll. 7(6/7) :565-586. : ,. , .. ... Holti, It. H.,1943.:Ihe biology of sewage sprinkling filters. Sewage Works J. 15(1) :14 -29. , Keup, E. E. 1966. Stream biology for assessing sewage treatment plant efficienc34; Water and Sewage Works, 1 l3:11 -411. . Kolkwifz,R., and M. Madeonf 1908? Oekologie ,derpflanzlichen 'Saprobien... Beiiclitebetrtschen. Batailitchen Gesellschaft, I.' 26505-519. .., ., , Kolkwitz,R. and M. Marsson. 1909. Oekologie der. Tierischen ,Saproien. Internationale. Revue Gesamten'llydrohiologie L % Hydrographie, 2:1264 52. / , . ..d,,,, : w Lackey, L B. 195 6...Aquatic, bkitogy and, the water works engineer: Public Works 81:30-41,64. Na - Mackenthun, k, M. 1969. Thelarastieeof water riollution.biology. US; FWPCA, Washington:D.C. 281 pp. . . ' Mackenthun,K. M., and L. e./Keup..1970. BielogicalnrblemsO encountered in water supplies: JAWWA,2 6():58 205 26. r ... O'Connell, .1: M., and. N. 0../ Thomas. 1965. Effect of-, benthie algae on strearn dissolved oxygen. PA-oc. ASCg,1:SauitIET:Div. 91%1-16, . -'1. '. - ' , ,, ',. : . . . . .1. Odum, H. V.1957. Trophic structure and productivity of Silver Spring's, Florida. Ecoi. Modogr. 27I5-1,1.2...... Parrish, Lit', ang A. M. LueLs. 197.(h.The effects: f waste waters on Periphyton growths in the' Missouri River. (Manuscript).. U. S. l IWPCA Nat'l! Field Investigations Center, Cincinnati. . e.. , 2. .l . Na. i - 2 I 5 BIOLOGICAL METHODS Patrick, R. 1957. Diatoms as indicators of. changes in environmental conditions. In:Biological Problems in Water Pollution-- Transactions of the 056 Seminar, Robert A. Taft Sanitary Engineering Center, U. S. Public Health Service, Cincinnati, Ohio. pp. 71-83. W57-36. -- Rohlich, G. A., and W. B. Sarles. 1949. Chemical composition of algae and its - relationship to taste and odor. Taste Odor Control J. 18:1-6. _ 9 Saffermary, W.' S., A. A. Rosen, C. I. Mashni, and M. E. Morris. 1967. Earthy - smelling substance from a blue-green alga. Enviroh. Sci. Techndl. 1:429-430. Seigo, A. 1905. Uber den Ursprung der Fisclinahrung. Mitt.d. Westpr. Fisch. 17(4):52: Silvey, J. K. G. 1-966. Taste and odors - Joint discussion effects of organisms. JAWWA, 58(6):706 -715. Sladecek, V., and A. Sladeckova. 19164. Determination of the periphyton production by means of the glass slide method. H,ydrobiol. 23:125-158. Sladeckova, A. 1962. Lirnnological investigation meths for tie periphyton ("Aufwuchs") community. Bot. Rev: 28:286 350. Srameck-Husek, 1946. (O'n the uniform classification o animal and plant communitiesour,waters) Sbornik MAP, 20(3):13 Orig. in Czech. Strickland. J. D. II. 1960. Measuring the production of marine phytoplank ton. Bull. No. 122. Fish. Res. Bd. Canada, Ottawa 172 pp. (Review of methods of primary production measurement, many applicable fib periphyton analyses.) Thomas;, A(. A. 1968. Methods for slidattachment in periphyton studies. (Manuscript). U. S, FWPCA, Tield'Investiiations Center, Cinati. I 'Nor U. S. Federal Water Pollution Control Administration. 1967.Effects of pollution on aquati,life resources of the South Platte River in Colorado. Vol. 2. Technkal Appendix. USEVP,CA, Cincinnati. 85p0. Warner, R. W., R. K. Ballentine, and L. E: Keup. 1969. Black-water impoundment investigations. U. S. FWQA, Cincinnati, Ohio. 95 pp.: Weber, C. 1973. Recent deverdpmentsin the measurement of the response_ of plankton and\periphyton to changes in their en ironf ment. In: Bioassay Techniques and Environmental Chemistry. G. Glas, ed. AmtArbor Science Publishers, Inc., p 119-138. . 1-, C. 1.,-andlMeRarlg41A69. Periphyton biomass-chlorophyll ratio as amindex of svatr quality. Presented at the 17th Annual Midwerrthologltal S'ociety, Ky.,April, 1969. C. I:. and R. L. Raschke. 1966. Use of a'floating periphyton sample for, water pollution-surveillance. U. S',41413. CA, Cincinnati,. Ohi Westo, R. S, and C. E.Turner.1917. Studies on the digestion of a sewage filter effluent by a small nd otherwrse unpolluted stream. ,, - ___--4517.1nst.Technol.. Sanit: Res: tab. Sewage Expel-. Sta. 10: 1-43. . r Wisniewski, T. t:'4-1,94$: The chemistry and biology of milk waste disposal. J. Milk Fotia'Technol. 11:293-300'. , Ycking, O. W. 1945. A limn.ological investigation of pc riphyton in Douglas Lake, Michigan.. Trans. Amer. Microscop. Sot. 64:1 CI- 41, 0 -6 -4 th MACROPHYTII . MACROPHYTON o Page .0 INTRODUCTION I'd 4 1 ° 2.0 SAMPLE COLLECTION AND ANALYSIS . . . 1 '2.1 Qualitative Sampling 2 2.2_ Quantitative Sampling r 2 10 REFERENCES 3 7 n . MACIZOPHYTON, 1.0 INTRODUCTION -1. Sludge deposits, especially those undergoing Macrophytes are all aquatic plants. possessing arapid ,decomposition, usually are too unstable or multi-cellular structure with cells differentiated toxic to permit the growth of rooted plants. into specialized tissues. Included are the mosses, The ramparif:grwthd of some ,macrophytes has liverworts,and flowering' plants.Their sizescaused concern over reeentyears (Holm et al. range from the near microscopic watermeal td,1969): Millions of dollars are spent each year in massive cypress trees. The most commonly dealt controlling macrophytes that , interferevIzith with forms are the herbaceous water plants. irrigationoperation,navigation, andrelated Macrophyton may be conveniently divided recreational uses. Mechanical cutting, applica- into three majoi\growth types: tion of herbicides, and habitat alteration are the . Floating. These plants have true leaves and primarycontrolmethods. Mackenthun and -' roots and float on the water surface (duckweed, Ingram (1967) and Mackenthun ( 969) have re- watermeal, water hyacinth). :viewed and summarized control thniques. Submerged.' These plants are anchored to the Yount and Crossman (1970) nd Boyd (1970) substratum by roots and may be entirely sub- discussed schemes for using macrophytes to re- mersed or have floating leaves and aerial repro- move nutrients fromeffluents andnatural ductive structures (water milfoil, eel grass, pond- waters. weeds, bladderwort). Aquatic macrphytes are a natura component Emersed. These plants are rooted in shallow of most aquatiecosystems, and arpresent in water and some species occur along moist shore those areasstablefor macrophyte growth, lines. The two major groups are: unless the haitat is Otered: Furthermore, the Floating leafed plants (water lilies and water proper proportions oknacrophytes are ecologi- shields). cally desirable (Wilson, 1939; Hotchkiss, 1941; Plants with upright shoots (cattails, sedges, Penfound, 1956; Boyd, 1971). Boya (1970, woody shrubs, rice and trees. 197 1)introduced concepts of macrophyte The use of macrophytes in water quality matiagement opposed to the current idea of investigationshas beensorelyneglected. eradicatingaquatic macrophytes from many Kolkwitz and Marsson (1908) used some species aquatic ecosystems. Much additional research is In their saprobic system of water quality classifi- needed on the roe of macrophytes in -aquatic cation,- but they are rarely mentioned in most ecosystems, literature. A number of pollutants' have dramatic The objective of an investigation, dictates the effects on macrophyte growth: nature and methodology of sampling macro- Turbidityrestrictinglightpenetration can phytes. Critical factors are the time available,/ prevent the,growth of submerged weeds.. how critical the information is, expertise avail- Nutrients can stimulate overproduction of able, duration of the 'study, and characteristics of the waterway. ,macrophytes in numbers sufficient to,create nuisances or can stimulate excessive Plankton Techniques are few, and the investigator's best growths that effect an increase inturbidities, assetishiscapabilityfor innovating sound thus eliminating macrophyte growths. procedures. Ierbicidal compounds, if present at sublethal concentrations can stimulate excessive growths 2.0 SAMPLE COLLECTION AND ANALYSIS or they can, at higher concentrations, destroy Collectingrepresentativegenera fromthe plant growths. macrophyton community is generally not diffi- Organic or inorganic nutrients, or both, can culebecause of their large size and littoral habi- supportperiphytic algal and slime growths tats. Macrophytes may be .readily identified to sufficientto smother and thus destroy sub- genera and some to.species in the field, or they mersed forms. 4may be dried in a plant press and mounted for BIOLOGICAL METHODS .11, further identification. Small, delicate speciei dures adapted from terrestrial plant surveys are may be preserved in buffird 4 percent formalin applicableinthe. aquatic environment. The solUtion. Some of theore, useful taxonomic following references will be helPful in adopting a works for identification are Muenscher (1944), suitable 'technique: Penfound, 1956; Westlake, Eyles and Robertson (1944); F'ssett (1960) and 1966; Boyd,1969; Forsberg,1959,1960; Winterringer and Lopinot (1966). Edwards and Owens,' 1960; Jervis, 1969; Black- ,burn, et al.,(1-968. 2.1Qualitative Sampling Standing crop. Sampling should be limited to Qualitative sampling includes visual observa- small, defined subareas (quadrates) With conspic- tion and collection of representative types from uous borders. Use a square framework with the the study area. Report the extent of growth as poles anchored on the bottom and floating line dense when coverage is continuous moderate fpr the sides.- Collect theoplaiits from within the when growths are common, and sparse when the frame By hand or by usingYlong-handled garden growth is rarely encountered. The crop of plants rake.Forsberg (1959)has describedOther may be comprised of just one genus or may be a methods suchaslaying out tong, narrow mixture; if a mixture, estimate the percentage of transects. Obtain the wet weight of material after the individual types. . plants ha've drained for a standard period or- Sampling gear is varied ari4the choice of tools time, determined by the investigator. D the usually depends on water depth. In shallow samples (or subsamples for large species) or 24 water, a garden rake or similar device is very hours at 105°C and reweigh. Calculate thdry effective for collecting macrpphytes. In deeper weight of vegetation per unit area. water, employ grabs, such as the Ekman, to Planimeter accurate maps to determine the collect submersed types. In recent years, scuba total area of investigation. If additional boat or diving has gained popularity with many investi- air reconnaissance (using photographs) is done gators in extensive plant surveys. Phillips (1959) to determine type and extent of coverage, data providesdetailedinformation on qualitative collected from the subareas can then be ex- sampling. .$ panded for the total study area. Boyd (j969) , describesa techniquefor obtaining surface 2.2Quantitative Sampling coverage by maCIOPhytes in a small body of , Quantitative sampling. for macrophytes is water. usually tb determine the exttor rate of ' Productivity. Istithate standing crops at pit- growth or weight of growth per unit of area. The determined inte04s to relate growth rates to study objectives determine whether measure- pollution, such as'imtrient stimulation, retarda- ments will involve a single species or several. tion, or toxicity from, heavy metals and thermal Before beginning a quantitative investigation, effects.Wetzel (1964) and Davies (1970) develoP a, statistical design to assist in deter- describe a more accurate method with the use of mining the best sampling procedure, sampling a carbon-14 procedure to estimate daily produc- area size, and number/of samples. Often proce- tivity rates of macrophytes. 1.47 r, 2 11. M AC ROPHYTON 3.0 REFERENCES Blackburn, R. D., P. F. White, and L. W. Weldon. 1968. Ecology of submersed aquatic weeds in south Florida canals. Weed Sci. 16:261-266, - Boyd, C.-E. 1969. Production, mineral nutrient absorption, and biochemical assiilation by Justicia americana and Alternanthfra philoxeroides. Archiv. Hydrobiol. 66:139-160. \ Boyd, C. E. 1970. Vascular aquatic plants for mineral nutrient removal from pollutedated Econ. Bot. 24:95-103. Boyd, C. E. 1971. The limnological role of aquatic macrophytoiand their relationship tos.ieservoir management. In: Reservoir Fisheries and Limnology, G. E. Hall (ed.), Spec. Publ. No. 8. Amer. Fish. Soc., Washington, D.C. pp. 15,3-166. Davie. S. 1970: Productivity of macrophytes in Marion Lake, British Columbia. J. Fish. Res. Bd. Canada, 27:71-81. 'Edwards, R.. W., and M. Owens: 1960. the effects of planis.on river conditions. I. Summer crops and estiniates of net productivity 9f macrophytes in a chalk stream. J. Ecol. 48:151-160. 4 Eyles, 'D. E., and J. L. Robertson, Jr. 1944. A guide and key to the aquatic plants( of the southeastern United States. Public Health Bull. No. 286. O. S. Gov. Printing Office, Washington, D.C. 151 pp. ; Fassett, N. C. 1960YA manual of aquatic plants. Univ. Wisconsin Press, Madison. 405 pp. Forsberg; C. 1959. Quantitative sampling of subaqu'atic vegetation: Oikos, 10:233-240. Foisberg, C. 1960. Subaquatic macrovegetation in Osbysjon, Djurholm. Oikos, 11:183-199. Holm, L. G., L. W. Weldon, and R. D. Blackburn! 1969. Aquatic weeds. Science, 166:699-709. Hotchkiss, N. 1941. The limnological role of the higher plants. In: A symposium of hydrobiology, lithe. Wisconsin Press, Madison. pp. 152-162. AP Jervis, R. A. 1969. Primary production in the freshwater match ecosystems`of Troy Meadows, New "Jersey. Bull. Torrey Bot. Club, '...96:209-231. 'it, Kolkwitz, R., and M. Mirsson. 1908. Oekologie der pflanzlichen Saprobien. Berichte deutschen botanischen Gesellschaft26a:505-519.. Mackenthun, K. M. 1969. The practice of Water pollution biology. U. S. PCA, Cincinnati. 281 pp. Mackenthun, K. M., and W. M. Ingram. 1967. Biological associated problem in'freshwater environments. II S. F% 'CA, Cincinriati. 287 pp. 1 ' i Muenscher, W C. 1944. Aquatic plants of the United States.Comstock Pub. Co., Ithaca.,374-pp. . .. Penfound, W. T. 1956. An outline for ecological life histories of herbaceous vascular hydrophytes. Ecology, 33:123-128. Phillips, E. A. 1959.-Methods of vegetation study. Henry Holt & Co., New York, 107 pp. Westlake, D. F. 1966. The biomass and produj ti ivity of Gylceria maxima. I. Seasonal changes in biomass. J. Ecol. 54:745-753. Wetzel, R. G. 1964. A comparative study A the primary produCtivity of higher aquatic plants, periphyton, and.phytoplankton in a large shallow lake. Int. Rev. ges. Hydrobiol. 49:1-61. Wilson, L. R. 1939. Rooted aquatic plants,and their relation to the limnology of freshwater lakes. In: Problems of Lake Biology. Publ. 4, No. 10, Amer. Assoc. Adv. Sci. pp. 10/-122. -- . , is Winterringer, G. S., and A. C. Lopinot. '1966. Aquatic plants of Illinois. Ill. State Museum -Popular Ser. ypi.,.1a, Ill.tate Museum Division. 142 pp. . 7 Yount, J. L'., and R. A. Cro,ssma r. 1970'. Eutrophication control by plant harvesting...- JWPCF, 42:173-183. / ---,,.. tl 3 1-) /7 1 - 4 M If MACROINVERTE-BRAZES Page 11.0 INTRODUCTION 1 -2.0 SELECTION OF SAMPLE SITES 2 ° 2.1 Systematic Sam,plirii . .. 2 ------2.2 Random Sampling.` - e ..c. ' 2 2 A 2.3 Measurement of Abiotic Factors ' 2.3.1 Substrate 2 It 2.3.2 Depth 4 2:3.3 Current Velocity 7.77747!--.-,N.... 4 2.3.4. Salinity 3.0 SAMPLING METHODS t ah 3.1 Quantitative 5 o 3.1.1Definitions and Puspoe 5 3.1.2 Requirements 3 .5 r3.1.3 Advatagesn '?- 5 3.1.4 Limitations 6 .3.2 Qualitative 6 3.2.1 pefini io s and. Purpose '6 3.2.2 Requirements L. 6 3.2.3' Advantages 6 3.2.4 Limitations 6 f3.3. Devices ' , . 7 \ 3.3.1 Grabs 7.- 3:3'.2 Sieving Devices `:-J ( ti. 9 Coring evices 9 3.3.4 Artificial Substrates 10 3.3.5rt Nets 11 3.3.6 Photography -.... ., 12 . 3.3.7 Qualitative Devices 12 4.0 2,AMPLEPROCESSING '12 -1.1 Sieving w 12 4.2 Preservation ,c 13 3.3 Labelling 13 /4.1: Soting and Subsampling 13 4.5 Identification , 14 4.6 'Bioinass z., 15 5.0 DATA EVALUAT1aN ...... :.. . . . 15 5.1 Quantitative Data - e I ._ ' 15 5.1.1RePOrting Units 15 5.1.2 Standing Crop and TaxOnothic . .--:---. 015 l) Compoftn 4 5.1.3. Diversity ) 16 5.2 Qualitative bata -) 18 5.2.1Inditator Organism Schemps ...... C.N.. 18 5.2.2 ,Reference Station Methods 18 .- L 6.0 LITEWURE CITED ir.,k: 32 =1:0 TAXONOMIC BIBLIOGRAPHY 33 . . . 33 7. I:Cpleopteta .,.,. . .,. . -.0...... , L tJ Page 7.2Crustacea 34 7.3Diptera , 34 7.4Ephemeroptera 35 7.5Hemiptera 36 7.6Hirudinea 36 7.7,,Hydracarina 36 7.8Lepidoptera :'9 36 7.9Megaloptera 36 .. .10 Mollusca 36 .1.1 Odonata s\,,. 37 7 12 Oligochaeta 37 .7.13 Plecoptera . 37, 7.14 Trichoptera 37 7.15 Marine 38 -F I MACROINVERTEB RATES 1.0 INTRODUCTION A community of macroinvertebrates in an The aquatic macroinvertebrates, as discussed aquatic ecosystem is vary sensitive to stress, an'd in this section, are animals that are large enough thus its characteristics serve as a useful tool for to be seen by the unaided eye and can be detecting environmental perturbations .resulting retained by a. U..,S. Standard No. 30 sieve (28 from introduced contaminants.- Because of-the Meshes per inch, 0.595mm openings) and live at limited mobility of benthic'organisms and their least part of their life cycles within or upon relatively long life span, their characteristics are available substrates in a..bOdy of water or water a function of donditions during the recent past, transport system. including reactions to infrequently discharged Any available substrate may provide suitable wastes that would be difficult to detect. by habitat IrCcluding bott9,rn sediments,, submergesperiodic chemical sampling. logs,debris, pilings, .pipes,. conduits,' vascular Also, bpcause ofthi.e. phenomenon of "biological magnification" and relatively long- aquatic plantS, filamentous algae, etc. , . The major taxonomic groups included in fresh term retention' of contaminants'y benthic water..are the insects, annelids, molluscs, flat- organisms, contaminants suchaspesticides, worms, roundworms,/ and crustaceans` The radioactive maten s, and metals, Are only major groups in saltwa,ter are the molluscs, peribdically dischaed or whicare-present at undetectablelevelinthewater, may be annelids, 'crustaceans, taelenterates, .porifera, and bryozoans. detected' by chemicanalyses of selected com- Benthic macrOinvertebrates can be defined by ponents of the macr invertebrate fauna. location and size but not by position in the In pollution-orited studies. of niacroinverte- trophie structure since-they -occupy virtually all bratecommunities,therearebasicall two levels. They may be omnivor s, carnivores, or approachesquantitative andqualitativthat herbivores; and in a well-bala ced 'sYstem, all.may be -utilizedsinglyorin..cOrnbition. thyee types will likely -be prese t. They include Because of the basic nature of this decision, the deposit, 'and detritusfeederg',parasites,.section of this manualrelAing to sampling scavengers, grazers, and pjedatorS. methods and data evaluation of macroinverte- Speciespre§ent, distribution, and abundance hrates is, arranged on the basis of whether a quantitative or qualitative apbroach is used/ of aquatic macroinvertebrates may be subject to A wide seasonal `varations. Thus, when conducting Ideally,the, design of macroinvertebr to comparative studies, the investigator- must be studies should be bdied.upon goalor quite careful tiavoid the confounding effects of objectives; however, the ideal.m4t frequently these: seasonat, changes. Seasonal,: variationare betempered bytherealities "of avail brke particularly _important- ,infresh-water habitats resources,timeamitatiohs impoSed on the 'dominated by aquatic 'insects king several life study, 'arid the characteristics of the habitat'to inselecting stages, not all of which are aquatic.. . bestudied. To aid the most The tm'acroinvertebratesareimportant advaltageous sampling method,ample sites,. mertibtrs of the food Web, and their well-being is and data 'valuation, the reader of this section reflected' in the well-being Of the higher forms should be familiar with the 'material in the ..such as ish7. Many. invertebrates, such as the "Introduction"ot, thismanual,partiyularly marine and fresh-water!sliellfish, are important th(34e, portions outlining and, discussing require-, commercial and recreational species. Some, such rnents of the various types of field studies:ring,. asmosquitos; black f0s, biting midges, andwhirhatiinvestigaffor may become involved. '- 'Asiatic clams', are Of considerable public\healt,h To supplement the material corftaine significance or are simple pests ;-and many forms manual, a number of bac references shOuld be .,are imppitant' for digesting organicmaterial and avvableto investigatoof the benthic co fecycling nutrients. pity, partifularly.itoose ,engased in wafer A BIOLOGICAL METHODS ;, pollutionstudies.TheseincludeStandard macroinvertebrate communitieis are so closely. Methods (2), Welch (57), Mackenthun (37), related to physical factors such as substrate Kittrell (291.,ynes (26), and Buchanan and type, current velocity, depth, and salinity, a Somniers (9). design using simple random sampling is seldom meaningful. Therefore, the investigator should 2.0 SELECTION OF SAMPLE SITES stratifythe habitat on the basis of known As discussed and defined more fully in the physical habitat differences and collect samples, section on biometrics, sampletes may be bythe random gridtechniqtie within, each selected systematically' or by vario s randomiza- habitat type. tion procedures. eis alluded to above, and regardless of the method of sample site .selection, the biologist must consider and accoukt for those natural 2.1Systematic Sampling . environmental variations that may affect .the Unless ;the data are to be utilized for quanta -ta- distributionof organisms. Among the more "ive. eValuations, some typeof syqeinatic important natural environmental.. variables in mpling is. generally employed for Synepti fresh-water habitats are substrate type, Current surveys, and reconnaissance. studies.Line velocity, and °depth. In estuaries, the salinity transects established at discrete intorvals across a gradient is an additional, variable that must be river or stream'and sampled at quarter points or accounted for. more frequent intervals are a form Qf systematic ` sampling and serve-as an excellent means of 2.3 Measurement of Abiotic Factors delimiting and mapping the habitat types. In lakes, reservoirs, and estuaries, transects May be 2:3.1 Substrate o, established along the short or Iongaxis or May Substrate is one of the most importapt factors radiate out from a pollution source. If a random controlling the characteristics of the cdmmunity start point is used for selecting sampling sites of aquatic Vacroinvertebrates found at a given, along'the.transects, the data may be anienable to location in a body of water (49). Over a period quantitative evaluation (see Biometrics Section)`'pf time, the natural substrates may be greatly AS will be d' ed, however, the confounding Altered by the discharge of particulate mineral or effects of c anges physical characteristics.of organic ratter 'and the location and expanse of the environmenhalg the transect must be fully various substratez,types (silt, sand, gravel, etc.) recognized anda ounted for.: . may change because of normal 'variationsinc. In another fo. f"sYsleailosrh sampling, the ..,. _hydrolobe- fa4ors such as current velocity and . investigatdr, .usingvariety. ofgqar, consciously- sTream flow. The biologist, Therefore, must be selefits and intensiverY Samples' all. recognizable cognizant. of c.hangesinthe nature and habitattypes.As-:previiouslywention011, this Rroperties of the substrate 1741.ich may provide form of sample site- sefectioiV. Viseful fOr clues on fiAe quality and quantit4of pollatan. ts synoptic surveys and for -COrityparative studies and consider factOrs which affect the normal where. qualitative comparisons. Are being made. distribution of the' benthic fauna.Q.' . . . Where the pollutant has a dirsat effect on the 2.2 Random Sampling characteristics of the subStrate-Jhe effects-bf For`conductingr quantitative studies, where- a'changes in water quality may' be inseparable measure of preciSion must be obtained, some from the effects,of changes in the substrate. In type of randomization ,groc durcr., mastbe-. Cases where substrate deterioration has occurred, employed inseleciing'sarnplin This selec- faunal` effects may be so obvious -that extensive tiOn may be carried'Out on the the. .area sampling may not be, required, and special atten- under study (simple Iandom sampling),.. or the tion should be given ,tothe Physical and/or randomizationprocedure may be conducted chemical characterization of the deposits. inclependently. on selectedstrata(stratified In nducting synoptiC surveys or other types random s'ampling). Because the.charActeristics Qf ofttalita ve studies and taking into account 1"") MACROINVERTEBRATE SAMPLING the limktati ns. of available"- sampling devices, favor of the more productive areas of "deposi- sarnplin404should be selettted to include all tion" on the inside of bends or in the vicinity of ° available".;wbktrates. If these qualitative samples-obstructions.- Just the _opposite situation may are to be used for determining the effects ofoccur in- many of the swiftly-flowing upland pollutants where the pollutant does not have a streams, where most of theeffort may be. direct affect on the substraE, the investigator devoted to sampling the productive rubble and must bear in mind that only the fauna from sites gravel riffle areas instead of the pools. having similaf substrates (in 'terms of organic ,.Because of the importance of substrate (in content,particlesize,vegetaiive cover, and terms of both organic content and particle size) detritus) will provide valid data for comparison. in Macroinvertebrate studies, it is suggested that sufficient samples be colleqted to conduct the , For quantitativestudies,' itissometimes following minimal analyses and evaluations: necessaryintheinterestof economy and .1 efficiency and within the limitations of the avail- ' able, gear, to sample primarily at sites having In the field, classify and record,-on' suitable forms,the mineral and organic "Matter substrates, Which normally support the most content of the stream, Ialce, of estuary abundant and varied, fauna, and devote a mini-. mum effort to thos'substrates supporting little bottom at each sample site on a percentage or no life. For histarice, in many large, swiftly- basis with the use of the categories shOwn in Table. I. Although the categories given in flowing rivers of theMidwest-and Southeast, the Table areas of "scour" with a substrate of shifting sand 1 may not apply universally, they- or Jiardpanmay be almost deyoid of macroinver- Id be applicable to most situations with only slight modification. tebrates; sampling effort may be reduced thele in TABLE 1, CATEGORIES FOR FIELD EVALUATION OF_SOIL CHARACTERISTICS* ,y. Type Size or characteristic Inorganic Components Bed rock or solid rock Boulders >256 mm (10 in.) in diameter Rubble 64.to 256 mm (21/2 to 10 in.) in diameter Grav'el' 2 to 64 mm (11112 to 21/2 in.) in diameter Sand 0.06 to 2.0 mm in diameter;grity texture when ribbed lietween. fingers. Silt 0.004 to 0.06:mm in diamcr Clay <0.004:mm in diameter; srn oth,islk feeling when rubbed between fingers Marl Calcium carbonate; usually gray; often contains fragments of mollusc shells or Orarct; effervewes freely with hydrochloric acid Organic Components Detritus Accumulated %vtgod, sticks, and other-undecayed coarse plant mateiials Fibrous peat Partially decomposed plaqnt remains; parts ofplant,readily distinguishable Pulpy peat Vdr; fittely-divided plant remains; parts of plantsnot distinguishable; variesin color from peen to brown; vario greatly in consistence often_beint,,_,serni-fluid . . MuFlt. Black, figiy. divided orgAic matter; completely defmpo 'ed 1 1 *Modified from Roelofs, L. W. 1944. Water soils in rcla,tion to lake productivity. Techf Cull. 19Agr. Ex2. Sta.,State ColleetS Lansing, Mich. . ...-- r \ 1 ( ob. ,---,, QJ ,../ . ,BIOLOGICAL METH40S In the laboratory,' evaluate, the inorganic sampling sites for both qualitative and'quantita- components by conducting a' wet and dry live studies, depth must be measured and incyd- p artiblesize. analysis on one or more ed as an independent variable in the study design. samples and preferably on replicate samples from each sampling site with. the use of 2.3.3 .Ctirrent velocity standard sieves and following the modified` Current velocity affects, the distribution of Wentworth classification shown in Xable organisms Al lotic vnvironments and alOng.the Detailed procedures for sediment analysts''Windswept shores or lditic environments,oth are found in IBP handbook No. 16.* directly (because of differing species require- m eaft s)',, 'a n d: indirectly L (sortingof, bottom Sediments). Therefore, It -isof critical TABLE 2.SOIVPARTICLESIZE ''.imporlance that velocity' be consideredwhen CLASSIFICATION* sampling sites are selected, and when data are analyzed. ,Only sites/ with comparable vefbeity U.S. standard sieve Name Particle size shOtild° be compared. At the actual time of (mm) 'series # sampling, determine velocity at .eachsamPle site Boulder >256 Rubble .64-256 by. using a suitable current measuring device. Cciarsegravel 32-64 ' "The TSK flow meter 'listed. in the appendix. is Medium gravel 8-32 t . suitableif modified by the.'addition of .a Fine gravel 2-8 10 Coarse sand 0.5-2 35 ;stabilizing fin arid Medium sand . 0.25-0.5 120 ti Fine =id 0.125-0.25 230 At depths greater ;than 57 feet, use tile Very fine sand 0.0625-0.125 Silt 0.0039:0.0625Centrifuge (750 rpm, 3.mit)t 'two point method (1); take readings at 0.2' Clay <04.0039 Evaporate and weigh residue and 0.8 of the depth beloW the surface. The -.0. *Modified from Wentworth (58); see Cummins, K. A: 19e average of these two observations is taken An evaluation of Some techniques for the collection and analysis as the mean velocity: of benthie samples with special emphasis on lotic waters. Amer. At depths less than 3- feet, the 0.6-depth Midl.Nat. 67:477-504... \ method/(1) is used; take, readings at 0.6. of t' Standara sieves with 8-mm diameter openings are commonly the dePth below thesurface. available., , s , . - Where, artificialcial substrate samep, beigg t Jackson, M. L. 1956. Soil chemical analysis. Univ. Wisconsin . Press, Madison. utilized, ta-l-e-ifie reading direr y. bpgtream Of the sampler and at the saMe'deptti,:' . Determine the organic -content by .drying 2.3.4 and ashing17,presentative 'sample of the Salinity is an 'mportant factor in rnarinc;and sediments; use the methods .outfined in the estuarine envirmerits. The salinity of sea water Pianktc6,Secti?t370. . is approxim. ely 35 parts per thousand; salinity,, 7 of .fiieSh at ris _ generally a few parts per 2.3.1 Depth million., In etsaries,,,Where sea-mater and fresh Depth indirectly affects the distribution ofwater meet,ere may be wideuctuations of aquatic macrbinvertebrates, as a result of its salinity with tidand river disch .This area: influenceon the availability 3 of lightfor plant may: be inhabited, to'some extenebYboth freih: grOwth, .on water SemPeraturer on the zonation and salt.-waterms, but the numbei of speCies of- bottom deposits; on the water chemistry is usually less than that that oc urS-under more (particularly wcygdn); and phototactic responses,stable conditions of Salinity (3 Since move- of organisms.In: .regal d' ",to the selection ofment,. as well as general loon 'of many *Fiblme, N. A.,an AI, D. Macknfre. 1971.ethodscbt the species, is governed'by tides(and salinity, these study of marine ilenthas./International Biological Program, Davis Must be' taken into accoun mining Corn"p4iiy Phil,alielphia. 346 pp. loampling time and.locatibn., .6 ,, MACROINVERTEBRATE SAMPLING 5 , -pi _ p - . Bedause of the extrem 'Spatial a d tempoi-41 o,by large; number' of individuals, AenlY distri- fluctuations of salinity stuarie, ,rapid. bqed in%the substrate:. Convcorsely,'a large coef- instrumental methods of meisur meat are more .:fi4ent "of` would be expected if :the , A desirable 'than slower, morerecise Chemical fatind consists of a. large. number of S'pecies with . =1 'tietliods-(3,8): patchy distribution of To sbtain Wide-range, temperature- ompensated con: , the' same level. of precision; at a 'given. level . ductivitysalinomete,rs - are recommended for probability, a larger number =of replicateS would deterniining hotti horizontal and vertical salinity .requireCU:ifi. the latter cag4han in the farmer!' profileidt higl-slack and loW:slack tide levels in Id' general,the. smaller the sufficearea the°;!.-arleaof 'estuary, or 'reach ofrives .being encoMpaped by a,samPling deVice, the larger the %studied:,, number :of, sainplesmequired to 66tain a desired. , lefelof., precisioii ..rCaribe 3 Kt:4A AVPLING.METHODS , ilicrefped :.collecting larger samples, or by. increasing the nunjbers 'of sarnfles collected: 3*102-21iNTITATiVE. Art Objective,qtiantitative apprOach neces- . , 31.1Pefinitions and purpose tsi ates that a Measure of the precsion of the , estimates' be Obtained thus, replicate SaMpling Alth9144,the data may be evaliatelk in various ways,a quantitative method essentially involv'es in each habitat or Strainin selected ,or study is ;dr.triQ numbers or biomass- an absolute requirement. For measurement of precision"' three 'replicates absolute' (standing" crop) "Ot: the: valious cOmponents of are an minimum:: (A series of single samples taken at the ma'crQinvertebrate community pet- unit area discrete afonda transact cionot reprgsent-. inall a p6ttion:..of the habitats lePliCate,.sampleS. of. benthic ottani ms unless it s..,t (including art-411yintroduced habitatsiin the an be demonstrated that the physic I character------.ensySlerri- be studied, and-provides :ninforma- tion orthe species composition, richness of istics of`' the -habitat klo° not change along the -transect.) sa6es'and distribution bf among the species. , ft is preferakile,.-if data are available (or can be 'Obtained by recbnnaissafice or ,expfbratory - 1,° , _' . studies); to determine the number of replicates '.1.1 .Tv ,_.,,R,equirements on theebis .91. the desired leVel. of precisiqn, as estimates by Using devices* _ . that sam,ple ,a unit area or volume or habitat", discussed.'the Biometrics Section. such as a 'Surber .squate-foOt sampler, :which in -; use pr4sumatily collects114iorginisrns, enclosed 3.1.3 Advantages 1 within the fratne' Of. the: sanpler, or th artificial InadditiontoproViding Arne data substrate.Samplet hiving,'a fixed volume or obtained' from qualitaftie study, the standing exposing a fixed aniouniof,surface. crop data generated. by a quantitative study pro- fri''the',.sfudy' of inacroinvertebrate- papula--vide a means of comparing the, ptodtietivity of tions; the Sampling' precision°is aifetted by a different environments; and ifa ;measure of .,i!, number of factors; including:. Size, Weight, and turnover. is available, the actual'oductiOn can .construction Of the:Sampling device the type of b corn uted. r subst4-ate, anethe distribtitiOn of organismsin ',The use of 'quantitative- sampling clevi4S in arid on,the,silbstrate:iFor example, it 4 expected. arefully chosen .habitatsis reconnneNed that the of Standing crop drawn froh a because they reducn sampling bias resulting from.. series St samples,will be, more precise (Mre a differences in expertise of the sample collector. o we4: 2c,Oefficient of v-ariltiOn) hen 'alio, , The data:train prerly designed quantititivf community Consists oil a few specieS,rresented studies are amenable to the use of iin.plbiit 5 , ,-- BtoLocicAL MI nibps powerful statistical tools that aid in maintainin echniquesquire experience and a high level of the objectivity of the data evaluation process. The measures of precision and probabilityate- conducting comparative studies. Of the ments that ph be attached to quantitative data owur.benthos, a major pitfall is the confounding reduce. the possibilities of bias in the data. yak!- e co thedifferences inphysical habitat atiOn process and make the results of.di ferent am different stations being studied. This investigators more readily comparable. danger isparticularly inherentin qualitative The ach7antages, then, of quantitative methods studies when an attempt is made to systemati- are: callycollectrepresentative specimens, of all speciespresentatthe sampling statues or They provide a measure of productivity. eaches of 'river bein'g compared. Unfortunately, The, investigator can measure precision of differences in habitat unrelated to the effects of estimates and attach probability statements, , introduced contaminantsmay render such com- thus providing objective comparisons. parisoAs meaningless. Minim4e this pitfall by The data. of different investigators may be CarefAy recording, in thefield, the habitats compared. froth which specimens are collected and then basing comparisons only on stations with like J. 1.4Limitations. habitats in .vv h the same amount of collecting Presently, no sampling devices Age adequate to:effort has bee xpended. sample all types ofhabitat; so when _quantitative. 3.2.3 Advantages devices, are used, 'only selected portions of the environment may be sampled. ' Because of wide latitude in Collecting tech- Sampling precision is frequentlx_so low that niques, the types of habitat t at can be sampled pro itive numbers of rtplicate samples may be are relatively unrestricted. A uming taxonomic reqiffed to obtain mearlingful estimates. Sample,expertise is available, the processing af-qualita- processing and analyit,,.are slow and time- tive samples ..is often considerably faster \than. that required for quantitative samples. . consuming.. 1n4 some, lases tl'iere fore, time limi- tations placed. do a Oftdyay prohibit the use 3.2.4 Limitations of quarititative techniques. Collecting techniquesaresubjectiveand: 3.2 Qualitative. depend onthe-.. skilland experience of the individual who makesthefieldcollections. 3.2:1Definitions and -purpose t Therefore,resultsor' oneinvestigator2re difficult to compare-with those ofianother. The objective of qualitative-studies is to deter: discussed elsewhere; the drift of organisms mine the presence or absence of forms having info he satriple area may bias the evaluation of . Varying .clegrees of tolerance to contaminants qlitaliVe data and Tender comparisons andtoobtain iriformat du"richness of eaningless. S;:finPles are'cibt.cd with the use of a o informationon standing crop or produc- "Vapety of collecting methods and gear, ,tn can be :generated from a qualitative study. iitiuyof which are not a 1)16 to quantitation . On h unit-area basis. Wh ducting qualitative 3.3° Device's studies, an attempt is usually made to colle4t aIJ species present by exhaustive samplirig in all 3.3.1Grabs b available habitat types. atf 'devices designed to penetrate the 'substratey viYtue of their own weight- and h. 2 Requirements leverage*, rid have spring- or gravity-activated. ',. Recognizing and locating various ty`Pes of;cloSing m Ilenisms, In shallow waters, some of ha lig t a is'wherequalitative .samplescanbe these deviceS flay be-rigged on poles or rods and collected' and : selectingsuitablec011octing phySically pushedintothesubstratetoa :-. 4 C..., MACROINVERTEBRATE GRABS predetermined depth.Grabswithspring- maxittium depth of penetration before closure., , activaid closing devices include the Ekinan, In soft substrates, for which this grab is beSt Shipe,k,andSmith-McIntyre; gravity-closing.suited, the penetration -.is quite deep and the grabs incde the Petersen,* Ponar, and Orangeiangular glosure of the spring loaded jaws has Peel: Excelltiitt desCriptions of these devices are .veryli,teeffect on the volume oP sample given in Standard Methods (2) and Welch (57).-.collected. In essence this means that if the depth ...1._Grabs_ are7useful for sampling at all depths/in of penetration is 15 cm,, the organisms lying at tlakes, estuaries, and rivers in substrates ranging that depth have the same opportunity to be from soft muds through gravel. sampled as those lying near the surface.. Inadditiontothepreviouslydiscussed In clam-shell type grabs, such as the Petersen, problems related to the patchy distribution of Ponar, Shipek, and Smith- McIntyre, the original organisms in nature, the number and kinds of penetration is often quite shallow: because of organisnis collected by a particular grab may be the sharp angle of "bite" upon closure, the area.. affected by: enclosed by the jaws decreases ,'atincreasing depths, of substratepenetration.Therefore, .7deth Of penetration within the enclosed area, organisms found at angle of 'closure greater depths do not have ampqual Opportunity completeness of closure of the jaws and loss to be sampled as in the caseof the Ekrnan grab of sample material during retrieval and other sampling methods described in the . creation of a "shock" wave and consequent next section. Th%problem is particularly true of "wash-out" of near-surfac'e organisms the Shipek sampler the jaws do not penetrate stabilityof samplerat'thehigh-flow the substrate before closure and, in profile, the al velocities often encounte in rivers. sample is'essentially one-half of a cylinder. Probably one of the most frusta ting aspects Depth of penetratiOTh is a very serious problem of sampling macroinvertebrates ithvarious and depends ontheweight of sampler as types Of grabs - relates to the problem of incomt opposed to theparticlesize and) degree of plete closure'.of the,jaws: Any object such as compaction of thebottom sAiments. The clump'S of vegetation, wOOdy debris, and gravel Ekman gab is light in weight,a d most useful that cannot be sheared by the closing action .fOr eampling soft,finely j ed. substratesof the jaws.often,prevents complete closure. In composed of varying propor ions- of fine sand;the order of: their decreasing ability to shear clay,)silt,pulpypeat, and Muck.: For clay obstructing materials, the-coinmon grabs may be. hardpan' and coarse substrates, such as coarse ranked: Shipek, Smith-McIntyre, Orange ?ea.'. ,'Sands and gravels, the heavier grabs such as the Ponar, Petersen, and .Ekman. If 'the -Ekrnan is orange peelor clam shell types (Ponar,"Petprsent,.filled, to within more than 5 cm of the top, .Srifith,Mcfnt-yre)aremore, satisfactor there may beloss of substrate material on weights may be added' to aid penetra-,.'.retrieval (16), An aavantase of the' Ekman grab Lion of the substrate and to .add stability in isthat the surfam.... of the sediment can ,b6 heavy currents and rough Maters, examined upon retrieval, and only those samples BecauSeiof differences in t,4 depth of gene - in Aibli the sediment surflicegi is undisturbed tration and the angle of "bite': upon clOstire, should be retained. !. data, from the different grabs are not coinpar- All grabs and corers produce .a/"shock" wave able. The. Ekman essentially encloses a square, as they descend. This disturbancecan affeCt the which' isequalin area from the ,surface to efficiencji of a sampler bi'causing an. oittWard wash (blow-out) Of flocculent materials ne,arthe *Fo.clist Modification of the Petersen grab desuibect314 Welch t (57). mudwater intercei ,:that. may result- in 2 7 4 BIOLOGICAL METHODS adequate samplinge7' near-surface organisms On the basis of-the calculdtions in Table '4, such asphantorr( midgelarvae, and ,some there appear to be no consistent differences in Obironomid midges. The shock wave of the the precision of, estimates collected by Ekman, Ekman grab is Minimized by the use of hinged,..Ponar, and Petersen grabs in mud. or sand sub- Iniely-opening top flaps. The Ponar -grab is a--strates. The poor closure ability of the Ekman in modifiedPetersen `withside-urtains and a coarse substrates such as gravel is demonstrated screen on the 'top. The screen aws water to by the large C valuesfor the Ekmaras compared, pass and undoubtedly reduces shoek wave; with values for the' Petersen and Ponlig graver" however, divers haVe "observeblow-out with substrates. this deVice (16), Another way of demonstrating the reliability prab-cofleetea . samples provideavery of. grab sample estimatesof macrobenthos iropre.cis& estimate of the numbers of individuals standing crop is to calculate, at a given proba- and., inil.'nbors of taxa of aqua& macroinverte- bilitylevel,the range of values around the brates. Asuminary of data frOm various sources sample mean in whic,4 the true mean should lie shows that the 'mean coefficient of variation (C) if a.,,given number 6,f replicate samples were for numbers -of individuals collected. by Ponar,. Ffom the data shown in Table 3 for Petersen, and Ekman. grabs was 46, 48, arid .50gthe Petersen, Ponar, and'Ekman grabs in various percetit, respectively (Table .3). In most of .the types of substrate, coefficients of variation near studies on which the.41eulations,in Table-3 are 50 percent for numbers of individuals and 35 based, 0/ level of replication ranged from tier:, percent fo numbers of taxa should be expected to six samples. Estirnatiorp,of number of taxa with 3 to 6 replicates. With the use of these aremoreprecise;forPonar,Petersen, and expected values, the true mean for numbers of Ekman grabs, the mean calculated C was 28, 36, individuals and, number of.taxa of Macroinverte'-. and 46 percent respectively (Table 3). liqrates should lie within plus or minus 36 percent "_ ITABLE 3.IVIEAN AND MODAL VALUES FOR COEFFICIENTS OF VARIATION* (EXPRESSE6 'AS PERCENTAGE) FOR NUMBERS OF INDIVIDUALS AND NUMBERS!. . OF TAXA OF MACROINVERTEBRATES COLLECTED.BY VARIOUS DEVICES Individuals Taxa . Sampling R emarks. device Mean Modet, Mean Modet GP Rock-filled 32 -21-i0 .. 20 11-20 22 sets of samples with 4-6 reps. per set .(52) and __, barbeque . 2 sets of sampl6 having 15 and 16 reps. (13) - basket Ponaf 46 41-50 i28 11-20 12 sets of samples-with ,12 reps. per s 16, 31). Petersen 48 51-60 ",36 21-30 21 sets of samples with 3-'6 reps. per s 1, 5 54).. I,T§ Ekman 50 41-5.0 46.,: 3-1-40 27 sets of samplelvith 3 -12 reps. pot set48., 16, 3I; 4* 45. 53). (--- . ,, Surber 50 , 41-50 60 sets of saMples having 6 reps. per setp0); Corer:- '50 7 sets of samples having,10 reps, per set (8), . . , Stovepipe 56 31-40 38 . 21-30. 32 sets of samples having 3-4 reps. per set:(53). *:ociticient of.variation = (s.taildard-devativ-x 100)/mean. t Frequenty distribution based On 10`;', increments. " tOligi.Nhaetes only. 0. MACROINVERTEBRATE SIEVING A CORING DEVICES and 25 percent, respectively, of the'sample mean When using a sieving-type device for quantita- at a 95 percent probability level; ifj 0 replicates tive estimates, reliability may be affected by: were collected. (See Biorndrics.Section.) O Precision would; of course, be increased if adequacy of seating of the frame on the additional samples_ were collected, or if the substrate sampling method were -more precise. ' o backwash resulting., from resistance of the Sincetheassumptionsnecessaryforthe ,net to water-flowat high velocity orflow statisticakcalculations shown in Tables 3A and 4, this.may be significant arenot likely met inthe data of, different care used in recoVering the organisms from investigators collected from different habitats, the substrate materials the above calculations only provide'tka gross de$th to which thesubstrate is worked approximation of the precision to be exPecte4. drift of organisms from areas upstream of They do, however, serve to emphasize the very the samp,leskte imprecise nature of grab sample ,datii and the resultant, need for careful stratification of' the., To reduce the possibility of bias.resulting type of the habitat .sampled and sample' repli- from upset disturbance of the substrate, cation. always ,stand on thedownstream side of aAieving device and take, replicates in an upstfan or lateral direction. Never start in the upstream portion of a ,pool or riffle,and work in a tOwn- TABLE 4. MEAN COEFFICIENTS OF stream direction. VARIATION (EXPRESSED AS PERCENTAGE) The precision of estimates of standing. croN FOR NUMBERS OF INDIVIDUALS AND of maCrobenthos obtainedwith ,Surber-type NUMBERS Oil-TAXA OF MAC 0 NVERTE- sieving-devices Vtries widely and depends on a BRATES COLLECTED IN DIFFERENT hurriber of factors including the uniformity of. SUBSTRATES BY GRAB -TYPE DEVICES substrate and distribution Of organisms-therein, AND A 'CORER DEVICE* ':the care used .in .collecting samples, and level of Substrate sample feplictItion. . Sampling 4 device Mud Sand Gravel For a large series of Surber samples from Ind,. Taxa Ind. Taxa Ind. Taita southeastern U. S. trout streams, the coefficient Fkman 49 40 41 21 1.06. r 74 of variation(C) ranged from 11percent to Petersen 41 L 29 50 41 49 20 It# greater than 100 percent (Table 3%). The mean Ponar 46 . 25 .38 .33 , 48 19 value of C was near 50,percent,,and mote .than Corert 50. one-half of the C values tell between 30 and 50' ,f.rlculated froM data in ref9tenceN (8, 16, 3L 45, 53, 54). percent. These Values are si4ilar to the20 to 50 Vligochaeto only. / percent reported by, Allen (1) and for those.. A discussed above for grab sample'-data. .- 3.3.3Co. ring.devices \, 'X: 3.3.2Sieving devices Includedinthis 'categdiy esingle- and FOr quantitative... sampling, 'the vell-icnown multiple-head coring deVices,-:bular inverting ,_. Surbersquare-foot sampler (2, 57.) is the most devices, and open ended. tovepipe-type devices. commonly used .sieving device. This device can Coringdevicesare =, describedinStandard be used-only, in floying water having depths not Methods (2) and. Welch (5'7). Corers can be used- greater than 18 inches and preferably less thantat various depthsinany substrates thatis 12 inches. Wis.' commonly used for sampling the sufficiently compacted so that the sample is rubble ahr'graveLriffleS of small streams and retained; however, -theyarebest suited',; for may be used in pools where the water depth is samplingthe 'felatively homogeneous soft knot,too gre' secinfents. ofthey dedper portions of lakd. °BIOLOGICAL METHODS \ Because of the small area sampled;data from problems relating to depth of sediment penetra- coring devices arelikely ..toprovidevery tion, changest.in cross-sectional area with depth imprecise estimates of the standing crop oh.of-penetraticinimd escapement of organisms are macr6benthos. As the data in Table 3 illustrate, circumvented by stovepipe samplers, they are the variabilityin numbers of oligochaetes (arecommended for quantitative sampling inall dominant component the fauna studied) shallOw water benthic'habitatS. They-probably collected in corers is ilar to that for grab-type represent the only quantitative device suitable devices; however,e corer data Were calculated. for sampling shallow-water habitats containing from two tothr times-4S'. many replicate stands of rooted vascular plants and will collylct samples and were collected. from "a relatively organisms inhabiting the vegetative substrates_as. homogeneous substrate. . well as thoseli ing in sediments. The coef- for the stovepipe samples in Such a'cldhional replicationwitlik corers-is ficients of vaiiao -feasible because of the small amount of material Table 3 are comparable to the coefficients 'for ,grab -samples, although the stovepipe samples pe? samplethatmustbe L handled ° in . the laboratory.. Multiple-head corers! have been used were collected in heavily vegetated and conse- in an attempt toiredtice.the field saMptitt&efforttquently hi iable habitats'. thacgauSt 156- expended t6 collectlarge series: of core samples (19). ..-..... substrates The Dendy inverting sampler (57) is ahighly Tkieba. miittipje-plate sampler ,(23) and efficient coring-type device' used for sarriplingp.0,rock -fill baskaktsampler (21) have been depths to 2 Or 3 meters in nonvegetate. ,modified bnumerous workers (17, 40) and are strates ranging from soft muds through' coarse Widely usefor investigating' the macroinverte- sand. Because of-the small surface area saripled, brate community. Both samplers may be data obtained by ,this sampler suffer'From the suspended from asurfacefloat or may be same lack of precision (51) as the coring devic.es modified for use in shallow streams by placing described ave. Since the peer- sample procesing them on a rod that is driven into the stream time is redgad, as with thecorers, large'series of,bottom of anchored in a piece of concrete (") replicates can be collected. The Daidysailer A multipie-platesamplersirhllar tot is highly, recommended fOr use in habitatsfor described b5 Fullner (17), except with circular Willa it is suitable. - plates and spacers, is recommended for use by FPA biologists. This sampler is constructed of , Stovepipe-type devices include the Wilding 'sampler (2; 57) and any tubular materiahstich as 0.3-cm tempered hardboard cut into 7.5-cm 60to75 cm sectionsoftandard :.diame`tercircular . plates :and2.5-cmcircul4ir, diameter stovepipe (51) or 75 t,:m sections of_spactrs. A total of =14 platesand 24 spacers are 30- cm- diameter aluminurmirri ati6n pipe fitted required for each, sampler. The hardboard plates with handles..In use,:the irriga io p or CDIT1- and are placed on a 1/4-inch (0.(s'15 mercial- stovepipe ismanuallypriced t eyqbolt so that there are eight single spaces, one substrate; after Which:! the: CO_ained v'egetatiOn dole ispac.,e,twotriplespaces,and two and coarse. substrate !;natt4ials aet .reTriPied by Aruple ispa9esbetween_ theplates.This hand.The remainitig,.rOieria#Larejereate'dly tamplce has an'effective surface area(excluding stirredo into suspension; 'PernOtd. With the bolt) of 0.13 square meter and conveniently handled dipper, and poured through a wooden- fits into a wide-mciuth glass kr plastic jar fot framed loating sieve. BecAnse.ofklhe laboriousshipment andstorage.Caution shouldbe and' repetitive 'irocess of stirring, dipping; and exercised in the .reuse of samplers that may have sieving large volumes of,material,the collection been subjected to contamination bytoxicants, of a saluple often fequires,20 to 30 minutes,,, oils, etc.. J. The use of stovepipe samplers, is limited to The'rocls, basket sampler is a highly effeciivel standirke or slowly movinwaters having tioevice _7(f(71,rstudying, themacroinvertebrate maximum, depth. of 'less 't an- 60 cmSince community: Al chrOnnenlated basket MACROINVERTEBRATE ARTIFICIAL SUBSTIEATES AND DRIFT NETS (2) or comparable enclosure filled with 30, 5 to Samples usually contain negligible amounts 8-cm-diameter rocks or rock-like materialis of extraneous material, permittirig quick recommended for use by EPA biologists. laboratory processing. To 'reduce the number of organisms that escape when the samplers are retrieved, the Limitations of artificial substrate samplers are:- multiple-plate-sampler and the rock-filled basket sampler should be enclosed by a dip net con- 'The need folha longexposure period makes structed, of 30-mesh or finer grit bolting cloth. the samPlerstAinsuited for short-term survey Artificial substrate samplers, to a great extent, studies. depend on chance colonization by drifting or Samplers and floats are sometimes difficult swimming organisms;..vand, thus, the time of atittior in place and may presenta exposure may be critical to. the develOpment of navigation hazard. a relatively abundant. and diverse community of .Samplers are vulnerable to vandalism and organisms. Adequate data are currently unavail-. are often lost. able to. determine the optimum exposure period, Samplers provide no = measure of the which is likely. to differ in different bodies of condition of the natural substrateat a water and at different times of the ,year: Until station or of the effect of pollution on that more data become available, adoption of a substrate, including settled solids. 6-week exposureperiod(2)isprovisionally Samplers only record thecommunit'that recommended as standard. If study time limita- de'velops during the sampling period, thus tions reduce thisperiod, the data must. be reducing the value of the collected fauna as evaluated with caution and, in no case, should indicaSors of prior conditions. data be.-compared from samplers exposed for diffekentlirne periods (43). Two other objections often made to the use In ,deepee,,,waterS, artificial substrate samplers of artificial substrate Samplers are. that they are should be, Suspended from floats and should be selective to certain types a fauna and the data well up in the photic zone so thh-periphytit obtained do not provide a valid measure of the growths candevelop ,and provide\ .foodfo'i productivity of a particular. environment. The grazing forms Of macroi0ertebrates.:Unless the validity of the lane': objection depends on study waterisexceptionally turbid, a1...2,-meter objectives and may be of minor consequence in to.(4-foot) depth is recommended as sti*:ird. If many pollution-oriented studies. The selectivity they water isless than 2.5 meters de4, the of artificial substrate samplers is a trivar:objec- sampler should be suspended from a float half- tion, since all currently available are Way between the water surface and, the stream selective. The selectivity of conventionalconventional bed. sampling devices other than artificial substrates ,In some situations,artificial te is directed toward those organismS that inhabit methods arCthebest means of.: co ing the types of substrate or substrates for which a quantitative -studies of the ability of aix, atic'particular type of sampler is designed. 'environment to ,support a diverse4Ssemblage of \ macroinivertebrate .organisms. Advantages of the method 3.3.5 Drift nets Nets, having a 1.5 by 30cm upstream opening aro The confounding effects of, substrate 'differ- and bagAength of m (No. 40 mesh , ences are reduced. nttting)-.a ;14- recommendedfor,small,sYvift A highe( level of precision is obtained than streams. In laige:; deep rivets with a current of i.41:' other sampling devices (Table 3). approximate) _03 rneters.per second (mps); varrtitativelycomparable datac nbe nets having. *, ;eni Qf 0.093 m2 are obtained in environments- from whiji it is mended (2). or e nets in flowing water'' virtually impbtsible to/obtain.samplefi. with .vg...(current not less a0.0'15-pips) for front I to . :conveptional devices. 24' hou 'Ys, depending on-the density of bottom 4f BIOLOGICAL METHODS' 1.1 fauna and hydrologic -conditions. Place the top the quantitative devices discussed previously, plus . of the nets just below the surface of the water to hand-held screens, dip nets, rakes; tongs,Apost permit 'calculation of the flow through the nets hole diars, bare hands, and forceps can be andto lesien the chance for collection ofused. For deep-water collecting, som.i of the floatin rrestrial insects. Do not permit the conventional grabs described earlier are normallyv nets to ch bottom. In large rivers, maximum,.required. In water less than 2 meters deep, a catches are obtained 0.3 to 0.6 meter above the variety of gear may be used for sampg the bottom in the shoreline zone at depthsnot).sediments including lodg-handled dip nets and exceeding 3 meters. post-hole .diggeis.CollOptions..from vascular Drift nets are useful for collectingmacro- plants and filamentous algae may be made with invertebrates thal migrate or are dislodged from a dip net, common garden rake, potatofork, or the substrate; they are particularly well-suitedoyster tongCollections from floating debris for_synoptic surveys because they are light- and rocks may be made by hand, using forceps weight and easily transported. Thousands of to catch the smaller, organisms. organisms including larvae of stoneflieS, . lh shallow streams, short sections of common mayflies,caddisflies,and midges' and other wirklow screen may be fastened beten. two Diptera, may be coliedted in a.sampling, period piles and held *place at right angl`to the -^ckf only a few ;hours. Maximum drift 'intensity Water flow to collect organisms dislodged from occurs between sunset and midnight(55). Elliot upstream materials that have been agitated. (14) presents an excellent synopsis of drift net methodology. 4.0 SAMPLE. PROCESSING N. 3.3.6 Photography 4.1Sieving ;. The use of photography is mainly limited to Samples collected with grabs,.,tubar devices, environments that have 'suitably clear water and and artificial:substrates contain var. amounts are inhabited by sessile animalsand rooted of finely divided materials such as mpletely plants. Many estuarine habitats, such as those decomposed organic material, silts; clays, and . Containing corals, sponges, and attached algal finesand. To reduce sample volume and forms, fall in this category, and can be photo- expedite sample processing in the laboratory, graphed before, °during, and after the introduc- these fine4s should be removed by passing the tion of stress. The technique has been used with sample through a U. S. Standard No. 30 sieve. Success, in south Florida to evaluate changes Sieves may range from commercially con- brOught abotit by the introduction of heated structed modelsto, homemade sieves framed 4effluents. w,ith wood or metal.Floating sieve with . The techniqueforhorizontal underwater',wooden frames reduce the danger of accidental phdlosusing scuba gear involves$1acing a Photo- loss of both sieve and sample when workingover graphically identifiable marker in the habitat to the side of a boat in deeper waters. A good sieve be photographed and an additiOnalnearby -Contains no cracks or crevices in which stall" marker on which the camera is placed each time organisms can become lodged. a photograph is taken. By this'means, identical If at all possible, siqving should be done in thei .areas can be photogaphed repeat,edlyover a field immediately after sample ,Collection, and period of time to evaluate ori,site chaftges in -while the captured organisms. are alivef'OnCe sessileformsat --bothaffected and control preserved; many organisms become quite fragile stations. Vertical, overhead photos may anfl if subjected to sieving will be up and .',.taken under suitable conditions.. " lost'or rendered unidentifiable. 1? ".accomplished,by one of 4 Siefing. may 3.3. 7 Qualitative,de vices several -techniques ependingiuporriire refereli,Ce- The investigator. has an unlimited Choice of of the individual ogist. Iii one technique; the _gfar for collecting qualitativesamples. Any of sample is place irectly. into a sieve, and the 4 1.°12 ( AviACROINVERTEBRATESAMPLE PRoCiSSING., sieve is then partially submerged in water and external labelswiththelog number and oagitatecf untilallfine materials have passed notations such askl of 2, 2 of 2, are ,helpful fOr through. The sieve'is agitated preferably ina tub identifying sample containers in the laboratory. of water. Minimum information required on the sample A variation of this technique is to place the labelis a sample identification (log) number. original sample in a bucket or tub, add screened The log number identifies, the sample ina bound water, stir, and pour the slurry throligh a U. S. ledger where. the name .of water body, station Standaid No. 30 'sieve. Only a moderate amount number, date, sampling devicecusea),name of of agitation is then required to completely 'clean sample collector, substratecharaaeristics, the sample. Since this method requires consider-,-depth, and other environmental informationare ably less effort, most biologists probably prefer'placed. it. InibOac of the above methods,remove all the large f pieces -of debris and rocks from samples 4.4Sorting and Subsampling collected, clean catefuly; and discard before the For quantitative studies, sort and pick all samgile is stirred or agitated. samples by hand in the laboratory usinga. low-7, e artificial substrate samplers are placed in -powerscanning lens.To pick organisms a bucket or tub of screened water and are efficiently and accurately, add. only very small dismantled. Each individual piece of substrate amounts of detritus (no moreflan a heaping material'is shaken and then cleaned gently under tablespoon full) to Standard-sized (25 X 40 X 5 water with a soft brush (a soft gra en aineVpaii-Sfilied .4 pro x im a tely brush is excellent), examined visually, and lai ,one-third full of water., Sma4inse.9ts and worms aside. They water in the bucket otub is then will float free of: most .debris when. ethanol- poured through a U. Sr/Standard No. 30 sieve to -preserYed samples are transferred to the wafa, remove the fines. ,0' filled pan. AnalYsis time for samples containing 4.2 Preservation excessively large numbers of organisms Can be -Fill. sample containers no more than one-halfvsubstantially reduce'dzif the satnplesare sub-' Vaull of sample material (exclusive of the preserv- divided Wore SortingTfhe Sainple is thoroughly ative). SuPplementarsaniple containers are Iised mixed and distributed evenly over the bottom of for samples with large volumes of material. a shallow tray: A diyider, delineating one-quarter Obtain ample nuM5efs,and kinds of sample sections, is placed in a .tray, and two :opposite' containers before the coftction trip: allow two'quarters are sorted. The' two remaining quarters or three1-liter containers per grab sample, a are combined andAstored. for futUre reference or 1-litercontainer for most artifiCial substratediScaiaed (57). The aliquot tobe sortedmust be samples, and 16-klrr screw-cap vials for' miscel- do *alter than one-'quarter of the original '1aneaus Collections. sample; otherwise considerable error may result Pr4erve_the sample'in 70 percent ethanol. A /1. in estimating the total numbers.of oligochaetes 70 percent"ethanol solution isapproximated by or other organisMs that tend to'cluirip. Thesame filling The ;One-half-full bottle, containing the procedure , may be followed forindividUal sample and aiii911 amount of rinse water, with taxonomic groups, such as midges, and woi 95 percent ethanOlVo not use formalin. that may be present in largeiiurnbers. :Numerous to iques other than hand-pitking 4.3 Lab ling have been propo ed 'to recover 'organisMs frOm 4the sample, inclu ing.,sugariolutions, salt. solu- .Make mple labels of waler-resistant ,,paper tions, st ; efeet city for unpresgrved*mples and place inside the sample 5ontainer. Write all fi b blirtair through sample in a information on the label with a'oft -lead t `he, etc. The of lacy techniques is Where the volume of sample is §o,grot.: that a Acted both by the CharaOteristi s of ;the sub-;'`, sevpial ,o,ntainers are nesded, strafe material and th4 typeiof'..or rhs. No 111V 13 ;' .r, a ;1`' BIeLDGICAL METHODS .4 , technique,' or combination -.of teclinique, will thereof on glass slides for examinaion at. high completelysort outor make more readily magnification.. Small whole insects or parts discernible all types of organisms from 411 types thereof may be slide - mounted directly., from of substratematerial.Inthe end, the total water or 70.percent ethatibt preseryatiye ifCMC- sample must be, examined. If techrkKins aremounting mediaisused.- Label theslides routinelyconducting 'thepicking '4'peration, immediately with the- saMple, log number and these techniques may lead to overconfidence the name of the structureVnounted. Euparol and Careless examination of the remainder of the Mounting metliiim tirray be Pr-eferable tg CMC sample. If used with proper care, such aids are for mounts to be kept in a reference collection. not,objectionable; however`, they are not recom- Place specimens to be mounted in Euparol in mended as.standard techniques. 95 percent ethanol before mounting. As organisms are picked from the debris, they To clear'op que tissue, heat (do not boil) . sho9,k1 be sorted into major categories(i.e., small cruele (5-ml capacity) containing 5 fo insect orders, molluscs, worms, etc.) and plaCed percent. KOH solution _ (by weight) until- it into vials containing 70 percent ethanol. All vials beComeS transparent. The tissue can be checked from a sample should 'be labeled internally with periodically under a stereoscopic microscope to the picker's name and the lot number and kept determine flit is sufficiently cleared. Then trans- , as a unit ina suitablecontainer' until the fer the tissue stepwise to diStilled, water and 9\5 organisms are identifiedr and enumerated, and percent ethanol for 1 minute each and mount the data, are recorded, on the bench 'sheets.. A with CMC 'orEuparol. Severe' different typical .laboratory bench sheet for fresh-waterstructures can be heated simultaneously, but do samp,eSls shown in the Appendix. not reuse the KOH solution. 4 The above methods work well for clear' ing and 4.5Identification mounting midges, parts'of caddisflies, mayflies, The taxonomic level to .which: animals arestoneflies, other insects,crustaceans, and identifiedepend; on the needs, experience, and'molluscs; however, worms, leeches, and turbel- larians require more specialized treatment befort -availableresources. However, the taxonomic. level to which identifications are carried in each mounting (10, 47): Major group should be constant throughout a Larval insets often co the majority of given. study. The accuracy of identification will macroinver. ebratesc ectedinartificial depend greatly on the availability of taxonomic'substratesamplers and tom samples.In literature. A laboratory .libraryof baSic certain cases, identifications are - facilitatedif taxonOrOic references is essential. Many of the exuviae, pupae, and adults are available. Collect basic :references that should be available. in a exuviae of insects with drift nets or by skimming benthOs laboratory are listed at the end of the the water's surface with' a small dip net near the chapter. . shore. Obtain adults with sweep nets andtent For comparatiVe 'purposes and quality control traps in the field or rear larvae to maturityin the checks, store identified specimens in a reference laboratory.- collection.' Most identifications to order and stereoscopic c'The life history stages of an insect can be ' family can be madP under .a positiyely.associated only if specimens .are reared microscope (up to 50X magnification). Identifi- individually. Rear small larvae individually in 6- _ cation to geniis and species often requires a cOm- to 12-dram vials half filled withstream water pound microscope, preferably equipped with and aerated with the us; of a fine-drawn glass.. phase Contrast (10, 4* and 100X objectives,lot tubing. Mass ;rearing can .be carried out by- Nohiarski (interference phase) optics:: placing the larvae with, stiolis and rooks in' an To make species ideraifications,itis often aerated aquarium. Usemagnetic Stirrerainside neeessary, to mount the entire organism orparts Of the aquarium (41) to proiri.4'current,. i Oil ' 4 tO t / f' ti ''.11vIACROINVKATE TA EVALUATION - 4.6 / Bitiinass uniform IconVentio Mast be (istab i hod thr thpj Macroinv.eftebrat biomass ',(weight .of.-,,units of -data, repor ed. rOuthispurpose, EP'A organisms pet Unitarea) isa uStiful quarititatWe biologists .-shotila,acliteretO the olloWingunits: ..;; estimation. of standingcrop. To determine wet Weights., soak the organisms in distilledwater for Data from _devices samplint,a unit area of Anninutes, centrifugefor 1 minute at 140 g in bottomwillbe 'repor d in'grams '-dr wire_nlesh co es, "and weigh tothe nearest 0:1 weight or ash-free dry wei ht'per square' mg. Wetweight,however, is not recommended meter (gm/m2), or number's ofindividuals as a useful, parame ter unless, bya deterthination per square meter, or both: of suitable conversion factors, itcan be equated Data fromMultiplater snmplerswillbe to dry weight. eported in terms of thg.totalsurfaccarea f the plates in grzims dry weight To obtain dry" -weight, oven dry theorganisms *. or ash-free to a constant weight at 105°C for'hours-6.r.., .ry weight. or numbers of individuaIs' per vacutim'dry .at 105°C for 15to 361ninixtesi4 meter, or both. I }:2 atmosphew.. Cool to roomtemperature in a J.Pata from rock- filled basket samplers will desiccator and' weigh,' Freeze drying (-55°C,10 'e reported as grams dry weightor numbers to 30 microns pressure) has adv4ntagesover oven iduals per sampler, or both., drying because the organisms remain intactfor further identification,and reference, preservatives 5. 1 .2 Standincrop and taxonomic composi- are not needed, and cooling the material in ..tion desiccAors .after drying is not required.The Standin oto and numbers Of taxa iNaapom- main dis4dvantage of freeze drying is the.length munity are ,highly seti've to enviroeVntaf of time (usually 24 hours) required for dryingto perturb Lions resulting'iribm the introdUction of a constant weight. contaminants. These parameters,particularly To 'completely incinerate the organic material, standing crop, may varyconsiderablyin ash at 550°C for ...°1hour. Cool the ash to unpolluted habitats, where theYmay range from ambient temperature ina desfccar and weigh. the typically high standingcrop of littoral zones Expkess the biomass as'ash-freey weight. of glacial lakes to the sparse fauna of torretitial soft-water .streams. Thus, itis important that 5.0 DATA EVALUATION .comparisonS; are made only between trulycom- parable environments.Typicalresponses of 5.1Quantitative Data standing crop or, taxa to various types of,,stresS. are: . 5.1.1Reporting units :114` Data fliom quantitative samplesmay be used to obtain: Standing crop Stress (numbers or - 7 Number of biomass) taxa total. standing crop of individuals,or Toxic substance Reduce:, 'Reduce biomass, or both Per unitarea or unit Severe temperature r vohime or sample unit, and alterations Variable RcAluce numbers or biomass, or both, of individual Silt Reduce taxa per unit area or unit volume 44cduce or sample Inorganic nutrients . Increase unit. often no Data, from quantitative samples may also lie detect- able evaluated in the same 'manner as discussed for' change qualitative samples in part 5.2. Organic nutrients (high .02 demand) Increase . Rcducc For purpOses of comparison and to provide Sludge deposit1 datausefulfordetermining priguctionl, (non-toxic) Increase Reduce . I l B1gLOG1C/IL METHODS . _ . . r abundance; there will be 'relatively 2 Prganicl_nutrients,'and sliid4e:s:depotita:re- fre,-*,,, numerical g'quently,44ociated. The responses'shori.:are lic few Ipecies withlafge--riUmbers--ofindividdals. . . niiinbers of 'species represented by, , . $' simplesimple ot liked and may virydepend-' -and large ,i no mean$ only 'a, --few individuals. Manyforms of stress ing on anumber.offaCticrs including A I tend to reduce divmity4bymaking 'the environ- of stressesAing together or Iment unsuitable, for somespecies or by 'giving a combination advantage. in oprtosition, , other species a competitive indirect ,effects, such as forexample the The investigator must be awaythat there are Adestruction of highly productivevegetative naturally occurring e4CtrenleVivironments in 'substrate, by temperature alterations,sludge which -thediveisit macroinvertebrate deposits, turbidity,:'Chemical weedcontrol, communities may be,1*; asfor example the - the physical charactFistics of thestressed profundal fauna of?'deep. "lake or the- black, environment iltirticularly in relation tosub- fly-dominated communities of thehigh gradient, Ftiether-- strate and current velocity: bed rock section of a torrential stream. fr a more, becausecolonizationis by chance, Data on standing crop and numbersof t xa diversity maybe highly variable in asuccessional. may be Ores ntedin simple tabular f or community; for this reason,diversity 'indices pictorially withbar andlinegraphspie Calculated from the fauna ofartificial substrate diagrams, andistogramS.- Whatever the method samplers must be evaluatedwith caution:- These of_presentation, the number ofreplicates and confounding factors can -bereduced by_c`qmpar-: be shown in the ing diversity in similar habitats'and by exposing the sampling variability. must long enough for a tables or graphs.Samj'ling variability may be artificial substrate samplers shown as a range ofalues or as a calculated relatively stable, climak communityto develop. asdiScussedinthe S Sc. S-1 standard deviation, n1 dices,' such "is ---,"rid -- where Biometrics SeCtion of this manual.' . N L,og N I.ogN. Data on standing crop' andnumber of taxa, are S = number of taxa and N =total number of amenable to simple but.powerful .statistical ,individuals, are merely additional meansoesum- techniques of evaluation:Under grossly Aressed maizing data on total numbersand total taxa in sitilAtions, such analyses. may be unnecessary; §ingle numerical formfor. evaluatio however, in some cases, theeffects o environ- summarization. Theyadd no new dimension t mental perturbations may be so sue in co: the methods of datapresentatiorynd .aal pariSon with sampling 'variation thstatisth discussed above and, inaddition\ are highly, compay.isa,s,Are a' helpful ancluecess tool for influenced by sample size..Sample sizp in this th.eevalua tiveprocess., Forth purpose, context relates to the totalnumber of o anisms biOlogiSASftaged -in studies of macroinverte- collected (an uncontrollable.Variable most brates shouldsfAjnifiarizethemselves with the ..,,croinvertefrate sampling),nbt to tharea or 'simple, statistical toolsdiscussedinthe141urRe of habitat sampled. Do' not usesuch Biometrics Section of this manual. indices -for summarizing andevaluating data-ori-- , aquatic macroinvertebratecommunitte 5.1.3Diversity (ofspecies, additional tool for There are two -,components INversity indices are an. diversity: measuring the quality of theenvironment and the structure of a the effect of induced stress on richness ,of species conimunity of) macroinvertebrates:Their use is. distributKn of individuals among,the based onNthe generallyobserved phenomenon that relativelyundisturbednvironmeh species. support communitieshavinglargenumbirs Itis' immediately obvious .tIatthe second Species with noindividual species present in dimension that was not Overwhelming Abundance. If thespecies; in such componeirt adds 'a new considered. in' the methods forevaluating data a community .areranked on the basis 'at their 0 r") % M'ACROINVERTESIZAT.E SPECIES DIVERSITY, ssed iibove. The distribution of indivi'dUals N logy 0. among the -sPecies-may' = 222,0795 Morn Table 5) readily-presentethirb ni log ni= :132:1321 frequency' distribution fables dr graphs;but' for 3.32,1928 \any "appreciable ").1.1,u,nberof samples, such (222:0795 - 139.1391) . 109 Methods ofpresentation are so Voluminousqhat = 0. 476 X 82.2404 theyare virtually, Amposstble to, cornpare, and ° interpret.. .. ,, Al? 44?!z; htlices of diversity basedon information Mean diversity, d, as calcu,late e affected.t theory, as originally proposed by Margalef (39) by richnessof Species an 11`, the distribut and ,strbsequently utilized bynumerous workers,:tion of individuals .among the species andmay include\both components oftspeciesdiversity, 4is range front zero to 3.:32I'928 log N.: $, enumerated above. Additionally,a measure ?of To, evaluate the component\of diiersity d'ueto the component of diversitydue to the distribu-. the. ,distributionof individuals among the tion orintlividuals among the speciescan readily species, comparethecalculatedcT with a . beextracted frbmtheoverallindex.For hypothetical maximum .d based.`onan arbitrarily , purposespf, uniformity,. the Shannon-Weaver selected distribution: Themeasvreof° functionisprovisioIly recommended forredundancy proposed by Wargalef-(39) is based. calculating me' rnrcliversi y d. . -Off theratio between d and a hypothelical The machine -forum presented by Lloyd, Maximum computed' as .though all speciesWere Zar, and Karr,(34) is: equally'abundant. In nature, equality of spqQies. isiquite unlikely, so Lloyd and Ghelardi (33)' C proposed the term ccequitabilitiC find compared-, =-- (N logo N.- E ni logi'0ni) with a maximum baSed on the clistributn- . obtained from MacArthur's (36-).. broken sti model. The MacArthur model results ina distri where ,C = 3.321928 (coriverts base 10log to bution quite_ fetquently observed in nature rase 2 [bits} ); N = total number of individuals; one with a ferrelatively abundant species an and ni = total nuniber. of individuaM in the ith.increasing numbers of species represented Species; When their, tables (reproduced inTable only a few individuals.:6ample :..dataare not' 5) are used, the calculations.'are simple- and,expected to' conform to the..MacArthur model, straightforward;as . shown -by We following since it is only being-used:as a yardstick against example: / which the distribution of abundances is being compared. Lloyd and Ghelardi (433) deviseda Number of individuals ni logi0 ni table. for determining equitability by comparing;. in each taxa (ni's),. grom Table 5) the number of species (s) in the sample with-the number of specie's' expected. (s') froma corn- 41 66..1-24l .munity that conforms to the 'MacArthur infidel.,. '5, 3.4949 In the table (reprOduced as Table 6 of this 18 22.5949 Section), the proposed meastirriof equitability 3 1.4314 is: k .0000 29,5333 e = .0000 s .6021 .1 12 12.9502 where s = number of taxa 'in the samplegend 4 2.4082. thetabulated. Value.,For the example given TOtal. 109 139.1391 above (without interpolation in the tablie):1-. Total ntimber`o'f taxa, s...= 'e 0 8 Total mimber of individuals, j\1 109... N. . 17 ri ditability. "e,'!as calculated, may range schemes advanced for ifig analysisof qualifati e data may be gr ed in twoscategO 'es? from 0 to 1 except in the unusual situation , ., where the distribution .in the -sample is more 512.4 ihdic or-organism sche equitAle than the distribution resulting from :thelVlacArthut. Model.- Such an eventuality .will 'For thistechnique, individu9lto valueof, e- greater' than and thisclassified on the basis. of the, rtole 'F'result, intolerance to variouslevels of p e occasionally occurs in samples containing only a 2 48) Taxi are c aSsifi few cimens with several taxa represented.'wastes (4, 5, v30, cCordiig totheir/Piesentor: absence in di The ate of a and e improves with increased eterinined by fiel sampl apd samples containing, lesS than rent::gnvironments 100 specimens should be evaluated with caution, studies. :Beck (6)'.reduced databased' on t e if at all. . . presenee .or absence of indicator organisms to a simple numerical form fot easein presentatio When Within (59) 'evaluated values calculated froth data that numerous anthorshad collected Reference station methods . from a variety of polluted and ungiolluted Comparativeor. control station iitho waters, he, found that in unnolluted waterseTi'vas donipare therqualltative characteristics .:of n e polluted ,generally between 3 and 4; whereas-in. fauna inelean',waterhabltats with those if water, a was generally less than However, fauna in' habitats, ,suklect to.stress. Patrick(4 ) corrected datd,ftom southeastern IJ.5. watersby Compared stations ba'sis of Tidiness f ERA biologists has shown that whereegrada- spe.cies and WurtzMW used indieator.organis ass tion is, at slight to moderatelevels, d tacks the inComparinl siations.j , sensiti ; to demonstrate differences, Entlit If adequate background data areavailable to abili the contrary, has been found to be.lan 'experienced investigator, both ofthese. to h- very ;n tive tp cyen slightleyis of degrada-.niqUes can prove quiteueitilparticularlY or' tio. levels below 0.5 have not been the` purposeof demonstraling the effects of encountered in southeastern streams knownto grosS to rhOclei-ate organic contannnation on he be unaffected by oxygen-demanding wastes,andsmaaoinvertebiate point-nullity.. To 'detectore in sucli`streains, e generally range'stween 0.6r. subtle chwhgeo in themaCtoinvertebrate cOm- and .O.S: EVen slight leveli- of de:-.4-,4a0n have Munity, collect quantitative, data onnumbers been found to reduceequitabl1ity below 0.5 and bioinass of organisms. Data on the 'Presince of,.'' generally to a range, of 0.0 to 0.3. toletant and intolerant taxa and richneSsof for ev Agency_ biologists are encouraged tocalculate species may. be effectivOy summarized both mean diversity a and :equitability a..for-a-lion :and-presentation by means of line .gra samples collected in the course of macroinverte-a bar graphs, pie diagrams, hiStograms, or pictoral diagrams (27). brat& studies. (IF the mean and range of values anthors of repro -;- found by different, sampling methodsand under The claisificatiOn by various varying levels aqd types ofpollution... are sentative mactoinvertebrates, accoger to their reported to the Biological .Methods)3ranch, tolerance Of organic Ivrtes.is'preSented in. form in 7. In most cases, the taxonomic,nomenclature these data will, be incliided in tabular authors, future revisions of thisSection.) Used in the table ithat.of the ,original . ;The pollutionalclassifications of the: authors wiere arbitrarily placed` eategOries.' S.2, Qualitative Data pleraiit, facultative, and intolerant --defined.as:, qualitative data ,result follows: As previously de ,. . -Vont. samples collec h no 4. estimate, of muneri ance "br biomacan : Tolerant;hganisms frequently associated ut consists oflist of with Brosorganic) contamination and; are ". ° calotilated. The o able. 6 fthriving, under taxa collected in the' various habitatsof the generall .- .environ'inent' king studied. The numerous anaerobic conditions,\ MACROINVERTEBRATE INnICATOR-ORGANISMS -Facultative: Organisms kiVinia wide-rage 'absent because of discharge of toxic of tolerance find:frequently ,are associated ubstance3,orfwaste heat. ^ Wititmoderate levels oforpnic contarninar ,Because evaluations are baseon the mere . .. $p,A bon., presence. "Of absence or orgarfistris, a' single r %;(1,14) Intolerant:, .,.00anisms 'tliatare not found -§pecimen, has. as -much weight large associated with evdn',.moderee 'levels of population. Therefbre; data ,for r-thethe original organic contaminants and are, gently cla,ssification and from. field studies'enay intolerant even. Moderate retOctiods'In -biassed by the drift of *organisms into the dissolved oxygen Mudy area. The presence -or .abseneeof'a particular.taxa . o. WhenIvahiating qualitative terms may depeild pore. , on.c,haracieristr9or the suChas that contained in Table 7, the environment, such° as velOcity and subsItate, tiga or ghOuld _keep in mind the pitfallS than- on the level of dagr4datiori sy organic "iitioned`earlier, as well 4s.t1lef011owirig: 'wastes... This affeCtsr.both fhe ori al place . hient of thetaxainthec ficatory Since tolerant species may be- found .in both scheme agii its' presenciri pies. elean artdclegradedthabitats, a simple ret,?ord_ Technique.; istotally subjecti a d quite of' Weir Xesen oriaBsendes is of0signiFi- dependent qipon the skill and experience oft..' . cance.The,r IndicatoiCorgankrn:;, , tli6 individual .*ho makes the.,,,Tield coffee-. technique, b-:provide positive evidence I tions.4,Theref9re, re§uits of one investigator billy- one CO dttibn,-clean v,talterand arediffictiltto "compare With those; of oilyif 44ixa 'olassified, as intolerant re another, - 'particularly ;where data are co exception to thi,s"rule .W0 Id: niarized in an indek suchas that Proposed' occur;jib t serf f live. speties mai; be;ibta I ' A - ' u iu u INN u u u u cNoi u Noi ubotu CIF u-Yoi 0E9191 6660'116 99)9'06 a11'61 19 9019'91,1 11 011011 18 861011 9000191 1'916 81 110).61 ' cuns 19 ' 99, 6196'051 6996'991 116 699601 6601'11 9610'09 10 811E761 1811'991 6012/16 01 1611'11 911E'9f N 1091'111 1/11111 1111766 IC 0616'N 111219 0616'09 611 ' 1111'911 1169.111 001/116 11 1011'61 mra 116E01 1. 6111111 8180111 1611'(16 N 181691 0111'116 016031 N , 111161 0161'011 8111'811 1611.611 1041'81 01016 /6 0160'51 1199,081 996L'111 66 11100) 911016 161'10 9696096 91 60(6'1) 6910'91. 016119 16 1690'191' 611901111 1910911 0111'181 0196196 LE 1861'1 51011 616666 16 1185'116 11E816 16 1. 1110'011 1E80'181 96, 181e4 8110'09 96 1966611 0061'061 1111'111 bf 96069) 616Pb 081106 16 1686'161 ,6911'161 1181'186 09 9116.11 Hoy OENr 116'661 1011'661 1896'986 It, 116'69 1114'99/' 6119'901 86 66 ,-0016.161 6191'161 1111'161 1) /111.16 1911'89 9/99'011 001 0016./61 000011 0000'001 1111416 I661111 I , 101 1116'661 1961'101 II 91116 6111'11 I ZINC '`!111901 1118'101 1916'111 6) 11110.96 9166'11 0066'111 101 '6186'191 601 8666'691 1111'101 6101'11) 9) 90111; 699'91 1611'111 901 8110'991 114/'601 0911'611 ) if 111)'66 9896'81 11101'111 0160191 6111'111 9916'811 Of 6660'19 9669.01 6619161 901 1660'011 1189111 9160E1 61 1181.19 9618'18 1186'611 9099'01 11 'tit 1 1111'11Z p tato NICK 0616'191 801 1''611"11 /666'911 9061'99 1990'18 9601111 111PILl 601 6661.911 6610'111 9041'169 npa '5 SNOI1,HNflt1 0H 1V) DNIIVI 11 ' 9906'19 1161'60 1111'01 011 zfav 6101'611 ES 60E969 998E'16 4611'161 walla (INV lad 6310'1N 1166'191 AlISHAIO 111 19111181 S31038 110,4) K 6696'11 16616 090191 111 6161'181 0111'011 -GNUS I'S 160161 1186'991 APIIM INOCINVII (MIMS SII 6611'16 6166'1E1 8606'919 51 61011 1068'16 Ul 0611' 1116'191 0131V 3SVEI 1189111 8411'191 011V ?EH Lc 116911 6990001 6911111 III 0'991 III !warn emu 1)11181 .31f1V1 RIV 1DV311a11 01 Bs 11/6111 611/101 119E11 911 9016'061 1111'6E1 5066161 66 0111'09 100'101 1610181 '16I 1116111 0161.006 1T NI 3I-11 1-11119I3 INVDIAINOIS 09 1016111 1699'901 6601'681 ill 0816.906 91E1161 811 1019'161 0ISS11418J1-11IM 19 COLD 1606'901 ')1191111 (130a101101) 611. 191L'961 1066'911 6669716 'HMI? 19 616110 11111'111 1681.661 Dll KI9161 L109.611 1651'815 141011i, `cAorn 'IN (NV `11SVX (.8961 19 11614 1856.111 6016101 OLIO'Zg. 19 1101'68 6666'611 198111 I11 l 6166'101 6565111 6610'166 191606 16111./11 9119111 NI , 111 61160'151 6411'166 99 661116 6699'011 0906'811 11'01 6111'101 69l161 )611616 19 619516 0111'111 0119'611 HI 8111401 8E11'191 0619'616 89 6166'96 9010111 0066'811 CI I 9/1 161.6'1I1 1919'191 1968'165 69 11E1'86 '911 1111'661 111 063'611 16111'/91 1001796 u u 01 4010'001 6%1'611, 1101'811 u u bob U 001 u uboi uboiu doi RI 6111'691 1196'896 , '91 IL 1616'101 1611111 1011'111 119116111 0000' 0000' 91 1016'01 8510 I 6:1 09.1,11 1991'111 0119'116 9IK'Ll 611/'01 11 0191101 6411'1E1 1/1E111 0101' 1109' 1181' 61 6911'11 0E1 1018'611x, 9110'111 6066006 6691'61 6161.11 IL 6059'101 9110'911 0961(61 1811' 1161'1 6189' 91 9011'11 161 111 0816' 111 5196' 1151196 91,1601 1061I/ IL 9616'101 1611'0E1 0E6161 1061.1 0101'1 6611'1 11 115;11 6010'111 IL 9966'601 9619'011 1609191 161 851411 )1116.666 1610'0 6161'6 111'1 81 611, 1900 6166'1,1 R9E'81 , 1111.911, 11W101 1616'666 9L I I 11/1'1 8116'111 6918'891 611 ENE 6899'1 X1169'6 61 1180'11 1961'11 N9011` 6661'811 N60 909 OER 11 6191111 8661'611 160111 111 11011 16161 6665'1 01 ,1981'01 9010'91 16119'119 6E1 8611'061 1666'181 1801'61 6'616'96 81 0110111 1186.111 641 SSO9') 1111'1 9116'9 11 99'h'11 fOL0'619 1811' 961 " 1196'111 6191'061 8060'11 Ei(6'61 19961 61 9166'911 6116'611 86Sr'c 188611 1t61'8 11 1001111 1011'161 1E81'619 08 1969811, 1/91'161 101'681 411 0000'01 0003'01 11 11 6111 1611.11 0619'19 0010'911 1601'161 1116119 18 1691'011 6186111 1116'661 811 " 11 1/61'61 161116 96111) 1109'1 1669'11 6616'11 . 6E1 0186'061 1640'161 1611'819 68 ES8'81 ZO 0119'111 1116111 0011001 109911 1%611 9646E1 51 9061'61 11 6161'001 ,,118'119 91 160 N13.16 18 1960/1 6111'661 11/9'601 11616 110'11 1161'91 91 960991 1611'11/ to) TABLE 5,, (Continued) n log n1 n log n,n logs n n log n1 n log n n n 141 n log nl n log n 143.2185 3051399 651.2991 n log2 n n 196 3712990 454.7397 log n! n log n n Idg' n 142 10441849 255 245.4306 905,6149 657,7130 304,5252 6136677 1471,8161 199 372,5959 457.4718 312 ,44.3226 778,1762 141 1051.6604 256 )9411,8118 247,5860 301.2151 506.9334 6663027 200 616.3094 ,1484,7026 313 974,0969 460.2060 616,0102 781.1054 10585478 257 1949.2031 144' 249.7443 110.8042' 509.3433 619.35211 1492.5988 670,8213/41 201 377.2001 114 649.3151 462.9424 10612471 784.0359 1957.6825 145 251,9057 '313,1984 258 511.7549 622.1979 677,3692 r. 1500,3047 315 202'' 375304' 465.6810 651.8134 786,9678 146 1073.513 259 1966.0900 254,410 315,9955 683,9258 514.1682 625.0446 1308.4201 203 316 654.3131 11.8129 468,4211 120.8812 109.9011 197+,5056 147 256.2374 260 516,3832 .318.5956 1 627.8931 690.4979 204 14.1226 1316,14311 317 471,1646 1 .2159 656.8142 792.8338 1982.9293 148 214076 321.1987 \ 697.0853 267 518199 630.1432 05 3861343 1524,2795 318 473.9095 1993.5622 659.3166 795,7718 1991,3610 149 225806 325,8048 262 521.4182 633,5943 703.6880 206 1532.2234 319 388.7482 476.6566 1102,9202 661.8204 798.7092 150 263 1999.0007 262,7569 326.4137 7113060 523.8381 636,4414 1540.1769 207 391.0642 320 479.4059 1110,2897 661.3235 801,6480 2008.2484 151 264 526.2597 161,9359 329.0255 711)9390 639.3034 1548,1397 208 393,3822 321 482.1572.1117.6709 666,8320 804,5881 2016,7041 152 ,267,1177 265 528,6830 6411602 331.6402''7231871 . 1536.1119 209 395.7024 322 669.3399 484.9106 1123.061 801.5296 2025,16711 153 269.3024 266 531,1078 645,0185 334,2578 730,2501 210 1564.0936 323 398.0246 481,6661 611.0491 810,4714 134 1152,4675 267 20334394 211.4899 336.8782 533,5344 647.8783 1572,0015 736,9280 211 , 324 400,3489 496.4236 1139.8829 674.35% 813.41662942.1189 155 .268 173.6803 339.5014 749,6207 535,9623 650,7401 1580.0847 212 402.6752 325 676.8715 493.1832 1147.5998 816.3621 2050,6064 156 275,8734 342.1274 269 338.3922 653,6034 150,3281. 213 1588.0943 326 4051036 495.9449 1134.7t79 679,3847 819.3089 157 270 2039,1016 218.0699 344.7561 757.0501 340, 136 656.4682 1596,1130 214 407.3340 321 681.8993 498.7085 1162,103 822.2571 2067,6048 158 280,2679 347.3818 271 M3. 566 659.3341 763.7867 , 215 1604.1410 328 409,6664 501.4743 1169,6379 684.4132 825,2066 159 272 2016.1157 282.4695, 350.0221, 54 12 661.2027 770.5377 216 1612.1782 329 412.209" 504,2420 666,9324 828,1575 117746 273 2084.6345 160 284,6735 352.6592 777.3092 548.1273 665.072#1620.2243 211 414,3373 , ' 330 ,4509 501.0118 1114.6121 831.1096 161 203,1611 286.i3. 355,2990 784,0830 214 550,5651 667,9437 218 4161758 16282800 331 509.7835 1192.1066 691.9707. 834.0631 2101034 162, 289. 273 553.0044 6708163 357.9414 790,8170 219 1636,346 419,0162 512.5573 332 694.4918 031.0178 163 1199.6116 276 2110,2375 291.3020 360,5866 797,6852 535,4453 673.6909 220. 421.317 1644.4182 333 697.0143 515.3390 1201.1271 839.97392118.7874 164 2933168 '363.2344 277 557.8878 '676.5669, 801301 221 1652,3009 334 429.7031 518.1107 1214,6547 6995380 842.9313 165 ,278 2127,3149 295,7343 15.8849, 811.3438 560.3318 679.4445 1660,5927 222 426 0451 335 702.0631 520.8901 1222,1926 845.8900 2135,9102 166 297 .9544 '3683379 279 162.7174 '682.3236 818.1941 223 1668.6934 336 428.3977 523,6720 1229,7415 704.5894 80.8500 167 280 1144,4631 321771 371.1936 825.0582 565.2246'685.2042 1676,1031 224 430480 337 707.1110 768 526,4556 1237,3011 831,8113 2153.0636 302 4014 313.8520 281 567.6733 .0865 8 831.9362 225 1684.9E17 338 433.1002 529,2411 1244.8716 709,6460 8.34.7730' 169 282. 2161.6518 304.6303 376.5129 838.8280 570.1235 6919702 226 4344543 1693.0492 339 532.0285 .1252,4528 712.1762 857,7377 2170.2177 170 306,r, 379.1763 283 5723753 693.8356 845,7334 227 1701,1856 340 4371101 5341179 1260,0447 714.7076' 660.7028 171 2178.8510 309.0938 381,8423 852,6524 286, 373.9287 6917424, 1709,3309 228 440,1682 ' 341 717.2401 537.6091 1267.6473 863.6692 2187,4620 172 311,3293 283 577.4833 699,6308 11,5109 mum 229 1717.4850 342 442.5281 540.4023 1275.2606 719,7744 866.6369 2196.080 773 3133614 30,1820 286 579,9399 702.5207 866,5311 230 1725,6179 343"722.3091 444,8898 543,1971 1282.8844 .6059'2204,7067 174 315.8079 989,8556 287 582.3977 705,4121 873.4906 231 1733.0196 344 447.2534 545.9514 1290.5184 724.8463 872.5761 175 2213.3403 314.0509 392.5317 880.4634 5641371 708.30501742.001 4 232 449.6189 rr89 345 Al 548.7932 1298.1637 727,3841 875.54762221.9814 320,2965 395.2102 887.44% 587.3100 711.1995 1750,1893 233. 451.9862 346 151,5939 1305.8192 729,9232 878,3203 22306299 177 322,5444 391.8913 290, 589.7804 714.0934 894.4489 234 1758.3871 347 454,3555 5543961 1313,43 732,4635 881.4443 178 291 2239.2860 924.7948 400,5748 901,4615 592.2443 716,9929 1766.5937 235 .4517265 557.202 348 755,0051 884.4696 179 1321.1613 2241.9495 327.01774,403,2607 292 351.7097 719,8918 1774. "0 908.4871 236 349 459244 560.0072 1328.84 137,5479 887.4461 180 293 2236.6204 329,3030 405,9491 915,5251 557.1766 7221922 1783,0327 350 237 , 461.4742 562115413363448 740.0920 gi.42182265.2988 181 3313606 445,6398 294 399,6440 725,241 922.5774 238 1791,2651 351 463.8508 565,625311344.2521 742,6373 893.40282273.9845 182 333.8207 411.3330 295 602.1147 728,5975 925E419 239 1199.5061 352 466.2292 568.0 1351.9696 745.1838 896.3830 2282.67* 183 336,0132 414.0185 296 604.5860 ,9361193 240 731.5023. 1807,7557 353 468.6094 571,2507 1359.6973 747.7316 899.3645 2291.3780 114 297 338.3480 416.7265 943.8096 607.0388 734.4067 1816.0138 241 470.9914 354 514.0661 1367.4352 70,2806 902,OffST22300.0858 135 ,296 340.6152 419.4268 950.9125 609.5330 737,31611824.2803 to 242 473.3152 355 576.8833 1375,1833 752,8308 905.3311 ,8009 186 342.8847 422.1294 910282 299 612.1;0137 7401257 243 1832.5554 356 475.7d 579,7023 1382.9415 755,3823 908.3162 23a.5233 187 345.1565 424,8344 300 614.4858 743.1364 965,1564 244 1840.8389 357 478,1482 582,5231 1390.7 757.9349 911.3026 2326.2531 188. 347,4307 301 616.9644 427.5417 972.2973 245 746.0185 1849.1308 480,5374 585,3457 358 760. .; 914.2901 189 1598.4881 302 2334.9900 349,7071 , 430.2513 979,4506 619.4444 741,9621 r 1 246 , 1857.4312 359 777 482.9283 5811100 7406,2764 763.0439 941.17892343.7342 190 351,9859 432,32 303 621,9258. 751,8771 ro 986.6164 247 1863.7399 360 483,3210 00,9961 1414,0747 165.6002 920,2689 2352.4857 191 154,2669 435 774 3E4 624,4087' 734.7936 trl 123,796 246 1874.0370', 361 481.7154 593.8240 1421,3829 768.1517 923,2601 2361.2444 192 156.5502 9 305 626,8930, 737.7113 120.9852 '249 1882 3824 362 490,1116 596.6536 1429.7010 7717164 926.2525 2370.0102 193 358.8358 306 629.3781 160.6306 1008.1 1890.7162 363 250 492.5096 599,4850 773,2764 929.2401 494 1437.5291 307 2378.7832 361.1236 443,83 5 1015.4031 1 631.11659 7E3,5515 1899.0582 251 . 494.9093 602.318t .364 775.8375 932.2409 195 1443.3669 2387.5634 363.4136 446.5567 1022.6304 SOB 041544 166.4136'1907.05 252 497.370- 605 15, 365 778.3991 935.2369 196 1453.2146 2396.3508 365,7059 449.2822 369 636.8444 769.3972 1029.8698 253. 1915.7670 499.7138 607,9895 366 780.9632 938.2341 191 3680093 1461.0710 310 2405,1453 m 452.0098 1037,1213 639.3357 772,3221 1924,1337 254 502.1106 367 783.5279 610,8278 1468,9392 941.1324 2413.9469 311 641,8285 775.2485 1932.527 360 716,0937 944.23202422.7556 3100! 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LL4 55152101 £559'1151 415'5511 1116'615. 1191'1011 5595'0685 561 1/60'551 11151191 051 8110156 CES 1510'6551 u £699'6111 51617965 811 9516'1101 5991'0951 91511515 85513'1011 '9995'6685 £65 5019'51851 1151'1191 9010'0916 151 1969.55.7' 95 Z+101051 £8'51f1 If 105166C 611 561'1101 '551 01551165 1111'0151 1155'956 6181'1011 6951'8065 A KC 1659.1191 551 Ltc lfii'1551, 596619+1 '6151'5001 514'1651 . 1 OOP 5115'0901 856'9051 19150515 6915'51811 11115'856 0916'0111 1111'1165 161 8915'16E1 950'5691 551 OCLI'1C1I 6091'6911 1151106 181 156'5801 9111'0651 5995'09K ILE5'0611 IS1S.156, 1500'11111 0568.9565 961 016616E1 0910191 151 65 9900'0151 595Z111 106115+. 561 1659'5901 1855'5651 5561'6915 551 505'15 5590'(111 0190'9E69 bb 1 ,TABLE 5, (Continued) n log nl n log n n log2,n '? "loan! n1ognn n 597 ' n 1399.1100 1657.2567 4600,5020 log nl n log n n log2 n 6213W, 62 1841.3878 re log n1 n log n 598 51843706 n log2 n 1402.5467 1660,4673 711 1720.7210 2627.6193 4610,6213 655 57826769 768 1561,9824 1844,6380 3194,9439 1884.2611 2215.9574 63931376 599 712 1405.5211 1663.6787 4620.1457 1723.5735 2030,9637 5791222 '658 1564.1993 1847, 9 769 1887.1470 600 3203.3254 2219.2773 6404.6710 1406,1023/1668907 713 1726.420 2034,2528 4630.8746 657 5883,9055 110 1367.6169 1851,1404 5213,7092 1890,0335 2222,5978 6415.5081 601 714 1410:8811'1670,1035 4641,0081 1729,2802 2037.5405.5814.5257 631 1370,4331 171 1854,9926 3226,0973 1892.9205 2225,9189 6426.3489 952 1413.6606 16733171 715 1172.1346 2040.8288 46513463 659 5025.1500 772 1573.2540 1837.6453 5236,4891 125,8082 2229.2403 6437,1935 609 1416,4411 116 172.9895 1676,5313 4661.2891 660 2044.1177 38311783 1576.0735 773 1898,6963 604 18601990 '5246.8865 2232.3627 6448.0419 1419.2221 16793463 '4611,4369 717 1737.8430 227,4012 gi 1578.8938 246.4105 774 1861.1532 5237.2815 / 1901,1831.2235,0055 6458.894 '605 1422.0039 718 1740,7011 1682.9620 4681.5886 662 2050.6913 5857.0468 1581.7146 1867.4080 5267,6927 775 1904,4744 606 119 2239.2088 '6469,7498 1424.7863 1686,1784 1743.5578 2053.9681 4691,7432 60 561611810 776 1511.5361 1870,6635 5278.1022 1907.3642 2212,5327 607 120 6480,6094 14273695 1689.395 4701.9065 1746.4152 207,27942783312 664 1587.3583 1873.9196 777 1910.2547 608 5288.5161 22418571 64914727 1430.3334 1692.6194 4712.0724 721 1749.2731 A01713 51819794 665 1590,1811 778 1913.1456 609 1877.1764 3290.9341 2249,1821 6502.3396 1433.1 7 1752.1316 1695,8319 4722.2429 666 20631 58,63162638 1593.026 1..1,4338 '3309,3564 779 1916.0372 2252.5077 610 7f5 6313,2103 1431,9234 1699.0512.4132.4180 1754.9968 .2067,1570 667 3910,2877 780 1595,8287 1883,6919 3319.7830 1918.9293 2255.8338 6524.0847 611 1438.7094 724, 1702.2712 4742.5977 668 1157.8505 2070,4301 3920,9478 1598,6535 1886.9501 5330.2138 781 1921.8219 2259.1604 6534.9628 612 1441,4962. 725 1705,4919 4752.7819 1760.71098 2013.7450 3931.6111 669 1601.4789 1890.2101 782 613 5340.6489 1924.7151 2262.4877 6545.8446 1444.2836 1708.1133 4162 9107 726' 1763.5118 2071.0101( 670 1604.3050 5942,2797 1893,4701 5351 1 783 1927..0 2265.8154 614 127 6556.7301 1447,0718 4711,9354 4773,1641 1766.4333 2680.33331 671 5952.9517 764 1607A17 1896.7308 5361,5317 192.50322269,1438 6567.6193 615 1449.8607 728 1715,1982'4789,3620 1769.205 20831316 59636213 672. 1609,9591 785 1899.9921 .53119794' i933.3981 2272,4727 6578.5122 616 1432,6303 729 1718,3817 4793,3645 1772,122 2016,9283 , 5974.3073 673 786 1612.7811 1903.2541 5382.4513 1936.2935 2273 8021 617 1453.4403 730 6589.4088 17216059 4.803,7715 1773,0215 2090,2257 5984.9910 674 1613.6158 787 1906.5168 3392.8875 .1939.1895 2219.1321 6600,3090 618 1458,2313 731 1124,8309 4813,961\ ,1777.8834 2093,5236 3995.6786 675 1618.4451 168 619 1909,7800 5403,218 1942,0860 2202.4626 6611.2129 1461,0232 1728,0363 732 .1780.7499. 2096,8221 424.1992 616 6006.3181 789 1621.2750 1913.0440 54113121 044.9;331 2285.7931 620 1463.8156 733 6622.1205 1)31.2828 4834.4198 1783.6150 2100,1212 6011.0656 677 1624.1056 790 1916.105 5424.2812 1947.8807 2289.12546633.0317 621 1466. 7 1714.5099 734 1786,4807 44050 610 2103,4209 6027.1650 1626,9368 1919.5737 791 1950.7789 622 5434.1542 735 2292.45166643.9467 1469.4025 1737.7376 4854.8146 1789.3470 2106,7212 679 1629,7681 6038,4683 792 623 1922.83% 5445.2313 1953.6776 2295,7903 6654.8652 14723970 1740.9660 4865,1 736 1792.2139 21100221 680 1632.6012 6049.1754 , 793 1926.1060 5455.7125 1956.5769 2299,1236 6665,7875 W 624 1474,9922 137 1793.0814 1744.1952 48719475 681 2113,3235 609,8065 16314344 1929.3132 3466.1981 794 1959.4761 2302,4575 625 1477,7 738 6676112 01741,4250 4885.5906 1797A1 2116.6256 682 1638,2681 6070,6015 795 626 1932.209 276,6877 1962,3771 2305.1918 6681.6129 1480,5846 1750,6555 139 180017t 4895.8383 683 2119.9282 6001,3203 196 1641.1026 1933.9093 5481.1815 1965.2780 2309,1268 627 740 6698.5761 123.3819 1753.8867 4906.0905 1803.6873 2123.234 684 1643,9376 6092.230 797 1939,1784 5497,6744 1968,11%2312.4623 67139.229 628 .1486,1798 741 006.5571 1757,1187 4916,3410 685 '2126.5353 6102,7697 1646.7733 1942,4480 798 1971.0814 629 5308,1116 4 2315,7983 67204534 1488.9785 142 1809,4275 1760.3312 4926,6082 686 2129.8397 6173.5002 1649.6096 1945,7183 199 630 5318,6878 1973.940 2319.1349 6731,1915 1491.1778 1763.5845 743 1812.2985 49366137 687 2133.1447 6124,2345 16524466 1948.9893 5529,1982 800 1916.1871 2322.4120 631 744 6742,3452 494.5179 17661115 49473437 1815.1101 2136,4503 6134.927 1655.2842 1952,2608 801 1979.7907 632 5539.7128 2325.8096 6753.2965 14973716 745 1818.0422 1110,0532 4937.4182 689 2139,1564 6145.7143 1658,1224 1955.5330 3330.2314 802 192.6949 2329.1418 67223154 633 1500.1800 746 1820,9150 11732183 4967,6916 690 2143,0631 6156.4600 1660.9612 1958.8059 3560,7542 803 1985,5996 2332.4866 634 1502.9821 147 67752100 1776.5246 4911,924 1823;1883 2146.3705 6167.2105 691 1663.8007 804 633 1962.0793 5371.2811 1988,5049 2335.8258 672,1722 1505.7849 748, 1826.6622 1719,7613 428.2682 692 21,496784 6177,9642 1666,642 1965.3514 805 1991,4106 636 5211122 2339.1657 6797,1380 1508.5883 1782,9987 4998.5604 ,149 1829:5367 2152.9869 691 129,4816 6188.7216 806 1968.6281 5592,3474 1994.3170 2140063208,1074 637 1511.3924 .150 1786,2366 5018571 694 1832,4117 2156,29596199.4829 1672,3229 1971.9035 807 638 ,5602. 1997.2239 2345.8469 68190804 1514,1973 1789,4756 5019.1581 751 1835.2874 21596056 695 1675.1649 6216.2480 ,808 1975.1794 5613.4299 2000.1313 2344,1E184 6830,0569 639 1317.0028 732 1792.712 3029,4636 696 1838.1636 2162,9158 6221:,0110 1678,0075 809 2003,0392 640 1978.4560 5623.9774 2352,5303 68416371. 1319,8089 1795,9552 5039.7734 7331 1841,0404 2166,2266 697 1680,1507 6231,7898 810 1981.7312 562.522 210.9477 2335.8729 6852.0209 641 1322;6138 734 1799.1960 5030.17877 698 1843.9178 2169,5380 6242.5674 1683.6946 19850111 5645.0845 811 2008.8567,2359,2159 642 755 6863,0382 1525.4233 1802,4315 3060.4064 1146,1957 ;172.8499 699 1686.5391, 6253.3469 812 19862895 5655.6442 2011,7663 2362.5593 6873,9992 643 1528.2316 756 1805,6796 5010.1294 700 1849,6142 216.1625 6264.1311 1689,3842 1991162 813 2014,6164 2365,9036 644 5666.2019 6884,9937 , 1531.0404 737 1852,5533 1808.9223 3081,0368 101 2179.4156 6274,9191 1692,2299 1994,8483 5676,7748 814 20174870 2369.2483' 6895.9918 645 1533,8500 738, 1855,4330 1812.1660 5091.3886 702 2182,18926283,7110 1695.07621998.1286 5687.3477 815 2020.4982 2372592 646 1516,6602 759 69069935 1815,4102 3101.7248 1858.3132 2186,1035 6296.5066 703 1611,9232 816 647, 2001,4096 5697.9296 2023,4099 2375.9391' 017,9987 1539.4711 1818.6551 5112.0653 t760 1861,1941 2189,403 , 704 1700,7708 6301,3060 817 20046911 5708.3031 2026,3221 2379.2854 6929,0014 28 1542,2027 761 18219006 5122.4102 1864.0754 2192,7337 705 1703,6189 6316.1093 2007,9133 4719,211 818 2029.2348 2382.6322 6940,0198 619 1545.0950 762 1866,9574 1823.1468 5132.7594 706 2196,0497 6328,9163 1106.4678 2011,2561 5729'6758 819 2012,1481 2E5,9795 650 163 6951.0357 1547,9019 1828,3437 5143.1130 1869.8399 2199.3662 ' 707 17093172 6339.7271 820 2014.3395 6740,220 2035,0619, 2389.32736962,0551 631 1550.7215 764 1831,6112 5153.4709 708 1872.7230 `2202.6833 6350V 1712.1672 821 2037,9763 652 2017,8235 5750.8642 2392.6737 6973.0781 1553.5,357 1834.8894 165 187.6067 5163.8331 7709 2206.0010 6361.1600 1715,0179 2021.1082 5761.4644 822 2040911 2396,024 653 766 69841047 1556.3506 1818.1383 5174.1997 1878,4109 2209.3192 710 1717,8691 6372.1821 823 2024,3934 5772,064 2043.8065 2399.3741 6995.1347 767 18113757 2212.6380, 6383.0079 824 2346.7225 2402.7240 7006,1683 Jr' 1 gigvi '(pontipuo)) . u u u 20( iu u8oru gtiol U, u 20j u U iu u u u u 0609.1591 01111961 1800'109 ' u b0r 8or 0669'1619 ; u u bur u u/Sofu 606 '08R 2 0100.1611 111111/1 01011691 240./01 966 1991.9061 1009'1969 Z0115611 6611'6009 U09.9;91 108 629.6101 £110'9051 1501.1104 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MACROINVERTfigR;kTE SPECIES EOUITABIltiTY LE 6: WIEDIVERSITYOF SPECIES, a- CHARACTERISTIC.OF-MacARTHUR!S. . MODEL FOR VARIOUS NUMBERS OF HYPOTHETICAL SPECIES' s'*. 4 1 1 0.0000 . 51, 5.0941 102 6.0792 205 re7.0783 2 '0.8113 52 5.1215 104 6.1069 210 7.1128 3 1.2997 53" 5.1485 106 ,6,1341, 215 7.1466 4 1,:6556 54 5.1749 1'08 6.1608, 220 7.1796 5 1.93'114 5.2009 110 6.1870 .225 7.2118 6 2.1712 56 5.2264 112 6.2128 230 7.2434 7 1.3714, '57 5.25,15 114 6.2380 235 .7.2743 8 .\..1-5-:-.16-5 58 5.2761 116 6.2629 240 7.3045 9 '.7022 59 5.3004 \ .118 6.2873 245 7.3341 10 2:8425 60 5.3242 6.3113 250 7.3631: 11 2.9701 61 5.3476 1224 '6.3350 255 7.39.I$' 12 .3.0872 62 5.37 124 6,3582 260 ' 7.4194 13 3.1954 63 5,3934' 126 -6-381 265 ,. 7.4468 14 3:2960 64-' 5.4157 128 6.4036 270 ' 7.4736 15. 3.3899 65 5.4378 130 6.425 275 '7.5000 16 3.4/80 66 5.45'94. 132- 6:4476 280 7.5259 17 3.5611 67 5.4808 134:. 6.44.91 . 285 7.5513 18 3.6395 68 4,5018 13.6, 903 290 .7.5763 , 19 3.7139 69 5.5226 6.5112' 2.95 7.6008 20 3,7846 70' , 5.5430 .140' 6.5318 300 7.6250 21 3.&520 71 5.5632 142 6.5521 310 7.6721 22 .3.9163 72 5.5830 144 6.5721 320 7.7177, 23 '3.9779 73 '5.6027' .146 6.5919 330 7.7620 24 4.0369 74 5.622Q 148 6.6114 310 7.8049 25 4.0937 75 .5.6411 150 6.6306 350 7.8465 26 4.1482 76 5.6599 152 6.495 360 7.8'1370 27 4.2008 ," 77 516.785 154 6.6683 70 7.9264 28 - 4.25,15 78' 5.61569 156 6.686Ja 7.9648 29 4.3004, 54150 158 0.7050 8.0022 30 4.347,8 80 5.7329 160 6.7230 8.0386 - Z, 31 ,4.3946 81 5.7506 162 6.7408 8.0741 32 4.4 82 5.7681 164 6,7584 420 8,1087 33 4.41'1, 83 5:7853 166 6.7757 430 .8.1426 34 4.5301 84 5.8024 168 . 6,7929 440 8.1757"--*/ 351 t. 4.637's, 85 5.8192 170 6.8099 450 8.2080 36 '4.6032 86 5.8359 172 6.8266-' 460 ,8.2396 37 '4.6417' 87 5. 524 174 6.841'2 470 . 8.2706 ' 4.679Y\ 88 5 87 176 ( 6.8596 480 8.3009 it 39 4.7157` Si:9/.,0344 , 178 6.87-58, 490 8.3305 J40 4.7513' 99 180 6.8918 500 - 8.3596' 41 4,7861 . 91 .. 45.9 a 6 9076 .550 8.4968 -42 4.8200' ,\ 92; 5.1320 184)_ 6.9233 600 18.6220 43 4.8531 5.9474, 186,_ 650 8.7373. 44 4.8856 94 5.9627' 188 6.9541' 700 8.8440 45 4.9173 95 5.9n8 190 6.9691 750 8:9434 46 4.9483 4 ; 192 6.9843 -800 , 7.b363 ,47 4.9787 97* 6.0075 .194 :6:5992 850 9.1236 ). 48 -5:0084 98 6.0221 196- 7.0139 900 9.2060 49 5.03751 . 99- '6.0366 198 4 95Q, 9.2839 504- 5'.0661 100 '6.051Q 200 7. 1000 93578 e. 4 *The Oat in this tablearc reptoctucetl, with nerm:s sion,wm 141 a c Ghektrci, Reference; 53. 102 3 , BiOLOGICALINETHODS TABLE 7.CLASSIFICATION, BY VARIOUS AUTHORS, OF THETOLERANCE OF' VARIOUS MACROINVERTEBRATE'TAXA TO DECOMPOSABLEORGANIC WASTES; TOLERANT (T), FACULTATIVE (F), AND INTOLERANT (I) . , . F Mai:-roinyer tebeate I Macroinvertebrate T .9 Porifera-1- Prosopora LI. 60 Demospongiae , , Lumbriculiclae Monaxonida thrudipea Spongillidac 42 It hynchobdellida Spongulafragills- 48 Glossiphoniidac Bryozoa Glossiphonia complanata 48 . Ilelobdella stagnalis '' 48;42. Ictoprocta st:o Phylactolaemata 7 ... ,.,,.(I. nepheloidea 48 , - . 60 . Plumatellidae , '---,, P1,4cobd clla montifera Plutnatellaatella repens . % . P. rugo 48 P. pr,inceps viz. mucosa 48, Placobdel s 42 . P. p. vat: inucom spongipsa 48 Piseieolidac Piscicola p nctata 60 . P. I),var.fruticosa ,. . 48 .48 Gmilhobdel a P. polyinorpha var.,repens . Crisratellidae r. Ilirudickae (iistatella meted° .0 51 Macrobdella 28 . 1.:ophirpOdidae g Pliaryngobdellida ..::Loplroporki.b.carter' '42 .' BrObdellidae Pectinate/la magnifica '48,42 Crpobdella punctata , 48. . /Dina parva --, 48 Endoprocta . s . Urnatellidac D. microStoma 48 1 42. Urnatelk gracilis . 48',42 Dina " Gymnolaemaht. Mooreobliella .microtoma'. Ctcnostomata -, llydracarina Paludjeel0ciac,' sA rthropoda ,-Paludicelehrenbergi 481 Crustitea CoelenteratA s e Isopo'da \. ' ..Asellidac ., llydrozga: 48 Ilydroida . ..i.7, Asellus intermedius Asellus 6U 42 5,4' flyttridae 42 _Hydra 2 Lircetis Clayidae Amphipoda 4 Cordylophora kwastris 42 Tplitridae' . il ya Bela az teca '5,3," I latybelminthes ,,.1 4,42 'Turbellaria , . . to 7 48 Triclaclida' ,, H.knickeYbockeri ... x.. .- I 'Planarkke , GIrrnmaridae 48 Gammarus h". . 42 .1Planaria 42 Nematoda 42'. .Crangonyxpseudogracilts N v - Decapoda matomorpha . . Gordidida Palinonidae,-v 48 Palaemonetes paludosus 5,3, Gordiidae . -. . 4 Annelid A r .48 Oligoehaeta 5.4 48 P. exilipes --Plesiopora . Astacidde 9 25 Naididae 48 Cambarus striatus Nais 42 C fodiens .. 1 . Dero 48 C bartoni bartoni \ Ophidonais 0. -, C b, cavatus \ -, C. tonasaugaensis Stylaria , n 42 . Tuttificidae , C.asperimanus Ti bifex.mbilex . 48,4.2 C. latimanus C acuminatus 1 TubtAx , 48,18,v Linmodrilus hoffmeisteri 48,3;42"- C. hiwassensis 1 L. claparcdiatlus 48 C. extraneus ',. 1 Limnodrilus '48,1,8,60 C. diogeries diogerzes BranchiuraLI sowerbvi 42 C. cryptodytest , . *Numbers refer to references enumerated in the "Li1crature section immediately following this table. .0°' tAlbinistic O 1' MACROINVERTEBRATE POLLUTION TOLERANCE, ; ontinued) qacroinvertebiatf , T 1 Macroinvertebrat T F 1 . . C'. floridanus '' Psilotanypus bellus 42. C'. carolinust _ 1 Tanypus stellatus 44,1218,60 5 4?lopkiilus longirostris 1 'T. carinatus' 42 Procambarus raneyi ... . I 1 -T. punctipennis 44,12 P. acutusacutus I ,... - 1 Tanypus 44,12 P. paeninsularzus Psectrotanypus dya 44,12 48 ;SP. spictilifer 1 Psectrotanypus 44 P.-versutus ' 1 , Larsia lurida 4 .. P. pubescens - -1-- Clinotanypus caligin sus 44,12 P.-litosternur _ 1 Clinotanypus 4 P. enopZ7s &num . 1 Orthocladius obumbrbtus 60 P. artgu,status ' 1 ' Orthocladius -..1 5,4860,42, P. senifooloe 1 , , 44,12 P. trucklentii4 . 1 Nanocladiu.1 4,42 P,adverza$ . 1 Psectrocladius nicer 42 P. pygmaeus$ f 1 P. juliari J. 42 14 P: pubischeide 1 Psectr cladius 4,44 P. barbatus 1 Metriocnernus lundbecki 4 P. howellae Cricotopus bicinctus 3,4, . P. troglodytes 1° 44,12 P. epicyrtus I C. bicinctus group 42 P. fallax 1 C. exilis 44 ' 12 P. chacei I C'. exilis group 42 P. lunzi 1 . C. trifasciatus 44 12 Orconectes.propinquus 42 C. trifasciatus group .42 0. rusticus 42 -,' C. politus 44,12 0. juverzilis' .' I C'. tricinctus 44 12 0. eriehsonianiis 1 C. absurdus 18,44, Faxonella clypeata -, 1 . 12 Insects . - .SriCotopus 44 Dip tera 'Corynoneura taxis 4 Chironomidae C'. scutellata . 44,12 Pentarzeura inculta 60. ( 3,4 Corynoneura 5,42, P. carneosa 60,4460,12 12 P. flavifrons 5 Thienernanniella xena 4,42 P. nreianops 44,12 Thierzernanniella 4,44 P. amerzcana 44,12 Trichocladius robacki 3,4 Pentaneur4 42,44 Brillia par 4 A blabesmyia janta. 3,4, Dia mesa nirariunda 18,42; 4.2 4 A. americana . ° 400 5 Diarnesa 0 s A. illiaoense 12 44 Prodiarnesa olivacea . 12 A. mallochi 42 4 Chironomus attenuatus group 5,4, 44 A. ornata 4 . 42,12 A. aspera 4 C. riparius 18,44, A. peleensis7 la 4 12 A. aurien±is C. riparius group __.-.--7- 42 .A:rhamplic 42 C. tentans 12 Ablabesmyia 42 C'. tentansplumosus - 60 Procladaus culiciformis 60, 44,12 C'. plumosus 48,18_, 48,12 P. denticulatus 42 60 Procladius 12 4,44, C. plumosus group. 42 .. 12 C. carus 4 Labrundirlia floridana 4 C. crassicaudatus 4 . L. pilosella - 42 C. stiematerus , 4 L. virescens 4 C. //a vus >° 60 Guttipelopia 42 C. equisitus 60 Con.chapelopia 42 . C'. fulvipilus 4 C'oelotanypus scapularis 42 C. anthracinus 12 C. concinnus 42 48,60, 44 C. paganus 12 . _ 44.12 C. staegeri 12 $Not usually inhabitant of open.water; are burrowers. 27 CI. A BIOLOGICAL METHODS TABLE 7. (Conti . . . Macroinvertcbrate/ T F I Macroinv rtebrate 42 Chirono mus 5 . 60 Cla otanYtarsus Kiefferullus dux 4 44,12 Micr4sectra dives . 60 12 42 Cryptochironomus fulvus .3,4 44,12 M. deflecta 44,12 C. fulvus group 42 M. nigripula 5 ° 9 C'. digitatus 48 12 Calop1ectra gregarius 44.12 C. sp. B (Joh.) Calopsectra , ------_. 44 12 , 1'5 Stempellinajohannseni - C.b-larina ...... -42 - C: psittacinus 60 Culicidac c 4 Culex pipiens 18,44 C'. naffs s 42 44 Cryptochironomus 5 Anopheles punctipennis C'haetolabis atroviridis 12 Chaoboridac , ' / 60,42 44 C'. ochreatus - 12 Chaoborus punctipennis' .42 Endochironomus nigricans 4,4244,12 # Ceatopogonidae 5,4 42,44 Palpomyia tibialis ... 60 Stenochironomus macateei . S. hikrris 3,4 Palpomyia - 48,60 Stictochironomus devinctus 4,12 'Bezzia glabra 44 44 S. varius . 44 Stilobezzia antenalis #4 42 Xenochironomus xenolabis 42. k ulidae , 44 42 . ula caloptera X. rogersi - 44 44,12 7'. abdominalis ; ' . - X. scopula 44 Pseudochironomus richardson 44,12 Pseudolimnophila futeipennis . 44 Pseudochironomus P 12 Hexatorna, . Parachironomus abortivus group 42 Erio'eera 60 4 P. pectinatellae 42 Psychodidac . 44 Cryptotendipes emorsus 42 Psychoda alternata --, 44 Microtendipes peclellus 44,12 P. schizura .-- 142 , Microtendipes 12 Psyc-hoda ,---6.0, 44,12 Telmatoscopus albipunctatus Paprendipes albimanus 44 Tribelos lucundus 12 Telmatoscopus . 42, 44 . 5,4 T. fuscicornis . :42 Simulidac 18,44 Harnischia collator - 42 Simulium v itratu in S. venustrum 44 H. tenuicaucta , .44 42 Simulium 3 Phaenopsectra o , ..... 42 Prosimulium johannseni 44 1 Dicrotendipes modestus 44. , D. neomodestus 44 42,12 Cnephia pecuarunt D. nervosus 42 12' Stratiomyiidac 4 44 D. incurvus 42 . Stratiomys discalis 44 D. fumidus , 42,12 S. meigeni Glyptotendipes senilis 42 Odontomyia cincta 44 . 4 G. paripes 4 - 12 Tabanidac 44 G. meridionalis 42 Tabanus atratui 18 48,4,.,./ 4,12 T. stygius 44 G. lobiferus 1 - 42 T. benedictus A 44 ' 44 G.-barbipes 42 T. giganteus z(2 7'. lineola , 44 G. amplus 44 12 T. variegatus Glyptotendipes 44 Polypedilum halterale . 42 ,12 Tabanus P. fallax 5,44, 4 Syrphidac 4 44 12 .SArphus cunericanus P. scalaenum 4 42 Eristalis bastardi 18,44 44 P. illinoense 3,4, 44,12 E. aenaus 42,44 E. brousi 44 P. triturn 42 Eristalis 44 - 42 P. simulans '42 ,- 12 Empididac P. nubeculosurn . 12 Ephydridae 44 Brachydeutera argentata 44 P. vibex 42 Polypedilum 48,44 12 A n thomyiidae 44,12 18 Lepidoptera Tanytarsus neoflavellus 5,4 T. gracilentus 12 Pyralididac f' 7'. dissimilis ' 42 Trichoptera 5 3,4 Hydropsychidac Rheotanytarsus exiguus 42 Rheotanvtarsus 42 'Hydropsyche orris 28 1 I "1/4.) a MACROINVERTEBRATE POLLUTION TOLERANCE TABLE 7. (Continued) . . .. = Macroinvertebrate T F I. Macroinvertebrate ' T I H. bifida group 42 1Caenidae H. simulans -. 42 Caenis diminuta 4 . . H. frisoni 42 Caenis . 42 48 H. incommoda 48 5,3,4 Tricorythidae 42 Hydropsyche . 5,4 Siphlonuridae Cheumatopsyche 5,18, Isonychia ..- 42 3,4, Plecoptera 5,4 r.. 42 Perlidae . Macronemum carolipa K 5,3,4 Perlesta placida 18 3 Macronemum V 1 42 A croneurk abnormis 42 Potamyia flava . 42 A. arida 42 Psychomyiidae Nemouridae Psychomyia 41- Taeniopteryx.,nivalis 42 Neureclipsis crepuseutaris 42 Allocapnk viviparia 18 .4, . PotYcentropus 42 5,48-, Perlodidae . 4 Isoperla hilineata 42, Cyrnellus fraternus 42 Neuroptera Ox.Pethira - 5,4 Sisyridae Rhyacophilidae Climacia areolaris 42 Rhyacophila 48 Megaloptera Hydroptilidae COry0alidae Hydroptila mrubesiana 42 CorWis cornutus 42 5,3,4. Hydroptila ' 5,3,4 Sialidae Ochrotrichia ' 42 Siaks infumata 48 - 'Agraylea 42 Siall'i 42 Lcptoceridae ' 48 40donata Leptocella 5,4 42 CaMpterygidae A thripsodes. 42 Hetaerina titia 4. Oecetis . , Agrionidae . Philopotarnidae , . 4rgia apicalis 42 Chimarr perigua ,4 . A..tr'anslata 42 ... 00- . Chimarra s 5,4 Argia ,,. 5,4 Bia6hycentridae ' lschnura verticalis 48 42 - B'achycentrus 4 Enallagma antennatum - 42 ..., Molannidae' 48 E. signatum 42 48 Epherquoptera Aeshnidae HeptageniVae Anax junius ,48 Stenonema integrutn . 32,42 Gomphidae . ) S. rubromaculatum 32 Gomphus pallidus .5,3,4 S. fuscum . 32 G. plagiatus , '48 S..pulchellum 32 I 1 G. externus # 48 , S. ares 32 . G. spiniceps' 42 S. scitufum . 42 G. vastus 42 S. femoratum 18,42' 32 GOmphus . , 5,4. S. terminatum .." 42 Progomphus 5,4 S. interpunctaturv(( 32,42 Droihogomphus 42 S. i. ohioense 32 Erpetogomphus . ' 42 . 'S. i. canadense . 32 Libellulidae S. i, heterotarsale 32 Libellula lydia' 18 S. exiguum 5,3,4 - Neuroc9rdulia moesta . 42 S. smithae 5,3,4 Plathemis 42 S.- proximum 3 Macromk rr 5,42 4 S. tripuncta turn' 32 Hemiptera 4 Stenonema 4 32 Corixidae :, 42 Hexageniidae s Corixa 18 Hexagenia limbata 42 Hesperocbrixa j 18 H. bilineata 60 48 Gerridae . Pentagenia vittgera i Gerris 18 Baetidae Belostomblidae Baetis vagans 42 Belostoma 18,3 . Callibaetis floridanus, Hydrometridae Callibaetis 8 Hydrometra martini 3 29 1 ri0. BIOLOGICAL METHODS TABLE-7. (Continued) MacroinverZbrate T ' F Macroinvertebrate T F I 4§ P. gyrina 28 ColeoNla 28 7 P. ocuto 28 Elmidae 28 28 Stenelmis crenata 18,50 P. fontinalis S. sexlineata 42,50 18 P. anatina 28 S. decorata 50 P. halei 28 Dubiraphia 42,50 P. cubensis 28 Pro moresia . 50 P. pumilia 3 Optioservus 50 Physa 5,4 ,4facronychusglabratus 50 Aplexa hypnorum 28 28 Anbcyronyx variegatus 50 Lymnaeidae . Microcylloepus pusillus 50 Ly4tea ovata 28 Gonielmis dietrichi 50 L. peregra 28 Hydrophilidae : L. caperata , 28 Berosus 42 L. humills 28 7ropisternus natator 18 L. obrussa 28 T,lateralis 3 L. polustris 28 28 T..dorsatis 48 L. auricularla 28 Dytiscidae L. stagnalis 28 28 Laccophilus maettlosus 18 L. s. oppress° 28 Gyrinidae Lymnaea 4 42 Gyrinus floridanys 3 Pseudosuccinea columella 28r Dineutus americanus 18 , Galb'a catascopium 28 , Dineutus ' 42 Fossaria modicella 28 Planorbidae Hollusca . Gastxopoda Planorbis carinatus N..28 Mesogastropoda P. trivolvis 28 Valvatidae R panes 28 'Volvata tricarinata 28 48,28 P. cornetts 28 28 V. piscinalis 28 "P. marginatus - 28. V. bicarinata 48 Planorbis 28 V. b. var. normalis 48 Segmentina armigera - 28 Viviparidae Helisoma anceps 28 48 ,',, Vivaparus contectoides. H.' trivolvis 28 V. subpurpurea . 48 Helisoma 3,4, ' Campeloma integrum 28 Gyraulus arcticus 28 C. rufum 28 Gyraulus '-'''' 28 C. contectus 2$ Ancylidae C. fasciatus 28 Ancylus lacustris - 28* 28 C. decisum 28 A. fluviatilis 28 28 C. subsotidum 48,28 28 4 . Ferfasio flAca Campeloma 60 F tyda j28 . Lioplax subcarinatus : , 48 -F. mularis 28 5,3,4 Pleuroceridae ) , Ferrissia 42 Pleurocera ocuto 48,28 Bivalvia P. elevatum 28 Eulamellibranchia P. e. lewisi , 28 Margaritiferidae Pleurocera 28 Margarittlerajnatiiiritifera 28 l°Goniobasis livescens ' 48,28 Unionidae a virginica ,. 28 Unio winplanata 28 Goniobasis 28 5,4 U. gibbosus . 28 28 Anculosa 28 U. batavus 28 Bulimidae U. pictorum 28 Bulimus tentaculatus . 28 U. tumktus 28 4 Amnicola emarginata , 48 Lampsilis lutgola , , 28 A. limosa , 48 L. alata 28 Somatogyrus subglobosys 48' L. anadontoides 28 t_. , Basommatophora - L. gracilis,° 48 48 Physidae , L. parvus i Physa integra 18,28 28 Lampsilis 48,42 P. heterostropha 28 28, Quadrula pustulosa 28,42 § Except rifflebeetlesY 30 MACROINVERTEBRATE POLLUTION TOLERANCE TABLE 7. (Continued) . 4 i 41 Macroinvertebrate T I Macroinver tqlSrate T F I ar Q. undulata .. 28 S. s. var. lilycashense ' 48 d Q. rubiginosa 28 . S. sulcatum -, 28 Q. lachrymosa 28 S. stamineum 48,28 Q. plicata- 28 S. moenanum" 28 28 7'runcilla donaciformis 48 , S. vivicolum 28 28 T. elegans 48 S. solidulum 28 Tritigonia tubercular; 91..28 Sphaerium / 42 Symphynota costata 28 Musculiunt securis - .V ,Strophitus edentulus 28 M. transversum 48,28 28 .. 'Arrodonta grandis 28,42 , M. truncatum 48 28 A. imbecillis 48,28 i Musculiuni 60 c A. mutabilis 2$ . Pisidiurn abditum 28 . Alasmodonta costa& 28 P. fossarinum . 28' Proptera alata . 42 . P. pauperculum crystalense - 48,28 leptodea fragilis 42 P. amnicvm 28" 28 A mblema undulata 28 P."casert7num Lasmigona complanata 28 P. compressum 48 28. Obliquaria reflex° L 60 P. fallax 28 Heterodonta . P.'henslorvamim .. 28. . Corbiculidae . /- P.,idahoensis* 28 Corbicuk manilensis 42 , P. complanatum 48,2848,28 GS phaeriidae 5,4 P. subtruncatum . 28 , Sphaerium notatum 28 Pisidium L. 48 S. co'ineum 28 Dresisseniidae. S. rhomboideum . 28 Mytilopsis leucophaeatus S. striatinum - 28 Mactridae , , S. s. var. corpulen tum . 48 Rangia cuneala 28 IDt 's BIOLOGICAL METHODS .0 'LITERATURE CITEQ s .1.*Alleit, K. R. 1951. The Horokiwi Stream -,a study of a troutpopulation. New Zealand Marine Dept. Fl Bull. #10. 231 pp. 2. American .Ptiblic Health Association. 1971. Standard methods for the examinationof water and wastewater, 13th edition. American Public. Health Association, New York. 874.pp. o 3. Beck, W. M., Jr., Biological parameters instreams..Florida State Board of Health, Gainesville. 13 pp: (Unpublished) -.,.. N 4. Beck, W. M., Jts,-4ndicator organism classification.- Florida StateBoard, of Health, Gainesville.-Mimeo. Rept. 8 pp. (Uribushed)shed) .1 . Acad. 5. Beck, W .,jr. 1954. Studies in stream pollution biology: I. A simplified ecOlogicaL classification of organisms. J. F Scie ces, 17(4):211-227. ., 't . . . 6. Beck, W. J., Jr. 1955. Suggested method for reporting biotic data. Sewage Ind. 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B. 1954. Speciation and distribution of the crayfishes of the Ozark Plateau and Ouachita Provinces. Kans. Univ. Sci. Bull. '36(121:803-918. - Williams, A. B. 1965. Marine decapod crustaceans of the Carolinas. USDI, Fish Wildl. Serv., Bur. Comm. Fish. 65(1):1-298. 7.3Diptera Bath, J. L., and L. D. Anderson. 1969. Larvae of seventeen species of chironomid midges from southern California. J. Kans. Entomol: Soc. 42(2):154-176. Beck, E. C., and W. M. Beck, Jr.,1959. A checklist of the Chironomidae (Insecta) of Florida (Diptera:Chironomidae). Bull. Fla. State r. Mus. 4(3):85-96. Beck, E. C., and W. M. Beck, Jr. 1969. The Chironomidae of Florida. Fla. Ent. 52(1):1-11. Beck, W. M., Jr. and E. C. Beck. 1964. New Chironomidae from Florida. Fla. Ent. 47(3):201 -207. Beck, W. M., Jr. and E. C. Beck. 1966. Chironomidae (Diptera) of Florida. Part -1.*Pentaneurini (Tanypodinae). BUIL Fla. State Mus. 10(8):305-379. Beck, W. M., Jr. and E. C. Beck. 196%- Chironomidae (Diptera) of Florida. 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Taxonomy and biology of ifearctic limnephelid larvae(Trichoptera), with special reference to species in eastern - United States. Entomologica Americana, 40!1-120. BIOLOGICAL METHODS i Flint, O. S. 1961. The immatAgeAtages of the Arctopsychinae occurring in eastern North America (Trichoptera:Hydropsychidae). Ann. Ent. Soc. Amer. 54 1):5-11"-N' . t Clint, 0. S. 1962.'Lakvae of the caddisfly Genus Riryacophik in eastern North America (TrichoOkra:Rhyacophilidi ). Proc. US NatL Mus. 113:465-493. . , - SR I Flint, O.'S. 1963. Studies of votropitaCcaddisflies. Part I, Rhyacbehilidae and Glossosoni4atidae (Trichoptera). Pr c US Natl. Mus. 114:453-478.. 4) - Flint, 0. S. 1964..Thetaddisflies (Trichoptera) of Puerto Rico.Univ.4'iierto Rico Agr. Exp. Sta. Tech. Paper No. 40. 80pp. . Flat, 0. S. 1964a. Notes on some.nearctic Psychomyiidae with special reference to their larvae (Trichoptera).Proc. US Nat. Mus. Publ. No. 34.91, 115:467-481. - 4.- Hickk N. E. 1968;Cadt3.i.s larvae, larvae of the British Trichoptefa..Associated Univ. Presses, Inc., Crandry..N. J. pp. 1-480. Leonard, l. W., and F. A. Leonard. 1949. An annotatedlist4 Michigan Tricptera. Mic us. Zool. Oct. Paper No. 522. pp. 1-35. Lloyd, J. T. 1921. North American cadakfly larvae. Bull. Lloyd LibrNo..21, E taw. Se Np. 1. pp. 16 -119. , A Ross, H. H. 1941. Descriptions and records of North American Trichoptera.'Trai er. t ntorrl Sol: 61135-129. . . Ross, H. H. 1944. The caddisflies, or Trichoptera, of lilinois. Bull.111.4.1at. Hiss, Surv. 23:1-326. ... Wiggins, G. B. 1960e A preliminary systematiC study of the North American larvae ,of the caddisflies, 'family Phiyganeidae (Trichoptera). Can. J. Zool. 38:1153.-1170.4 1 Wiggins, G.B. 1962. A new subfamily of phrYglineid caddisflies from western North America (tr.optera:Phryganeidae). Can. J. " .4 Zool. 40:879-891. , ,, _ 4Wiggins, G.B. .1963. Isarvae and pupaepupae of two North American limnephilid caddisfly genera (Trichoptera:Limnephilidae). Bull. ., Brooklyn Ent. Soc. 57(4):103-112. ' . 44, Wiggins, G. B.1965, Additions and revisions to the genera of North American caddisflies of the family Brichycentridae vejth special reference to the larval stages (Trichoptera). Can. Ent. 97:1089-1106. ., Wiggins, G: B., and N. H. Anderson. 1968. Contributions to the systematics of the caddisfly genera Pseudostenophylax and,Philocasca With special reference to the immature,s6ges (Trichop,tera:Limnephi)idae),Can. J. Zool. 46:61-75. ' .. Yamamoto, T., and G. B. Wiggins.'1964. A comparative study-of the North Americ4n species in .thecaddisfly genus Mystacides .(Trichoptera:Leptoceridae). Can J. Zool. 42:140571126. . /4 4I4 n--6 a I 7..15Marine ,.t. v ° Hartman, 0. 1961. Polychae toannelids from California, All= Hancock Pacific Expeditions. 25:1-226. Hartman; 0., and D. J. Reish950. The marine annelids of Oregon. Ore. State ColL Pies's, Corvallis, Ore. Miner, R. W. 1951. Fiel ook of seashore life. G. e. Putnam's Sons, Ndw York. . Sniith, R. I. 1964. Keys to the marine invertebrates of the Woods Hole region. Woods Hole Marine Biol. Lab., Cont. No 11. Smith, R., F. A. Pitelha, D.'P. Abbott, and F. M. Weesner. 1967. Intertidal invertebrates of the central. California coast.'Univ. Calif. Press. Berkeley. teP 38 r .9r ' V' FISH , Page t --- e.1.0 INTRODUCTION = 1 2.0 ,SAMPLE COLLECTION ' 1 2.1 .General Considerations 1 2.2 Active Sampling Techniques 2 - % 2.2.1 .Seirfes % . 2 . ,2.2.2 .Trawls - 3 2.2:3 Electrofishing ..z d 5 ..2 $ 2.2.4 Chethical Fishing 5 2.2.5 Hook. Line ...... ,. 6 - 2.3 Passie Sampling Techniques 7 :,.!.- , '-2.3.1 Entariglernent.Nets 7 -Y . i , -2.3.2 Enti-apment Devices:... . !..'...... 7 3.0 SAMPLE ESERVA,TION . 10 4°,0 SAMPLE ANALYSIS 11 , ,,, / \- . 4:4' °Data Recording .:-. , I 1 1- . 4.2 Identification '.. . .-...... $...... 11 . 4.3 Age, Growth, and .Concl:itiori 11 :2 . ', . -5,0 'SPECIAL .TECHNIQUES f 11 5.1. Flesh Taiirting 11 C. ': '5.2 Fish kill ltiive tigationsN: r .." 12, 6.0 .REFEgENC-ES--. 13- 7.d B-IBLIOGRAPHY '14 7.Gcneral,Refe ences ..... 14 ' .,1.2 E.lectrfitishin: 14 d 7.3. Chei-hr6a1FiS ing I5 ' $. ' 7.4 Fish Identification . 16 7.5 Fish Fills 1-8 : a ti FISH 1.0 INTRODUCTION and (8)palatability.Observations of fish Fo the public, the condition of the fishery is behavior can also be valuable in detecting en-. vironinentalproblems;e.g.ventilatiohrates, tile most meaningful index of water quality. position, in the current, and erratic movement. Fish occupy the upper levels of the aquatic food e collected, for use in laboratory web and are directly and indirectly affected byj Fish may als bioassays, for sue analyses to measure the con- chemical and physical changes in the en als and pesticides, and for ,ment. Water quality conditions that significan y centrations of histopathologic examination. affect the lower levels of the food web will Fisheries data have some serious limitations. affect the abundance, species composition, and Even if the species composition of the fish in a condition of the fish population. In some cases, specific area were known before and after the however, the fish are more sensitive to the pol- discharge of pollutants, the real significance of oriutant(s) than are the lower animals and plants; changes' in the catch could not be properly they may be adversely affected even when the interpretedunlessthelifehistories of the lower levels of the food web are relatively affected species were understood, especially the unharmed. migration,temperature Many species of fish have stringent dissolved spawning,seasonal and are gradient and stream-flow responges,diurnal oxygen and temperature requirements movements, habitat preferences, and activity intolerant of chemiCal and physical c ntami- patterns. Without this knowledge, fish presence nants resulting from agricultural, indus'al, and or absence can of be correlatedwith water mining operations. The discharge ofoderate quality. Of courge, any existing data on the amounts of degradable organicwash may in- the habitat and water quality requirements of fish Would be of crease the nutrient levels in great value in interpreting field-data. result in an increase in the standing crop of fish. Fisheries data have been. found useful in en- -This increase, however, usually occurs' in only forcement cases and in long-term water quality one or a.few species and rtsults in animbalance monitoring (Tao, 1965). Fishery surveys are in the population. The effects of toxic wastes of all fish to a costly, however, and a careful and exhaustive may range from the elimination search'should be conducted for existing informa- 'slightreductioninreproductivecapacity, tion on the fisheries of the area in question growth, or resistance to disease and par4tism. before, initiating a field study. State and Federal Massive and complete fish kills are dramatic'fishery agencies and universities are potential signs ,of abrupt, adverse, changes in environ- sources of information which, if available, may Mental,Conditions. Fish, however,can repopulate col- an area' rapidly if the habitat is notdestroyed, save time and expense. Most states require a and the cause of the kill may be difficult to lecting permit, and the state fishery agency must detect by examination of .the fish communityusually be contacted before fish can be taken in a field study. If data are not available and afield, after it has recovered from the effects of the and pollutant. Chronic pollution, on the otherhand, study must be conducted, other Federal is more selective in its effects and exerts its in- State agencies will often join the survey and fluence over a long period of ,time andcauses pool their resources because they have an reCognizable hanges in the species composition interest in the data and have found that a joint effort.is more economical and efficient. and relativg abundance of the fish. . The ptincipal 'characteristics of interest in 2.0 SAMPLE COLLECTION field studies of fish populations include: (1) species present,2) relative and absolute abun- 2.1General Considerations dance of eachspecies,(3) size distribution, (4) Fish can be collected actively or passively. growth rate, (5) condition, (6) success ofrepro- Active 'sampling methods -include the use of duction, (7).incidence of disease and parasitism, seines,trawls,electrofishing,chemicals, and A BIOLOGICAL METIIODS hook and line. Passive methods involve entangle- colle,ction methodsare very selective ment (gill nets and -trammel nets) and entrap- and do not tobtain reprosientative samples of the ment (hodp nets, traps, etc.) devices. The t al population. Active methods/are less selec- limitations in obtaining qualitative and( uantita- tive and more effiNent, but usually require more tive data on a fish population are gear selectivity equipment and manpower. Although lrie choice and the mobility and rapid recruitment of the of method depends on the objectives of the fish. Gear selectivity refers to the greatersuccess particular fishery investigation, active methods . of a particular type 'of gear in collecting certain are generally preferred. species, or sizes of fish, or both. All sampling gear is selective to some extent. Two factors that 2.2Active Sampling Techniques affect gear selectivity are:( I) the habitat or portion of,habitat (niches) sampled and (2) the 2.2,1) Seines adtual-efficiericy of the gear. A further problem is that the efficiency of gear for a particular /A haul seine is essentially a strip of strong species in one area does not necessarily apply tofitting hung between a stout cork or float line at the same species in another area. Even if non- the top and a strong, heavily-weighted lead line selective gear could be developed, the problem at the bottom (Figure 1). The wings of the net' of adequatelysampling,. an areaisdifficult areoftenof -largermesh, than the middle because of the nonrandom distribution of fish portion, and the wings may taper so that they populations. are shallower on the ends. The center portion of Temporal changes. rtri the relative abundan e of -the net may be formed into a,&ag to aid in con- a single species Can be assessed under a gi. n set fining the fish. At the ends Fftlr wings, the of conditions if that spe }Cies is readily taken with cork and lead lines are often fastened to a short, a particular kind of gear, but the data are not stout pole or brail. The hauling lines are then likely to reflectthetrue abundance of the attached to the top and bottom of the brail by a species occurring in nature. short bridle. IP* .4., ... Figure 1. The common haul seine. (From Dumont and Sundstrorth 1961.) 2 (. FISH SAMPLING Deepwater seining usually requires aboat. attached to the forward end of each wing ,and One end of one .ot the hauling lines is anchored are used to keep the mouth of the net open on shore and the boat pays out the line until it while itis being towed. The otter boards are reaches the end. The boat then changes dir6;Ction approximately rectangular and usually made of and lays out the net parallel to the beach. When wood, with steel strapping. The lower edge is all of the net is in the water; the boat brings the shod with a steel runner to protect the wood end of the second hauling line ashore. The net is when the otter slides along the bottom. The then beached rapidly. leading edge of the otter. is rounded near the The straight seines (without bags), such as th.e bottom to aid in riding over obstructions. ti common -sense minnow SeinCS, can usually be The towing, bridle or warp is attached to the handled quite easily.by two people. The method board by four heavy chains or short heavy metal of paying out- the seine and bringing itinis rods. The two forward rods are shorter so that, sithilarto the haul seine, except the straight when towed, the board sheers to the outsideand seineis generally used in shallow water where down. Thus, the two otters sheer in"opposit9- one member of the party can wade offshore'directions and keep the mouth of the trawl open with lines. and on the bottom. Floats or corks along the. Bag and 'straight seines vary considerably in headrope keep the net from sagging, and the dimensions and mesh size. The length varies weights on the lead-line keep the net on the from .3 to 70 meters, and mesh size andnet bottom. The entrapped fish are funneled back width vary with the size of the Nish and the into the bag of the trawl (cod end). depth of the water to be sampled. A popular small trawl consists of a 16- to - of the Nylon seines are recommended becm. 20-foot(5- to 6- m) headrOpe,' semiballoon ease' of maintenance. Cotton seines s ould be modified shrimp (otter) trawl with 3/4-inch (1.9 treated with a fungicide to prevent dec.. y. cm) bar mesh in the wings and cod end. A 1 /4- Seining is not effective in deep water because incli-(0.6 cm) bar mesh liner may be installed in the fish- can escape over the floats and ,under the the cod end if smaller fish are desired,,This small lead line. Nor isit effective in areas that have trawl uses otter boards, the dimensions ofwhich, snags and sunken debris. Although the results in inches, are approxi,mately 24 to 30 (61 to are expressed as number of fish captured, per 76 cm) X 12 to 18 (30'to 46 cm), X 3/4 to 1-1/4 unit area seined, quantitative seiningis very niches (0.9 to-31/2 cm), and the trawl can be difficult. The method is more useful in deter- operated out of ,amedium-sized Bloat. mining the variety rather than the nuMber, of The midwat,er trawl resembles an otter trawl, fiSninhabiting the water.. with modified boards and vanes for controlling 2.2.2 .Trawls the trawling depth.. Such trawls are cumbersome for freShwater and inshore areas. Trawls-are specialized submarine seines used in large, open-Water areas of reservoirs,kites, Trawling data are usually expressed in weight large rivers, estuaries, and in the oceans. They of catch per unit of time. may be of -considerable size and are towed by The use of trawls reqUires experienced person- boats at speeds sufficient to overtake and en- nel.Boats deployinglargetrawlsmust be close the fish. Three basic types arc: (I) the equipped with power winches and large motors. beam trawl used to capture bottom fish (Fi'gure Also, trawls can not be used effectively if the 2), (2) the otter trawl' used to capture near-.bottom. is irregular or harbors snags or other bottom and bdttoip fish (Figure 3), and (3).the debris. Trawls are best used to gain information mid-water trawl used to collect schooling fish'at on a particular species of fish 'rather than toesti- various depths. mate .the-overall fish population. See Rounsefell 2The'bearn trawls have a rigid opening and are andEverhart '(1953),Massman,Ladd and diffiCultto operate From a small boat: Otter McCutcheon (1952) and Trent (1967)for trawls have vanes or "otter boards," which arc further information on trawls. 319_9 BIOLOG&AL METHODS .1 . Figure 2. The beam trawl. (Fr Dumont and Sundkroin, 1961) Figure 3. The otter trawl. (From Dumont andSundstrom, 1961.) 4 N. FISH SAMPLING 2.2.3Electrofishing water, even though the voltage drops may be Electrofishing is a sampling method in which identical: Seehorn (1968) recommended the yse alternating (AC) or direct (DC) electrical current of an electrolyte (salt blocks) when sampling in is applied to water that has a resistance diffeient some soft waters to produce a large enough field from that of fish. The differencOn the resist- with the electric/ shocker. Frankenberger ance of the water and the fish to pulsating DC(1960), Larimore, D'urham and Bennett (1950) short and Lhtta 'and Meyers (J961) have excellent stimulatesthe swimming muscles for Frankenberger and toorient papers onboat shockers. periods of time, causing thefish Latta and Meyers used a DC shocker and Laril- towards and be attracted to the positive elec- 6), trode. An electrical field of sufficient potential more et al. anAC shocker.Stubbs to immobilize the fish is present near the posi- used DC or pulsed DC, and his (aluminui boat wired as the negative pole. In his paper, he tive electrode. also shows the design and gives safety pre- The electrofishitigunit may consist of a cautions that emphasize the use e treadle 110-volt,60-cycle,heavy-duty generator, an switch or "deadman switch" in cas worker electricalco ntrolsectionconsistingof a falls overboard. modified, commercially-sold,variable-voltage Backpack shockers that' are_quite useful for pulsator, and electrodes. The electrical control small, wadeable streams haYe been described by section permits the selection of any AC voltage Blair (1958) and McCrimmon and Berst (1963), between 50 to 700 and any DC voltage between as has a backpack shocker for use by one man 25 to 350 and permits control of the size of the (Seehorri, 1968). Most of these papers give dia- electricalfieldrequired'0/various types of gramslor wiring and parts needed. water. The alternating currant'serves as a stand- There are descriptions of electric trawls (AC) by for the direct current and is used in cases of (Haskell, Geduliz, and .Stiolk, 1955, and Loeg,' .extremely low water resistance. 1955);electric seines (Funk,1947; Holton, Decisions on the use of AC, DC, pulsed DC, or 1954; and Larimore, 1961)i-and a fly-rod elec- alternate polarity forms of electricity and the trofishing device employing alternating polarity selectionof theelectrodeshape,electrode current (Lennari,1961). spacing, amount of voltage, and proper equip- The user must decide which design is most ment depend on the resistance, temperature, and adaptabletohispariictilarneeds.Before 'total dissolved solids of the water. Light-weight deciding which designtouse,the biologist conductivity meters are recommended for field should 'carefully review the literature. The crew use. Lennon -(1959) provides a comprehensive should wear rubber boots and electrician's gloves table and describes the system or combination and adhere strictly to safety precautions. of systems that worked best for him. Night sampling was found to be much more Rollefson (1958, 1961) thoroughly tested and effective thar day sampling. Break' sampling evaluated AC, DC, and pulsating DC, and dis- efforts into -time units so that unit effort data cussed basicelectrofishingprinciples,wave are available fOr.comparisonpurposes. forms, voltage current relationships, electrode types and designs, and differences between AC 2.2.4 Chemical fishing and DC and their effects in hard and soft waters.' - Chemicals usedinfishsampling Include He concluded that pulsating DC was best in rotenone, toxaphene, cresol, copper sulfate, and terms of power economy and fishing ability sodium cyanide. Rotenone has generally been when -correctlyused.Haskell and Adelman the most acceptable because of its high degrad- (1955) found that slowly pulsating DC worked ability; freedom from such problems as precipi- best in leading 'fish to the anode. Pratt (1951) tation (as with capper sulfate) and persistant also found the DC shocker to be more effective toxicity (as with toxaphene) and; relative safety than the AC shocker. for the user, Fisher ,(1950) found that brackish water re- Rotenone, obtained fromthe)derrisroot quires much more power (amps) than fres (Deguelia elliPtica, East Indies) and cue. root "-.. BIOLOGICAL METHODS.. ,2 (Lonchocarpus nicour,South America),has A very efficientethod of applying emulsion been used extensifely in fisheries work through-.products is to pumthe emulsion from a drum out the United States.; and Canada since 1934 Mounted in the bottoin of a boat. The emulsion (Krumhblz, 1948). Although toxic to man and is suctioned by a venturi pump (Amundsoyi twat warm-blooded animals (132 mg/kg),' rotenone bailer) clamped on the oute7ard motdr. The has not been condideted hazardous in the con- flow can be metered by a valve at the drum hose centrations used for fish eradication (0.025 to connection This method gives good dispersion 0:050 ppm active ingredient) (Hooper, 1960), of the chemical and greater boat handling safety, and has been employed inwaters used for since the heavy drum can be jrnounted in the bathing sand in some instances in drinking water bottom of a boat rather than above the gun- supplies (Cohen et al:, 1960, 1961). Adding acti- wales, as required for gravity flow. ;.;*, vated i carbonnotonlyeffectivelyremoves Spraying equipment needed Ito apply. a rotenone, butitalso removes the solvents, rotenone emulsion efficiently varies according to odors; and emulsifs present in all commercial the size of the job. Fsir small areas of not more rotenone formulation than a few acres, a portable hand pump ordinar-, Rotenone obtaineas an emulsion containing ily Used fokarden spraying or fire fighting is sufficient. The, same size pump is also ideal for . approximately ,5 ercent active ingredient,is- recommended because of tl e case of handling. It sampling the population of a small area. is a relatively fast-acting toxicant. In most cases, A power-driven pump is recommended for a the fish will die within 1to 2urs after expo- large-scale or long -term, sampling program. A sure. Rotenone decomposes rapidly in most detailed description of spraying equipment can lakes- and ponds and is quickly dispersed in be found in Mackenthun and Ingram '(1967). streams. At summer water temperatures, The capacity of the pump need not be greater toxicity lasts 24 hours or less. Detoxification is than 20liters per minute. Generally speaking, a brought about by five principal' factors:dis- (1-1 /2 .P. engine is adequate. solved oxygen, light,alkalinity, heat, and turbid- The, power application of rotenone emulsives ity. Of these, light Ad oxygen are the most im- requires a pressure 'nozzle, or ,a sprdy boom, or portant ,factors. both, and sufficient plumbing and hose to con- nect with the pump. The suctio90 line of the Although the toxicity threshold for rotenone pump should be split by a "Y" to attach two differs slighly among fish species, it has not been intake lines.Orie lineis fisedto supply the widely Used as a selective toxicant. It has, how- toxicant ,from the drum, and the other line, to ever, been used at a conntration of 0.1 ppm of supply water from the lake or embayment. The the 5 percent emuision to crtntr1 the gizzard valves are adjusted so the mater and toxicant are shad (Bowers 1955).,,. , N drawn into the pumping system in the desired Chemical sampling is usually employed on a proportion and mixed., spot basis, e.g. a short reach of river or an ern- In sampling a stream, select a 30- to 100; bayment of a lake. A concentratio.npf 0.5 ppm meter reach depending on the depth and width active ingredient will provide good recovery of of the stream; measure the depth of the section most species of fish in acidic or.slightly alkaline selected, calculate the area, and determine the 'waters. If bullheads and carp are suspected of amount of chemical required. Block off the area being present, however, a concentration of 0.7 dpstream and downstream withseines. To ppm active ingredient is recommended. If the detoxify the are9tdownstream from the rote- water is turbid and strongly alkaline, and resist- none, use potassium permanganate. Care must ant species (i.e., carp anti bullheads) are present, be exercised, however,becausepotassium use 1-2 ppm. To obtain a rapid kill, local con- permanganate is toxic to fish at about 3 ppm. .entratiOns of 2 ppm can be used; however, cau- tion is advised becauSe rotenone disperseddispersed into 2.2.5. Hook and line peripheral wafer areas may kill fisiras long 4s the Fish collection by hook and line can be as concentration is above 0.1 ppm. simple as using a hand-held rod or trolling baited 6 1/4",461bv FISH SAMPLING hooks or other lures, or it may take the form of Stationary gill and trammel' nets are fished at long...Jr/at lines or set lines with many baited right angles,,to suspected fish movements -and at hooks. Generally speaking, the hook and line any, depth from the surface to the bottom. They methodis not acceptable for conducting a may be held in place by poles or anchors. The fishery survey, because it is too highly selective anchoring, method must hold the net imposition in the size and species captured and the catch against any unexpected water movements such -per -unit -of effort is too low.*Ithough it can as, runoff, tides, or seiches. only be used as a; supporting technique, it may Drifting gill or trammel nets are also set and be, the best method to obtain a few adult speck fished the same as stationary gear, except that mens for heavy metalanalysis,etc., where `they -are not held in ylace but are allowed to sampling with is impossible. drift, with the currents. This method requires constant surveillance when fishing. They are ,- 2.3Passive Sampling Techniques generally set for a short period of times, and if currents are too great, stationary; gear is used. 2.3.1EntangleMent tjets -) The use of` gill nets in the estuaries may Gill and trammel filets are used extensively to presentspecial 'problems, and consideration sample fish populations-in estuaries, lakes, reser- should be given to tidal currents, predation, and voirs, and larger rivers. optimum fishing time, and to anchors; floats, A gill net is usually set as an upright fence of. and line. r netting andhas,..,,auniform mesh size.Fish The gunnels of any boat used in a net fishing attempt to swim HiNigh the net anare caught operation should be free of rivets, cleats, etc., on 3 in the mesh (Figure,., ). Because th size of, the which the net can 'catch. mesh determines the species an ize of the fish to be cattglegill nets 'are considered selective. The most versatile type is an experimental gill 2.3.2 Entrapment devices net .consisting of five different mesh size sec--1 With 'entrapment devices, the fish enter an en- flogs. Genets can be set at the surface, in mid- closed area (which may be baited) through a w ter,or atthe bottom, and they can be o crated as.staticor movable gear. Gill nets series oflone or more funnels and cannot escape. 'made, of multifilament or monofilament nylon \ The hoop netand trap net are the most coih- afe recommended. Multifilament nets cost less mon types of entrapment devices used in fishery and are easier to pse, but monofilament nets surveys. These traps are small enough to be de- -generally capture more fish. -The floats anq leads ployed from a small open boat and are relatively usually supplied with the nets can cause net en- simple toset. They are held in place with, tanglement. To reduce this problem,' replace the anchors or poles and are used in 'water deep individual floats with a float line made with a enough to cover the nets, or to a depth upl core of expanded foam and use alead-core meters. , leadlineinstead of individual lead weights. The hoop net (Figure 6), is consTructed by The trammel net (Figure 5) has a layer of covering hoops or frames with netting. It has large mesh netting on each side of loosely -hung, one or more internal funnels and does not have smaller gill netting. Small fish are captured in wings or 'a lead. The first two sections can be the till netting and large fish are captured in a made square to prevent the net from rolling in "bag" of the gill-netting that is formed as the the currents. smaller-mesh gill netting is pushed through an The fyke net (Figure 7) is a hoop net with 4, opening in the larger-mesh netting. Trammel wings, or a lead, or both attached to the first-- nets are not used as extensively as are gill nets frame. The second and third frames can each in samling fish. hold funnel throats, which prevent fish from° Reslit for both nets are expressed as the escaping as they enter each sectipn. The oppo-f numbeor weight of fish taken per length of net site (closed) end of the net may be tied with a per day. slip'cord to facilitate fish removal. 7 . BIOLOGICALMETHOD I Figure 4. Gill net. (From Dumont and Sundstrom, 1961.) 1° Figure Trammel net. (From Dumont and Sundstrom, 1961.) 8 /' J c, 125 FISH SAMPLING rt. Figure 6. Hoop net. (Froin Dumont and Sundstrom,-1961.) Figure 7. Fyke net. (From Dumont and Sundstrom, 1961.) 9 1`) , . BIOLOGICAL METHODS Hoop nets are fished in rivers and other waters habittifs. Hoo and trap nets are made of cotton where fish move inpredictabledirections, or nylon,lit nets made of nylon have a longer whereas the fyke neris used when fish move-life and a e, ighter When. wet. Protect cotton ment is more random such as in lakes, impound- nets frodecay by treatnieq, Catch iskecorded ments, "and estuaries. Hoop and fyke frets can be as numrs or weight per i3pit of effort, usually. obtained with hoops from 2 to 6 feet (0.6 to 1.8 fish. per n t day. meters) in diameter, butany net over 4 feet (1.2 meters) in diameter is too large to be used ina 3.0 'SAMPLE PRESERVATION fishery survey. . - Preserve fish in the field in 10 percent forma- Trap nets use the same principle as hoop nets lin. Add- 3 grams borax and 50 ml glycerin per for capturing fish, but their construction is more formalin. Specimens larger than 7.5 cm complex. Floats and weights instead of hoops shouldbe,slit on the side at least one-third of, give the net its shape. The devices are expensive, the, leitgth of the lody cavity to permit the require considerable experience, andare fished preservative to bathe the internal,organs. Slit the in waters deep enough to cover them. fisoh the right ,side, because the left side Is'` One of the most simple types is the minnow generally used for measurements, scale sampling, trap, usually made of wire mesh or glass, with a and photographic records. single inverted funnel. The bait is suspended in a Fixation may take &Om a few hours with porous bag. A modification" oc this type-is the small specimens to a week or more with large slat trap; this employs long vitooden slats in a forms. Afterfjxation, the fish may be washed in cylindrical trap, and when baited with cheese running water or by several changes of water for bait, cottonseed cake, etc., it is 'used. very suc- atleast 24 hours and placed in 40 'percent 'cessfullyinsampling' catfish, inlarge rivers isopropyl alcohol. One change 'of alcohol is (Figure 8).. necessary to remove the last traces of formalhe Most fish can be sampled by setting trap and Thereafter, they may be permanently preserved hoop nets of varying mesh sizes in a variety. of in the 40 percent isopropyl alcohol. Figure 8. Slat trap. (From Dumont and Sundstr m, 1961.) 10 4 FISH IDENTIFICATION 4.0 SAMPLE ANALYSIS by the temperature of the water in which they live. Growth is rapid during the warm season arid 41 Data Recording slows greatly' or stops in winter.. .This seasonal 0 The sample records should include collection change in growth rate ot fishes is often reflected numberehame ocyiater body; date, locality, and in zones or bands (annual rings) in hard bony 'other pertinent information associated with the structures, such as scales, otoliths (ear stone), sample. Make adequate field notes for each col- and vertebrae. The scales of fish may indicate lection..-Wria with water-proof ink and paper to exposure to adverse conditions such as injury, ensure a permanent record. Place the label inside poor food supply, disease, and poisibly water quality. the container with the specimens and have the Note the general well being of the fish do label bear the same number or designation as the field notes, including the locality, date, and col- they 'appeemaciated? diseased from fungus? lector's name. Place a numbered tag on the out- have open sores, ulcers, or fm rot? parasitized? side of the container to make it easier to find, a Check the gill condition, also. Healthy fish will particular collection. Place any detailed observa- be active when handled, reasonably plump,`and lions about a collection on the field data sheet. not diseased. Dissect a few specimens and check Record fishery catch data in standard units such the internal organs for disease or parasites. The as number or weight per area or unit of effort. stomachs can be checked at this.time to deter- Use the metric system for length and weight mine if the fish are actively feeding. measurements. 5.0SPECIALTECHNIQUES 4.2Identification 5.1Flesh Tainting Proper identification of fishes to species is im- Sublethal coiiicentrations of chemicali, such as portant in analysis of the data for-water quality phenols, benzehe, oil, and 2,4-D, are often re- interpretation. A list of regional and national sponsible for imparting an unpleasant taste to 41, references for fish identification is located at the fish flesh, even when present in very low concen- end of this chapter. Assistance in confirmingtrations. Flesh tainting is nearly as detrimental queStionable identificationisavailable from to the fisheries as a complete kill. State, Federal, and university fishery scientists. A method has been developed (Thomas, 4.3 Age, Growth,' and Cohditiori 1969) in whiCh untainted fish are placed in cages 'upstream and downstream from suspected waste 43; 'Changes in water quality ,can be detected by studying the irOwth 'ram. of fishes.Basic'iouraes. This procedure will successfully relate- methodi used to deter the age and growth the unacceptable flavor produced in native =fish of fish include: if exposed to a particular waste soUree. To ensure uniform taste quality before, ejwo- Study of fllength- frequencies, and sure, all fish are held in pollUtion-free wateffor Study of seanal ring formations in hard a 10-day period. After this period, a minimum bony parts such s scales and bones. of three fish are cleaned and frozen with dry ice I as control fish. Teit fish are transferred to The length-frequency method of age deter- the test sites, and a minimum of ee fish are intation depends On the fact that fiSh size varies placed in each portable cage. The cages are sus- with age. When the number of fish per length pended at a depth of 0.6 meter or 48 to 96 interval is plotted on graph paper, peaks gen- hours. erally apiear for each age group. This method After exposure, the fish are dressed, frozen on works best for young fish. dry ice, and stored to 0°F until tested. The con- The s asonal ring-formation method depends trol and ex ed samples are shipped to a fish- on theact that fish are col- blooded animals !tasting pa el, such as is available. at tht food and the rates of their body pro esses are affected science andchnology- departnients in many of 11 'BIOLOGICAL METHODS ihekrnajor universities, and treated as follows: (a) result of a heated, water discharge, will often The fish are washed, wrapped in aluminum foil, result in fish kills. Long periods of very warm, placed on slotted, broiler-type pans, and cooked dry weather may raise water temperature§ above in -a gas oven at 400°F for -23 to '45 minutes lethallevelsfor particulr species. A wind- depending on the size of thefish. (b) Each induced- seic'he may be hazatdokis to certain sample is boned and the flesh is flaked and temperature=sensitive, deep-lake, cold-water fish, mixed to ensure a uniform sample. (C) The or'fish of shallow coastal waters. samples are served in coded cups to judges. Disease, -a dense infestation of parasites, or Known and coded references or control samples natural death of weakened fish at spawning time are included irr each test. The judges score the must always besuspectedascontributory flavor and desirability of each sample on a politt factors in fish mortalities. scale. The tasting agency will establish a point Explosions, abrupt water level fluctuations, on the scale designated as the acceptable and litirricages, extreme turbidity or siltation, dis- desirable level. sharges of toxic chemicals, certain insecticides, algicides, and herbicides may eaCh cause fish 5.2Fish Kill Investigatlons Fishmortalitiesresultfrom a variety of -R centinVestilationsinTennessee have 'ruses, some natural and some man-induced. shown that the leaking of small amounis of very Natural fish kills are caused by phenomena such toxic chemicals from spent pesticide-Containing':' as , acute temperature change, storms, ice and barrels used as floats fol:Or piers and diving rafts in snow cover, decomposition of natural organic lakes and reservoirs can produce extensive fish materials,salinitychanges,pawning mortalf- kills. ties, and bacterial, parasitic, ad viral epidemics. Fish die of old age, but the number so af- Man-induced fishkills may bettributedto flicted at any one time is usually small. municipal orindustrialwastes\ agriculfural All po's'sible speed must be in con-,. activities, and water manipulations.-inter kills ducting-the initial phases of anyany fish kill investi- occur in northern areas where ice Ottshallow gation' because fish disintegrate rapidly -in hot lakes and ponds becomes covered with snoW,,weather and the cause of,death may disappear or andtheresulting opaqueness stopsphoto- become unidentifiable within minutes. Success synthesis. The algae and vascular plants die in solving a fish kill problem is usually related to, because of insufficient light, and their decompo- the speed with which investigators can arrive at sition results in oxygen depletion. Oxygen deple- the scene after a fish kill begins. The speed,or tion and extremepH variation can be caused'response iff.the initialiinvestigation is enhanced" also by, the respiration or decay of algae and",through the .training ualified- personnel who higher plants during summer months-in very will report iminediatelY the location of observed warm weather. Kills resultini froih such causes kills, the time that 'the kill was firstobserved, are often associated with 4selies of cloudy days the general kinds .of organisms affected, ap esti- (--\-5 that follbw a period of hot, dry, sunny-days. mate of the number of dead fish involved, and Occasionallyfish may be killed by toxins any unusual phenomena associated with the kill. released from, certain species of living or de- Because there is always the possibility of legal caying algaethat reachedhighpopulation liabilityassociated with afishkill, lawyers, densities because' of the increased fertility,j,e-,judges; and juries may scrutinize the investiga- sulting from organic pollution. tion report. The investigation, therefore, must Temperature chynges, either ,natural or the be made witi> great care. 12 FISH KILLS 6.0 REFgRENCES Blair, A, A. 1958. Back-pack shocker. Ca . Fish Cult. No. 23, pp.33-37. Bowers, C. C. 1955. Selective poisoning ofzard shad With rotenone. PrOg. Fish-Cult. 17(3):134-135. Cohen, J. M., Q., H. Pickering, R. L. Wo aril, and W. Van Heruyelen. L960. The effect of fish poisons on water supplies. JAWWA, 52(12):1551 -1566. Cohen, J. M., Q. H. Pickering, R. L. Woodward, and W. Van Heruvelen. 1961. The effect of fish poisons on water supplies,,JAWWA; 53(12)Pt. 2:49-62. DumOnt, W. H.,,and G. T. Sundstrom. 1961. Commerciaishing ger,of:theUnited.States. U.S. Fish and Wildlife Circular No. 109. U.S: Government Printing Office, Washington, D.C., 61 Fisher, K. C. 1950: Physiological considerations involved inlectrical methods of fishing. ad. Fish Cult. No. 9, pp. 26-34. Frankenberger, L. 1960. Applications of a boat-rigged direct-current shocker ontlakes an streams in west-central Wisconsin. Prog. Fish-Cult. 22(3):124-128. ....- Funk,..I. L. 1947. Wider application of electrical fishing method a collecting fish. Trans. Amer. Fish. Soc. 77 :49 -64. Haskell, D. c., and W.(E-Acleirnan, Jr. 1955. Effects of rapid direct current pulsations on fish,ew York Fish Game J.2(1.):95-105. Haskell, D. C., D. Geduldiz, and E. Snolk. 1955. An electric trawl. New York Fish Game J./(1) 120:125.. . Holton, G. D. 1934. West Virginia's electrical fish collecting methods. PrOg. Fish-Cult. 16(1):.10 = - Hooper, F. 19,60. Pollution control by chemicals and some resulting problems. Trans. SecOn Seminar on Biol. Problems in Water Pollution, April 20-24, USPHS, Robert A. Taft fan. Engr. Ctr., Cincinnati, p241-246.. Krumholz, L: A. 1948. The Use of Rotenone in Fisheries Research J. Wildl. Mgmt. 12(3):305.3 Larimore, R. W. 1961. Fish p4ulation and electrofishing success in a warm water stream. JW*. Mgmt. 25(1):1-12. Urn-i-mre, R. W, L. Durham, and G. W. Bennett. 1950.`54 modification of the electric fistocker forAzdee.*Nork. J. Wildl. Mgmt. 14(3):320-323. ' LattaW. C.; and G. F. Meyers. 1961. Night use of a p,c electric shocker to c Ilect trout in mer. Fish.Soc. 90(1):81-83. Lennon; R. E. 1959. The electrical resistivity in fishing, investigations. U.S. Fish Wildl. Serv., pt. Fish. No. 287, pp. 1-13. Lennon, R. E. 1961. A.fly-rod electrode system for electrofishing. Frog.- Fish-Cult. 23(2):92-93 Loeb, H. A. 1955. An electrical surface device for crop control and fish collection in lakes. NeYork Fish Game J, 2(2):220-221. McCrkunon, H. R., and A. H. Berst. 1961. A portable AC D C backpack fish shockersigned for operation in Ontario streams. Prog. Fish-Cult. 25(3):159-162. Mackenthun, K. M. 1969. The - practice of *water pollution biology. USDI, FWPCA; 281 pp. Mackenthun, K. M., and W. M. Ingrani. 1967. Biological associated problems in freshwater envinmments, their identification, investiga- tion and control. u,spi, FWPCA, 287 pp. wl for-sampling young fisheVin tidal rivers. Trans. North Massman, W. H., E. C. Ladd, and H. N. McCutcheon. 1952( A surface . %, . *mer. Wildl. Conf. 0:386-392. . - . Pratt, V. 5.'1951. A measure of the efficiency of alyrnating and direct current fish shockers. Trans: Amer. Fish-Soc. 81('0:63-68. RollefSon, Iit.,D: 1958." The,i deVelopment and evaluation of interrupted direct current eleotrofishing equipment. Wyo.Game Fish Dept.-, Coop.oProj..1so. 1. pp. 1-123. ' ° Rollefson, M. D. 1961. The development of improved electrofishing equipment. In: Proc.Fortkirst Ann. Conf. West.Assoc. St. Game and Fish Comm. pp.218-224. 4 4 Rdunsefell, G. A, W. H. Everhart. 1953. Fishery science:Its methods and applications. John Wiley and'Sons, New York. Seehorn, M. R. 1968. An inexpensive backpaCk shocker for one man use.Iln: Proc. 21st. Ann. Conf. SoutheasternAssoc. Game and , FisliComm. pp. 516-5241 . .. Stubbs, J. M. 1966.4yectrofishing, using a boat as the negative. In: Proc. 19th Ann. Conf. SoutheasternAssoc. GaMe and Fish Comm. pp. 236,245. Tebo, Jr., L. B. 1965.-Fish population sampling studies at water pollution surveillance system stations on the Ohio,-Tennessee,Clinch and Cumberland Riiers. Applications and development Report No.-15, Div. Water Supply and Pollution Control,USFHS. Cincinnati. 79 pp. " Thomas, N. 1969. Flavor of Ohio River channel catfish (Icialarus punctatus Raf.),USEPA.Cincinnati. 19 pp. i-. ..'w "Trent, W. L. 1967. Athichment of hydrofoils to otter boards for taking surface samples of juvenile fisharid iiiir Clies. Sci. 8(2):130-133. 7 13 BIOLOGICAL METHODS !!!*1 7.0 BIBLIOGRAPHY 7:1General References Allen, G. H., A. C. D'elacy, and D. Gotshall. 1960. Quantitative sampling, of marine fishes - A problem in fish behavior and fish gear. In: Waste Disposal in the Marine Environment. Pergamon Press. pp 448-511. American -Public Sealth Association et al. 1971. Standard methods for' the exariiipation of water and wastewater. 13th ed. APHA, . e New York. pp. 771-779, t - B(eder, C. M., and D. E. Rosen. Modes of reproduction in fishet. Amer. Mus, Natural History, Natural History Press, New York. 941 pp. - . Calhoun, A., ed. 1966: hand fisheries management. Calif. Dept. Fish,and Came, Sacramento. 546 pp. Carlander, K. D. 1969. Handbook of freshWater fishery; Life history data on 'freshwater 'of the U.S. andCanada, exclusive of the Percifornies, 3rd ed. Iowa State Univ. Press, Amts. 752 pp. Curtis, B. 1948. The Life Story of the Fish. Harcourt, Brace and Company,New.York.,284-pp. Cushing, D. 1-1.1968. Fisheries biology. A study in.population dynamics. Univ.; Wis. Press, Madison. 200 pp. FAO. 1964. Modern fishing gear of the World:. 2. Fishing News (gooks)' Ltd., London. 603 pp. Green, J. 1968. The biology of estuarine animals. Univ. Washington, Seattle.,-401 pp. Hardy, A. 1965. The. open sea. Houghton Mifflin C.ompany, Boston. 657pp. . A° Hynes,11. B. N. 1960. The biology of polluted water. Liverpool Univ. Press, Liverpool. pp. . . Hynes, H. B. N. 1970. The ecology of running waters. Univ. Toronto Press.-555 pp,- Jones, J. R. E. 1964. Fish and river pollution. Butterworths, London. 203 Pp. Klein, L. 1962. River pollution causes and effects. Butterworths, London. 456 pp. Lagler, K. F. 1966. Freshwater fisheries biology. William C. Rrown Co., Dubuque. 421 pp.- Lagler, K. F., J. E. Bardach, grid .R. R. Miller., 1962, Ichthyology. The study of fishes. John Wiley and Sons Inc., New York and London.'545 pp. r Madan, T. T. 1963. Freshwater ecology. John Wiley and Sons, New york. 338 pp. Marshall, N. B. 1966. Life of fishes. The-World fiubl. Cd., Cleveland and New Yorlc. 402 pp.. Moore, H. B. 1965. Marine ecology. John Wiley and Sons; Inc., New York. 493 pp. Reid, G. K. 1961. Ecology of inland' waters and estuaries. Reinhold Ffnbl. Corp., New York. 375 pp. "Ricker, W. E. 1958. Handbook of computatiqns for biological statistics of fish pc/Pfdations. FishRes. Bd, Can Bull. 19. 300 pp. . Ricker, W. E. 1968., Mtithods for,,theavessment offish produetfon in fresh water. International Biological Program Handbook No. Blackwell Scientific Puklications, Oxford and Edinburgh. 326 pp. Rounsefell, G.'A., and W. H. Everhart. 1953. Fishery science, its methods and applications. John Wiley & Sbn, New York. 444- pp:' Rpttner, F. 1953. Fundamentals of limnology. Univ. Toronto Press, Toronto. 242 pp. Warren, C. E. 1971. Biology and waterpollutiOncontrol. W. B. Saunders Co., Philadelphia. 434 pp. Welch, P. S. 1948. LimnolOgical methods. MCGraw-Hill, New Yokk. 381 pp. t. 7.2Electrofishing : Applegate,. V. C. 1954. Selected bibliography on applications of electricity in, fishery science. U.S. Fish and Wildl. Serv., Spec. Sci. Rept. fish. No. 127. pp: 1-55. ,Bailey, J. E., et al. 1955. A direct current fish shocking technique, Prog. Fish-Cult. 17(2):75. Burnet, A. M. R. 1959. Electric fishing with pulsatory electric current. New Zeal. J. Sci. 2(1):48-56. Burnet, A. M. R1961. Anelectric fishnig rna\chine_with piilsatory direct current. New Zeal. J. Sci. 4(1):1514-161. Dale; H. B. 1959. Electronic. fishing with underwater pulses. Electronics, 52(1):1-3. Elson, P. F. 1950. Usefulpess electiofishing methods. Canad. Fish Cult. No. 9, pp. 3 -12. Halsband, E., 1955. Untersuchungen uber die Betaiibungsgrenzimpulzahelii vor schiedener suswasser Eische.rchiv. fur Fishereiwis- senschaff, 6(1-2):45-53. Haskell, D. Cf 1939. An electrical method of collectin fish: Trans. Amer. Fish.Soc. 69:210-215. Haskell, D.'C.,1954. Electrical fields as appliedito the operatidn'of electric fish shciekers. New York Fish Game J. 1(2):13.6-176. Haskell, D. C., and R. G. Zilliox. '1940. Further developments'of the electrical methods of collecting fish. Trans. Amer. Fish!' 70:404-409. . FISH REFERENCES Jones, R. A. 1959. ModificatiOns of an alternate-polarity electrode. Prog. Fish-Cult. 21(1):39-42. Larkins, P. A. 1950. Use of electrical shocking devicei. Cinad. Fish. Cult., No. 9,pp. 21-25. e. Lennon, R. E., and P. S. Parker. 1955. Electric shocker developmentson southeastern trout waters. Trans. Amer. Fish. Soc. 85:234-240. Lennon, R. E., and P. S. Parker. 1957. Night collection of fishwith electricity. New York Fish Game'.4(1):109-118. Lennon, R. E., and P. S. Parker. 1958. Application's of ,salt in electrofishing. Spec. Sci. Rept., U.S. Fish Wildl. Serv.No. 280. Ming, A. 1964a. Boom. type electrofishing device for sampling fish populations in Oklahomawaters. Okla. Fish. Res. Lab., D-J Federal Aid Proj. FL-6, Semiann. Rept. (Jan -June, 1964). pp. 22-23. Ming, A. 1964b. Contributions' to a bibliogrphy on tho construction, development,use and effects of electrofishing devices. Okla. Fish. Res. Lab., D-J Federal Aid Proj. FL-6, Semiann. Rept. (lan.-June, 1964). pp: 33-46. Monan, C. E., and D. E. Engstrom. 1962. Devel'opcnent ofa mathematical relationship between electri-field pareters and the electrical characteristics offish. U.S. Fish,Wildl. Serv., Fish. Bull. 63(1):123-136. Murray, A. R.-1958. A direct current electrofishing apparatus using separate excitation. Canad.Fish Cult., No. 23, pp. 27-32. Northrop, R. B. 1962. Design ofa pulsed DC-AC shocker. Conn. Bd. Fish and Game, D-.1 Federal Aid Proj. F-25-R, lob No. 1. Omand, D. N. 1950. Electrical methods of fish oollection. Canad. Fish Cult. No.'9,pp. 13-20. Petty, K. C. 1955. An alternat5-polaity electrode. New Yorkish Game I. 2(1):114-119. Rylir, C. E. 1953. The electric shocker in Tennessee. Tenn. Gar1-ie Fish Comm: (Mimeo). 12 pp. '',...... --.* Saunders, J.,W., and M. W. Smith. 1954. The effective use of a direct current fish shocker ina Prince Edward Island stream. Canad. . Fish. Cult., No. 16, pp. 42-49. _ Schwarti; F. 1,1061. Effects of external forces on aquatic organisms. Maryland Dept. Res. Edu.,Chesapeake Biol. Lab.,Contr.,Nb. _.' 168, pp. 3-26. A . Snrtith, G. F. M., and P.-F': Elson. 1950. Ai,D.C. electrical fishing apparatus. tanad. Fish Cult.,No. 9, pp. 34-46.. Sullivan. C. 1956. Importance of size grodping in population estimates employing electric shockers. Prog.Fish -Cult. 18(4):188-1.90. Taylor, G. N. 1957, Galvanotaxic response of fish to pulsating D.C. 1,Wi1dl. Mgmt. 21(2):201-213. s \ . Thompson,RI B. 1959. The use of the transistorized pulsed directcurrent fish shocker in assessing populations 8f resident fishes. In: Proc. Thirtyninth Ann. Conf. West. Assoc. St. Fish and Game Comm.pp. 291-294. Thompson, R. B. 1960. Capturing tagged red salmon wir13,p.ulsed direct current. U.S. Fish Wildl. Serv., §0c.Sci. Rept. Fish, No. . 355, 10 pp. ..- , (-- Vibert, R., ed. 1967. Fishing with electricity- Its ipplications tbiology and management. European Inland Fish. Adv. Comm. FAO, United Nations, Fishing News (Books) Ltd. London, 276 pp. Webster, D. 4., 1. L. Forney, R. H. Gibbs, Jr., J. H. Severns; and W. F. Van Woert.1955. A comparison of alternating anddirect electric currents in fishery, work. New York Fish Game J. 2(1):106-113. . Whitney, L. V.. and R. L. Pierce. 1957. Eactors controlling the input of electrical energy intoa fish in an electrical field. Limnol. ---Oceanogr. 2(2):55 -61. - ti 73 Chemical Fishing Hester, F. E. 1959. Ttie tolerance of eight species of warm-water fishes to certain rotenone formulations. In: Proc. 13thAnn. Conf. Southeastern Assoc. Game and'Fish Comm. . Krurnholz, L. A. 1950. Some practical considerations in the use of rotenone in 'fisheries research. J. WildAgmt., vol.14. Lawrence, .1. M. 1956. Preliminary results on the use of potassium permanganate to counteract the effects ofrotenone on fish. Prog. Fish-Cult. 180 1:15.21. McKee, J. E., and H. W. Wolf, eds. 1963. Water quality,aiteria. 2nef ed: Calif. Wata Quality Control Board Publ. 3A. Ohio River Valley Water Sanitation Cornmission. 1962. Aquatic life resources of the Ohio River.pp. 72-84. Post, G. 1955. A simple chemicalLest for rotenone in water,Prog. Fish-Cult. 17(4):190-191. Post, G: 1958. Time vs. water temperature in rotenone"dissip,ation. In: Proc. 38th Ann. Conf. Game and FishComm. pp. 279-284. Solman, V. E. F. 1949. History and use of fish poisons in the United States. Dominion Wildlife Service, Ottawa.15 pp. Sowards, C. L. 1961. Safety /as related to the use. of chemicals and electricity in fisherymanagement. U.S. Fish and Wildl. Serv.,But:" Sport Fish and Wildl.. Brinch Fish Mgt., Spearfish. South Dakota. 13 pp.' "Donee, H.A., and M. L. Hayes. 1955. Evaluation of toxaphene as a fish poison. Colo. Coop. Fish. Res. Unit, Quar. Rep. 1(34):31-39. Turner. W. R. 1959. Effectiveness.Of various rotenone- containing preparations in eradicating farm pond,fish populations. Kentucky . Dept. Fish and Wildl. Res., Fish.13u11. No. '25, 22 pp. Wilkins, L. P. 1955. Observations on the field U tie of cresol as a stream-survey method. Prog. Fish-Cult. 17:85-86. 15 BIOLOGICAL METHODS 17A Fish Identification General: - Bailey, R. M., et al. 1970. A list of common and scientific names of fishesfroni the United States and Canada. 3rd ed. Spec. Publ. Amer. Fish. Soc. No. 6. 149 pp. Blair, W. F., and G. A. Moore. 1968. Vertebrates Of Th. 'tied States. McGraw Hill, New York. pp. 22-165. Edp, S. 1957. How to know the fresh-water fishes. C. Brown Co., Dubuque. 253 pp. Jordan, D. S., B. W. Evermann, and H. W. Clark. 195 Check list of the fishes and fish like vertebrates of North and Middle America north of the northern bbundary of Venezuela and Colom . U.S. Fish Wildl.Ser., Washington, D.C. 670 pp. LaMo nte, F. 1958. North American game fishes. Doubleday,arden City, N.Y. 202 pp. Morita, C: M. 1953. Freshwater fishing in Hawaii. Div. Fish Game. Dept. Land Nat. Res., Honolulu. 22'pp. Perlmutter, A. 1961. Guide to marine fishes. New York Univ, Press, Nesit. York. 431 pp. Scott, W. B., and E, J. Crossz41969. Checklist of Canadian freshwater fishes with keys of identification.Misc. Publ. Life Sci. Div. Ontario Mus. 104 Thompson, J. R., and S. Springer. 1961. Sharks, skates, rays, and chimaeras. Bur. Comm. Fish., Fish Wildl.USDI Circ. No. 119, 19 pp. Marine: Coastal Pacific Baxter, J. L. 1966. Inshore fishes of California. 3rd rev. Calif, Dept. Fish Game,'Sacramento. 80 pp. Clemens, W. A., and G. V. Wilby. 1961. Fishes of the Pacific coast of Canada. 2nd ed. Bull. Fish.Res. Bd, Can. No. 68.443pp. McAllister, D. E. 1960. List of the marine fishes of Canada. Bull. Nat. Mus. Canada No. 168; Biol.Ser. Nat. Mus. Can. No. 62. 76 pp. McHugh, J. L. and J. E. Fitch. 1951. Annotated list of the clupeoid fishes of thePacific Coast from Alaska to Cape San Lucas, Baja, California. Calif. Fish Game, 37:491-95.. Rass, T. S., ed. 1966. Fishes of the Pacific and Indian Oceans;Biology and distribution. (Translated from Russian). Israel Prog. forSci( Translat., IPST Cat. 1411; TT65-50120; Trans Frud. Inst.Ok-eaual. 73. 266 pp. Roedei. P. M. 1948. Common marine fishes of California. Calif.Div. Fish Garne Fish Bull.No. 68. 150 pp. Wolford, 4 A. 1937. Marine game fishes of the Pacific Coast from Alaska to theEquator. Univ. Calif. Press, Berkeley. 205 pp. Atlantic and Gulf of1111co Ackerman, B. 1951. Handbook of fishes of the Atlantic seaboard. AmeriCan Publ. Co.,Washington, D.C. Bearden, C. M. 1961. Common marine fishes of South Carolina. Bears Bluff Lab. No.34, Wadmalaw Island, South Carolina. Bigelow, H. B., and W. C. Schroeder. 1953. Fishes of the gulf of Maine. Fish. Bull. No.74. Fish Wildl. Serv. 53:577 pp. Bigelow, II. B. and W. C. Schroeder. 1954. Deep water etasmobranchs and chimaeroidsfrom the northwestern slope. Bull. Mus. Comp. tool. Harvard College, 112:37-87. Bohlke, J. E.,and,C. G. Chaplin. 1968. Fishes of the Bahamas and adjacenttro,pical' waters:Acad. Nat. Sci. PifiLodelphia. Livingston Publishing Co., Wynnewood, Pa. Breder, C. M., Jr. 1948. Fiela book of marine fishes of the Atlantic Coast fromLabrador to Texas. G. P. Putnam and Sons, New York. 332 pp. Casey, J. G. 1964. Angler's guide to -sharks,ttf-the northeastern UnitedStates, Maine to Chesapeake Bay. Bur. Sport Fish. Wildl. Cir. No. 179, Washington, D.C. Fishes of the western North Atlantic.14948-Mem..Sears Fdn., Mar. Res. 1. Hildebrand, S. It , and W. C. Schroeder. 1928: Fishes of Chesapeake Bay. U.S. Bur. Fish. Bull.43:1-366. Leim, A: H., and W. B. Scott. 1966. Fishes of the Atlantic Coast of Canada. Bull.Fish. Res. Bd. Canada. No. 155. 485 pp. McAllister, D. E. 1960. List of the marine fishes of Canada. Bull. Nat. Mui-. CanadrNo. 168; Biol. See. Nat. Mus. Can.No. 62. 76 pp. Pew, P. 1954. Food and game fishes of the Texas Coast? Texas Game Fish Comm. Bull. 33. 68 pp. Randall, J. E. 1968. Caribbean reef fishes. T. E. FL Publications, Inc.,Jersey City. Robins, C. R. 1958. Check list'of the Florida game and commercialmarine fishes, including those of the Gulf of Mexico and the West Indies, with approved common names. Fla. State Bd. Conserv. Edue. Ser. 12.46 pp. Schwartz, F. J. 1970. Marine fishes common to North,Carolina. North Car.Dept. Cons. Develop., Div. Comm. Sport Fish: 32 pp. Taylor, H, F. 1951. Survey of marine fisheries of North Carolina. Univ. North Car. Press, ChapelHill. 16 1 FISH REFIRE NCES Freshwater: Northeast Bailey, R. M. 193g. Key to the fresh-water fisheiof New Hampshire.'4n:-The fishes of the Merrimack Watershed. Biol. Surv. of the Merrimack Watershed. N. H. Fish'Game Dept., BioL Surv. A:ept. Bean, T,H. 1903. Catalogto of the fishes of New'York. N. Y. State Mus. Bud. 60. 784 pp. Carpenter, R. G., and H. R. Siegler. 1947: Fishes of New Hanipshire. N.H. Fish Game Dept. 87 pp. Elser, H. J. 1950. The common fishes of MarylandHow to tell, them apart. Publ. Maryland Dept. Res. Educ.,No. 88. 45 pp. Everhart, W. H. 1950. Fishes of Maine. Me. Dept. Inland Fish Game. (ii). 53 pp. Greeley, J. R., et al. 1926-1940. (Various papers on the fishes of New York.) In: Biol. Surv. Repts, Suppl. Ann. Rept., N.Y. St. Gons. Dept. McCabe, B. C. 1945. Fishes. In: Fish. Sur. Rept. 1942. Mass. Dept. Cons,pp. 30-68. Van Meter, H. 1950. Identifying fifty prominent fishes of West Virginia. W. Va. Cons. Comm. Div. Fish Mgt. No. 3. 45 pp. Whiteworth, W. R., R. L. Berrieu, and W. T. Keller. 1968. Freshwater fishes of Connecticut. Conn. State Geol. Nat. Hist.'Surv. Bud. No. 101. 134 pp. Freshwater: Southeast Black, J. D. I 940..The distribution of the fishes of Arkansas. Univ. Mich. Ph.D. Thesis. 243 pp. Briggs, J.C:1958. A list of Florida fishes and their distribution. Bull. Fla. State Mus. Biol. Sci. 2:224-318. Carr, A. F., Jr. 1931. A key to the freshwater fishes of Florida. Proc. Fla. Acad. Sci. (1936):72-86. ) Clay, W. M. 1962. A field manual of Kentucky fishes. Ky. Dept. Fish Wildl. Res., Frankfort, Ky. 147 pp. Fowler, H. W. 1945. A study of the fishes of the southern Piedmont and coastal plain. Acad. Nat. Sci., Philadelphia Monogr. No. 7. 408 pp.. Gowanlock, J. ls.l.-1933. Fishes and fishing in Louisiana. Bud. La. Dept. Cons. No: 23. 638 pp. ,Heemstra, P. C. 1965. A field key to the Florida sharks. Tech. Ser. No. 45. Fla. Bd. Cons., Div. Salt Water Fisheries. King, W. 1947. Important food and game fishes of North Carolina. N.C. Dept. Cons. and 54 pp. Kuhne, E. R. 1939. A guide to the fishes of Tennessee and the mid-South. Tenn. Dept. Cons., Knoxville. 124 pp. Smith, H. 1970. The fishes of North Carolina. N.C. Geol. Econ. Surv. 2:x1;453 pp. Smith-V;>./7 W. F. 1968. Freshwater fishes of Alablina. Auburn Univ. Agr. Exper. Sta. Paragon Press, Montgomery, Ala. 211 pp. Freshwater: Midwest ,s Bailey, R. M., and M. 0. ARum. 1962. Fishes of South Dakota. Misc. Publ. Mus. Zool. Univ. Mich. No. 11 9.131 pp. Cross, F. B. 1967.,Handbook of fishes of Kansas. Misc. Publ. Mus. Nat. Hist. Univ. Kansas No. 45. 357 pp, Eddy, S., and T. Surber'. 1961. Northern fishes With special refernce to the .Upper Mississippi Valley. Univ. Minn. Press, Minneapolis. 276pp. Evernn, B. W., and H. W. Clark. 1920. Lake Maxinkuckee, a physical and biological suy. lnd. St. Dept. Cons., 660 pp. (Fishes, pp. 238-451). . Forbes, S. A.,and R. E. Richardson. 1920. The fishes of Illinois. Ill. Nat. Hist. Surv. 3 CXXXI. 357 pp. Gerking, S. D. 1945. The distribution of the fishes of Indiana. Invest. Ind. Lakes and Streams, 3(1):1-137. Greene, C. W. 1935. The distribution of Wisconsin Fishes. Wis. Cons. Comm. 235 pp. Harlan, J. R., and E. B. Speaker. 1956. Iowa fishes and fishing. 3rd ed. Iowa State Cons. Comm., Des Moines, 337 Hubbs, C. L., and G. P. Cooper. 1936. Minnows of Michigan. Cranbrook Inst. Sci., Bull 8. 95 pp. Hubbs, C. L., and K. F. tagler. 1964. Fishes of the Great Lakes Region. Univ. Mich. Press, Ann Arbor. 213 pp. Johnson, R. E. 1942. The distribunof Nebraska fishes. Univ. Mich. (Ph.D. Thesis). 145 pp. Trautman, M. B. 1957. The fisIOhio. Ohio State Univ. Press. Columbus. 683 pp. Vati Ooosten, J. 1957. Great Lakes.fauna, flora, and their environment. Great Lakes Comm., Ann Arbor, Mich. 86 pp. Freshwater: Southwest Beckman. W. C. 1952. Guide to the fishes of Colorado. (Jniv. Colo. Mus. Leafl. 11. 110 pp. Burr, J. G. 1932. Fishes of Texas; Handbook of the more important game and commercial types. Bull Tex. Game, Fish, and Oyster Comm. No. 5, 41 pp. Dill, W. A. 1944. The fe,gyy of the Lower Colorado River. Calif. Fish Game, 30:102 -211. LaRivers, I.,and T. J. Trelease.,1952. An annotated checklist of the fishes of Nevada. Calif. Fish Game,38(5:113-123. 17 1 r) BIOLOGIOAL METHODS Miller, R. R. 1952. Bait fishes of the Lower Colorado River from Lake Mead, Nevada, to Yuma, Arizona, with a key for their identification. Calif. Fish Game. 38(1):7-42. \1,9 Sigler, W. F., and R. R. Miller, 1963. Fishes of Uta ah St. Dept. Fish Game. Salt Lake City. 203 _ Walford, L. A. 1931. Handbook of common commercial and game fishes of California. Calif.Div:' Fish(ame FishBull.No. 28. 81 pp.. Ward, H. C. 1953. Know your Oklahoma-fishes. Okla. Game Fish Dept, Oklahoma'City. 4013P-S`' Freshwater: Northwest Baxter, G. T., and). R. Simon. 1970. Wyoming fishes. Bull. Wyo. Galpe Fish Dept. No. 4. 16a pp. Bond;C. E. f§61. Keys to Oregon freshwater fishes. Tech. Bull. Ore. Agr. Exp. Sta. No. 58.142 pp. Hankinson, T. L, 1929. Fishes of North Dakota. Pop. Mich. Acad. Sei. Arts, and Lett. 10(028):439-460. McPhail, J. D., and C. C. Lindsey. 1970. Freshwater fishes of Northwestern Canada ardl Alaska. Fish. Res. Bd. Canada, Ottawa. Bull. No. 173. 381 pp. - Schultz, L. P. 1936. Keys to the fishes of Washington, Oregon and closely adjoining regions. Univ. Wash. Publ. Biol. 2(4):103-228.' Schultz, L. 1941. Fishes of Glacier National Park, Montana. USDI, Cons. Bull. No. 22. 42 pp. Wilimovsky, N. J. 1954. List of the fishes of Alaska. Stanford ichthyol. Bull. 4:279-294. 7.5Fish Kills Alexander, W. B., B. A. Southgate, and R. Bassindale. 1935. Survey of the River Tees, 'Pt. 11. The Estuary, Chemical and Biological. Tech. Pop. Wat. Pol. Res., London, No. 5. Anon., 1961. Effects of Pollution on Fish. Mechanism of the Toxic Action of Salts of Zinc, Lead and Copper. 'Water .Pollution Research, 1960:83. Burdick, G. E. 1965. Some problems in the determination of the cause of fish killS. In: Biological Problems in Water Pollution. USPHS Pub. No. 999-WP-25. Carpenter, K. E. 1930. Further researches on the action of soluble metallic salts on fishes. J. Exp. Biol.S6:407-422. Easterday, R. L., and R. F. Miller. 1963, The acute toxicity of"molybdenum to-the bluegill. Va. J. Sci. 14(4):199-200. Abstr. Ellis, M. M. 1937. Detectionand measurement of stream pollution. Bull. U.S. B-4. Fish. 48:365-437. Extrom, J. A., and D. S. Farrier. 1943. Effect of sulfate mill wastes on fish life.. Paper Trade J. 117(5):27-32. Fromm, P. O., and R. H. Schiffman. 1958. Toxic action of hexavalent chromium on largemouth bass. J. Wildlife Mgt. 22:40-44. Fujiya, M: 1961. Effects of !craft pulp mill wastes on fish. JWPCF, 33(9):968-977. ,. Havelka, J., and M. Effenberger. r957. Symptoms of phenol poisoning of fish. Ann. Czech. Acad. Agric.Sci., U. Serv. Animal Prod. 2(5):421. Henderson, C., Q. H. Pickering, and C. M. Taxzwell. 1959. Relative toxicity of ten chlorinated hydrocarbon insecticides to four species of fish. Trans. Amer. Fish. Soc. 88\23-32. Mgtam, W., and G. W. Prescott. 19541. Toxic freshwater algae. Amer. Mid. Nat. 52:75. ( Joshes, J. R. E. 1948. A further study of the reaction of fish to toxic solutions. J. Exp. Biol. 25:21-34. ' Kuhn, 0., and II. \V. Koecke. 1956. Histologische and cytologische Veranderungen der fishkierne nach Einwirkung iin wasser enthaltener schaaigender Substanzen. Ztschr. F. Zellforsch. 43:611-643. (Cited in Fromm and Schiffman, 1958.) Mathur, D. S. 1962a. Histopathological changes in the liver of certain fishes as induced by 1311C and lindane. Proc. Natl. Acad. Sci. India, Sec. B, 32(4):429-434. Mathur, D. S. 1962b. Studies on the histopathological changes ,induced by T in the liver, kidney and intestine of certain fishes. Experientra, 18:506. Rounsefell, G. A., and W. R. Nelson. 1966. Red-tide research summarized to 1964, including an annotated bibliography. USD1 Special Sci-Rept:, Fisheries No. 535. _, . Schmid, 0. J., and H. Mann. 1961. Action of a detergent (dodec'yl benzene sulfonate) on the gills of the trout. Nature, 192(4803):675. Shelford, U. E. 1917. An experimental study of the effects of gas wastes upon fishes, with special reference to stream pollution. Bull. Ill. Lab. Nat. Hist. 11:381-412. Sluapek, K. 1963. Toxicity of phenols and their detection in fish. Pub. Health En . Abs,ts. XL1V( Abst. No. 1385. Smith, L. L., Jr. et al. 1956. Procedures for investigation of fish kills. A guide fo field reconn issance and data collection. Ohio River\ Valley Water Sanitation Comm., Cinciqiti. Stansby, M. E. 1963. Industrial fishery technology. Reinhold Publ. Co.,New York. Stundle, K. 1955. The effects of waste waters from the iron industry and mining on St*ian waters. Osterrich Wasserw. (Austria). 7:75, Water Poll. Abs. 29:105. 1956. 18 FISH REFERENCES US. Department of the Interior. 1968a. Pollution caused fish kills 1967. FWPCA Publ. No. CWA-7. US. Departthent of the Interior. 1968b. Report of the National Technical Advisory Commission. FWPCA, Washington, D.C. JJ US. Department of the Interior. 1970. Investigatifig fish mortalities. FWPCA Publ. No. CWT-5. Also available from USGPO as No. 0-380;257. Wallen, L E. 1951. The directeffectof turbidity on fishes. Bull. Okla. Agr. Mech. Coll. 48(2):1-27. Wood, E. M. 1960. Definitive diagnosis of fish mortalities. JWPCF, 32(9):994-999. , s 4 L9 BIOASSAY ,r BIOASSAY Page .'i 1.0: GENERA': CONSIDERATIONS 1 2 2.0 PHYTOPLANKTON ALGAL , 2.1 Principle 2 2.2 Planning Algal Assays 2 . 3 -2.3- Apparatusand Test' Conditions, 2.3.1 Glassware 3 2.3.2 Illumination 3 2.3.3 PH 3 2.4 Sample Preparaiion 3 2.5 Inoculum 3 2.6 Growth Response Measurements 3 2.7 Data Evaluation 4 5 2.8 Additions,(Spikes), 2.9 Data Analysis andInterpretation 5 2.10 Assays to Determine Toxicity s 5 :,. ... 3.0 PERIPHYTON 5 3.1 Static ,4 5 3.2 Continuous Flow 6 4.0 MACROINVERTEBRATES 8 5.0 FISH 8 6.0 REFERENCES 8 6.1 General 8 6,2 Phytoplankton Algal Assay 9 .---- 6.3 PeriphytOn 10 6.4 Macroinvertebrates 10 6.5 Fish 11 FATHEAD MINNOW CHRONIC TEST 1 BROOK TROUT CHRONIC TEST . -J 0 BIOASSAY 1.0 GENERAL CONSIDERATIONS The 48- and 96-hour LC50 valuesare 4tt, presently important for determining com- The term BIOASSAY .includes anytest in pliance with water quality standards as which organisms are used to detect or measure established by various pollution control the presence or effect of one or more substances. authorities. ,Short-termthresholdinfor- or conditions. The organism responses measured mation can tie derived by reporting LC50 in these test's include: mortality, growth rate,n values at 24-hour intervals to depyonStrate standing crop (biomass), reproduction, stimu- the shape of the toxicity curve. .lationor inhibition ,of 'metabolic or enzyme systems, changes in behavior, histopathology: Reports of LC50's should state the method and flesh tainting (in shellfish and fish): The of calculation used and the statistical con- ultimate puipose of bioassays is to predict the fidence limits when possible.t, response of native populations of aquatic organ- ismsto specific changes within thenatural. Rubber or plastic materials should be used environment. Whenever possible, ,therefore, tests° in bioassay equipment only after consider- should be carried out with species that are native ation has been given to the possibility of (indigenous) to the receiving water used as the the leaching of substances such as plas- diluent for the bioassay. Bioassays are important p, ticizers or sorption of toxicants. because in most casethe success of a water pollption control program must be judged in Test .materials should be aglministerpd in terms of the effects of water quality on the con- such a way that their physical and chemical ,dition of the indigenous communities of aquatic behavior approximatesthatinnatural. organisms. Also, in many cases, bioassays are systems. more sensitive than chemical analyses. Two general kinds of bioassays are recog- nized Biological tests can be cond cted in any kind of water with proper pr tons, and although most tests haye beenonducted in freshwater, laboratory tests conducted to determine the the same general principles apply to brackish effects-of a substance on a species; more or and 'salt waters: The literature contains a great less arbitrary conditions are employed; many formulations for artificial seawater. Of in situ tests conducted to determine the thes,,a modification- of the Kester et Q. (1967) effects of a specific. natural environment; formulation (LaRoche et al., 1970;Zaroogian,et the test organisms are held in "containers" al., 1969) seems to support the greatest variety through which the water circulates freely. of marine organisms. When metal-Containing wastes are to be bioassayed, omitting EDTA and controlling trace metals, as described by Davey Thee ral principles and methods of con- et al. (1970), is recommended. ducting. la ratorybioassayspresentedin Standard Methods fore Examination of Water Using a standard toxicant and a parallel 'series and Waste Water, 13th APHA, 1931) a standard medium is recommended to help applyto most bioassays, and the des d aess variations due to experiMental technique methods can be used with many types of aquati anthe condition of the organisms. Such tests organisms with on19,,,slight modification. aralso useful in distinguishing effects due to an The following are suggested improvements to tered character of the effluent from changes the methods given in Standard Methods, 13th inthe sensitivity of the organism, or from edition (APHA, 1971). clyges in the quality of the receiving water. ...BIOLOGICAL METHODS When making waste management decisions, it of thewaste discharge. A bioassay ,trailer is important' to consider and tentatively define(Zillich, 1969) has proven useful for this pur- the persistence of a 'pollutant. Materials that pose. In situ bioassay piocedures are also agood, have half lives less than 48 hours can be termedmethod for defining the impact to aquatic life as rapidly decaying compounds;those with halfbelow the source of industrial waste discharges lives greater than 48 hours, but less than 6 (Basch, 1971). months, as sloWly decaying; and those com- Biomonitoring, a special application of biolo- pounds in natural waters with half lives longer gical tests, is the use of organisms .to provide C than 6 'months, as long-lived persistent materials. information about a surface water, effluent, or ; mixture t thereof on a periodic or continuing,. Bi assays can be conducted over almost any basis. Rif: the best results, biomonitoring shpuld inrval of time, but the test duration must be maintain. continuous 'surveillance with theukor propriate to the life stage or life cycle of the indigenous species ina flow-through system test organisms and the objectives of the investi- natufai` gation. The purpose of short-term tests, such asunder conditions that approximate the acute mortality tests, is to determine toxicant environment. concentrations lethal to a given fraction (usually 50 percent) of the organisms during a short 2.0 PHYTOPLANKTON ALGAL ASSAY period of their life cycle. Acute mortality tests The Algal Assay Procedure: Bottle Test was with fish generally last about 4 to 7 days. Most published by the National Eutrophication Re- toxicants, however, cause adverse effectsat search Program (USEPA, 1971) after2 years of levels below those that cause mortality. To meet intensiveevaluation,during which excellent this need, long-term (chronic) tests are designed agreement of the data was obtained among the8 to expose test organisms to the toxicant overparticipating laboratories. This test is the only their entire life cycle and measure-the effects of algal bioassay that has undergone sufficient eval- the toxicant on survival, growth, and reproduc-uation and refinement to be considered reliable. tion. Sometimes only a portion of the life cycle The following material represents only a briZIf is tested, such as studies, involving growth or outline of the test. FOr more explicit details, see emergence of aquatic insects With fish, such the references. tests usually last for 30, 60, or 90 days and are often termed subacute. 2.1Principle Laboratory bioassays may be conducted, on a An algal assay is based on / the principle that "static" or "continuous flow" basis. The specific growth is limited by the nutrient that is present needs of the investigator and available test facil- in shortest supply Itith respect to the needs of ities, determine which technique should be used. the organism. The test can be used to identify The advantages and applications eif-ch have algalgrowth-limiting nutrients,to dairmine been ,described in Standard-Metliods, (APHA, biologicallythe availability of algal, growth= 1971) and by the National Technical Advisory limiting nutrients,to quantify the1;iological Committee (1968). Generally, the continuous- response (algal grOwth response) to changesin _flow technique should, be used where possible. concentrationS) of algal growth- limiting nutri- -Apparatus advantageous for conducting flow- ents, /and to determine whether or not various throu-ghtestsincludesdiluters (Mount and compoundor water samples are toxic or inhib- Warner, 19657 Mount and Brungs, 1967), valve itory to algae. controlling systems (Jackson and Brungs, 1966) and chemical metering, pumps (Symons, 1963). 2.2 Planning Algal Assays The biologicaleffectsof ,many industrial The specific experimental design of each algal wastes are beSt evaluated in the field; trans- assay is dictated by the.particularproblem to be porting large volumes of industrial wastes to a solved. All pertinent ecological factois must be laboratory for bioassay purposes can be imprac- considered in planning a given assay to, ensure tical. Testing facilities are best located at the site that valid results and conclusions are obtained. O 2 . ALGAL ASSAY 'Water quality may vary greatbi with time and you wish to determine the growth response locatiOn in lakes, impoundments and streams. If - to growth-limiting nutrients which have not meaningful data are to be obtained, therefore, been -taken up by filterable organisms, or if the_sampling program must take these variations you wish. to predict the effect of adding into account. nutrients to a test water at a specific time. autocraving autoclave samples if you wish 2.3 Apparatus and Test Conditions to determine th amount of algal biomass 2.3.1Glassware that cap be growh from all nutrilts in the water,,.inciuding .ose in -thelankton. Use good - quality borosilkate glasswar'e. When Autoclaving iOlubilizes the nutrients in the -studing trace nutrients, use special glassware indigenous filterable organisms and releases. such as Vycor or polycarbonate containers: them for use by the test organisms. Although "container size is not critical, the sur- face to Volume, ratios are critical 1?ecause of 2.5 Inoculum 4 possible carbon limitation. The recommended The algal test species may be one of .those sample volumes for use in Erlenmeyer flasks are: recommended in the Bottle Test or anothei that 40 ml in v125'1111 flask; 60 ml in a(250 ml,flask; has been obtained in unialgal culture. Grow the and 100 in a 500 ml flask: Use culture test, species in a culture medium that minimizes __ckl,sures....such...as loose-fitting aluminum foil or the intracellular carryover of nutrients in the inverted beakers to permit good gas exchange test species when transferred from the stock and prevent contamination. culture to the test water (Table I.) When taken from the stock culture, centrifuge the test cells Illumination and discard the supernatant. Resuspend the After inoculation, incubate the flasks at 24 ± sedimented cells in an appropriate volume of 2°C under cool-white fluorescent lighting: 200 glass-distilled water containing 15 mg, sodium ft-c (2152 lux) ± 10 percent for blue-green algae bicarbonate per liter and recentrifuge. Decant and diatom test species, and 400 ft-c (4304 lux) the su.perna resuspend the algae in fresh ± 10 ,percent far green algae test species. Meas- bicarbonatsolu on, and use as the inoculum. ure the light intensity adjacent to the flask at The amount ofoculintidepend.S upon the algal the liquid level." test speCies us .The following initial cell con- 2.3.3 pH centrations ar recommended: To ensure the availability of carbon dioxide, Test organism Initial cell count/nil maintain the pH of the incubating cultures Selenastrum capricornutum 1000/m1 below 8.5 by Using .the sample (volumes men- A nabaena flos-aquae 50000/ml tioned above and shaking the cultures -at 100, Microcystis aeruginosa 50000/ml oscillations per minute. In samples containing high concentrations of nutrients, such as highly- Ptepare test flasks in triplicate. productife surface waters or domestic waste effluents, it may be necessary to bubble air or an 2.6 Growth Response Measurements air/carbon dioxide mixture through the culture The method used to determine growth re- to maintain the pH below 8.5. sponse during incubation depends on the equipment available:-Cells may be counted with 2.4 Sample Preparation amicroscope, -using a he;Inacytometer or a Two alteate methods of sample preparation Palmer-Maloney or Sedgwiek-Rafter plankton are recom ended, depending upon the type of counting chamber...The amount of algal biomass informatioto be obtained from the sample: may be determined. by measuring the optical density $),f the. culture at 600 - 50 nlh with a membr ne filtration (0.45 pore diameter) colorimeter or spectrophotome er. The amount remove the indigenous, algae by filtration if of chlorophyll contained in t e algae may be 3 , -t. BIOL09ICAL METHODS TABLE 1. STOCK CULTURE AND CONTROL NUTRIENT MEDIUM. MACROELEMENTS: Final , Element Element Compound concentration furnished concentration (mg /1) (mg/I) NaNO3 25.500 7411 N 4.200 K2HPO4 1.044 P 0.186 K 9X168 MgC12 5.700 Mg MgSO4.7H20 14.700 Mg 1.450 S 1,911 CaC12-2 H2 0 4.410 Ca 21.203 NaHCO3 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, K2HPO4 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.) MICROELEMENTS: Wg/1) (Pg/l) H3B03 185.64 B 33 MnCl2 264.27 Mn 114430 ZnCl2 32.70 ° Zn - 15 CoCly 0.78 Co, 0.35, CUC12 0,009 Cu 0.003 Nat MO 04 -2 H2 0 7.26 Mo, "1 2.88 FeCl3 96 Fe 33 Na2EDTA2H20 333 C-) measured either directly (invivo) by fluoro- averaging the /Amax of the individual flasks. The metry or, after extraction by fluorometry or specifif growth rate,p,is defined by: spectrophotometry. If available, an electronic particle counter will provide an accurate and In(X2/X1) rapid count of the cells. All methods used for 12ti determining the algal bioniass should be relatedwhere: to a dry weight measurement (mg/1) determined in = log to the base "e" gravimetrically. (See the Plankton Section of the X2 = biomass concentration at the end of the manual. for analytical details.) selected time interval X1, = biomass concentration at the beginning 2.7 Data Evaluation of the selected time interval Two parameters bra used to describe the t2 7 t1 = elapsed time (days) betIVeen selected ;growth of-a test alga: maximum specific growth . 'determinations of biomass rate and maximum standing crop. The maximum Because .the maximum specific growth rate specific growth rate (lmax) for an individual (Amax) occurs during the logarithmic phase of flaskisthe largestspecific growth irate (p)growth (usually betkveen day 3 and day 5), the occurring at any time during incubation. The biomass must be measured at least daily. during pmax ,for a set of replicates is determined by the first 5 days of incubation. 4 ALGAL ASSAY The maximum standing crop in any. flask is specific growth rate 4Or each flask', and com- defined as the maximum algal biomass achieved paring the averages by a Students' t-test or other dining incubation. For practical purposes, the appropriate statistical tests. maximum standing crop is assumed to have been Data analysis for multiple nutrient spikingCan achieved when the rate of increase in biomass be performd .by analysis of variance, calcu- has declined to less than 5 percent per day. lations.. In multiple nutrient spiking, accounting for the possible interaction between different 2.8 Additions (Spilces) nutrients is important and can readily be done The quantity of cells produced in a given by factorial analysis. The smile methods de- medium is limited by the nutrient present in the scribed above can be used to determi?ie the lowest relative quantity with respect to the nutrient limiting growth of the maximum needs of the organism.If a quantity of the standing crop. limiting substance were added to the test flasks, cell prdrduction would increase until this addi- 2510 Assays to Determine-Toxicity tional supply was depleted or until some other As previously pointed out, the assay' may be substance became limiting to the organism. used to determine Whether-Ornot various com- Adding substances other than the limiting sub.) pounds or water samples .are eith'er toxic or stance would not increase algal growth. Nutrient inhibitory to'algal growth. In this bagerwthe sub- additions may be made singly or in combination, stance to be. tested for toxicity is added to.the and the growth response can be compared with standard algal culture medium in varying con- that of unspiked controls to identify, those sub- centrations, the algal- test species is 'added; and stances that limit growth rate or cell production. either the maximum standing crop or maximum In all instances, the volume of a spike should specific growth rate (or both) determined. These be as small as possible. The concentration of are then compared to those obtained in the spikes *ill vary and' must be matched to the standar 5 .1'12 BIOLOGICAL METHODS this method is recommended for static bioassays a paddlewheel.Duplicate chambers should with the periphyton. A be prcividedfor each conditiontested (Figures 1 and 2). 3.2 Flow }Continuous Current velocity 30 cm/sec: Any periphyton grow well only in flowing TeMperature 20°C water and can be studied only in situ or in arti- Light 400 fc,cool-white(daylight) ficial streams (Whitford, 1960; Whitford et al., fluorescent lamps 1964). The following procedure, which is similar , Culture mediumOptional to the method described by McIntire et al. a. Algal Assay-Medium (Table 1). (1964), is tentatively recommended at this time. b.'Natural surface water supply Where direct flaw-through is not Test Chamber , Twin, inter-connected the water exchange rate should channels, each approximately 4" 4" X . ensure a complete change at least six 36", with t'inches of water circulated by times daily. WATER SOURCE FLOWMETER -WATER SUPPLY CONTROL VALVE 2.5ddr-- I.D. RUBBER TUBING SCREW CLAMP PAPULE WHEEL 50 cm DIA. ELECTRIC VARIABLE SPEED MOTOR /' 441'11 TRAYS OVERFLOW 7 Icm I.D. TROUGH-DIMENSIONS RUBBER INSIDE WIDTH = .25cm TURING 1 INSIDE LENGTH = -Or INSIDE DEPTH 20Cm PINCH CLAMPS NPUT AND OUTPUT SAMPLE BOTTLES DRAIN' FLUM Figure 1. Diagrarh of laboratory stream, showing the padd e wheel for circulatiitrtlie water between the two interconnected troughs and the exchange water system. (From McIntire et al., 1964). PEitIPHYTON BIOASSAY 121tm FILTERED WATER SUPPLY LINE 25cm ANGLE IRON SCREW CLAMP ADJUSTABLE HEIGHT W ODD STRIP 2 gn.Fb TYe I,TUBING 1500 WATT . 6mm LUCITE TOP" INCANDESCErIT LAMP Jcm HARD RUBBER GASKET 3.2cm I. D. POLYETHYLENE Y PIPE LIP OF CHAMBER , WODO STRIP OVERFLOW HEAD it . SIDE 'VIEW 2cm X 3mm IRON STRIP JAR , 15 mm Y- SHAPED WASTE WATER WATER JACKET OVER FLO 11P CONNECTING CO2 COLUMN TUBES, 2cm IWO - STAA E TYGON PRESSURE REDUCING VALVE .CUBING GLASS RASC/i10 UBBER STO ERS STOPCOCK RINGS CHAMBER DRAIN CHAMBER - DIMENSIONS WATER1JACKET INSIDE WIDTH m,,513m C(1:14LDGA4GcELLED INLET AND OUTLET INSIDE LENGTH = 60 tm STEEL. OXYGE N ADJUSTABLE PORCELAIN STRIPPING HEAD CONTROL INSIDE DEPTH 17 cm COATED; COLUMN I50 mm. 2cm MARINE PLYWOD' WATER JACKET //' 'FLOWMETE I. D. X 1.5 LONG INPUT , ' SAMPLE BOTTLE 2cm f.D. TYGON PORCELAIN . TUBING COATED STEEL TRAYS-. CENTRItUGAL . 1 PUMPS CARBON DIOXIDE GAS ---- CYLINDER NITROGEN \ DIFFUSER. Jt PLAN VIEW' C AMBER BY- PASS NITROGEN GAS 6min RUBBER TUBING SCREW CLAMPS SCREW CYLINDER CSILABOTTLE,IE:trk INSTRUMENT PORT CLAMP Scm I D 75cm LG 2cm I.D. TYGON TUBING WITH RUBBER CHAMBER DRAIN STAINLESS STEEL STOPPER CURRENT DIFFUSION CHAMBER Figure 2. Diagram of photosothesis-respiration chamber, showing the chamber with its. circulating and -ex- change water systems, the war& jacket fbr temperaturirrtrol, the nutrient and gas'concentration control system, and the light source:, .Test organism(s) -' Optional; filamentous Test duration Two ,-weeks blue-green or green algae o.djatoms. ' ' Evaluation The effectsof thetest a: UnialgalCulture No standardtest condition are evaluated \it the end of the -organisms are available' test period by comparing the biomass and b. Periphytonb,community Use "seed" of. community structure in the test chambers periphyton froM the water cresource for with that of the; control chambers. (See; which the data are being developed. Periphyton Section for Filettiodology. Acclimatization Period The culture (or community) .should be allowed to develop a. Biomass . Use four of the eight slides; in the test. chambers for a minimum of two analyze individually., Weeks before introdUcing the, test condi- (1) Chlorophyll a (mg /m?.) tiori: (2) Organic matter (Ash-free weight, Maintaining test conditions Chemicals are g /m2) added to the water supply prior to flow b: Cell count and identilication Use four into the test chamber. Temperature control pooled slides.. rriajt be maintained by placing thermostat- (1) Cell density (cellS/mnri2) icallycontrolled 'heating(orcooling)- (2) Species propottional'count. elenien s in the channel. (3) Community diVersity(Diversity I Substrto " A minimum of eight I ". )( 3". Index). plain ss slides should be,,praced on the "Toxicity The toxicity of a chemical or bottom(.each channel. effluent is eXpre ed as the LC50, whiCh is BIOLOGICAL METHODS the 'concentration of toxicantresultdgin a and in the report of the National Technical 50% reduction in the biomass or Al count. Advisory Committee (1968): Sprague (1969, Community diversity is not affected in the 1970) has recently reviewed many of the prob- 'same manner as ,biomass and cellocOunts,.lems and the terminology associated with fish and would yield a much different value. toxicitytes Chronictestsare becoming increasingly 4.0 MACROINVERTEBRATES important as sublethal adverse effects of more and more toxic agents are found to be signifi- In general, most of the considerations covered cant. At present, a chronic fish bioassay test is a by, Standard Methods (APHA, 1971) apply relatively sophisticated research procedure and equally well to macroinvertebrate tests in fresh entails large allocations of manpower, time, and and marine waters. Recent refinements in acute expense. Important contributions in, this area and chronic methodology for ,aquatic insects, include those by Mount and Stephdn' (1969), amphipods, mussels, andDaphniahave been Brungs (1969), Eaton (1970), and McKim,et4/ described by Gaufin (1971), Bell andNebeker (1971). (1969), Arthur and Leonard (1970), Dimic,k and Two procedures for chronic toxicity tests Breese (1965), Woelke (1967), and Biesinger and using the fathead minnow,Pimephales promelas Christensen (1971), respectively.. Rafinesque,- and the brook trout,Salvelinus fontinales(Mitchell), developed by the -staff of 5.0 FISH, N Water Quality Laboratory, U.S. The general principles and methods for ,cute Envirmental Protection Agency, Duluth, and chronic laboratory fish toxicitytestare Minn., are presented following the references i presented in Standard MethodslAPHA,- 1971) this section. 6.0 REFERENCES 6.1General American Public Health Association. 1971. Standard methods for the examination of water and wastewater.13th ed..Amer. Public Health Assoc., New York. 874 pp. . . . Basch, R: 1971. Chlorinated municipal waste toxicities of rainbow trout and fatheadminnows. Mich. Bur. Water Mgtnt, Dept. Nat. Res., Lansing, Mich. Final Report of Grant Number 18050 GZZ feit the U.S. EnvironmentalProtection Agency. . Davey, E. W., J. H° Gentile, S. J. Erickson, and P. Betzer. 1970. Removal of trace 'metalsfrom marine culturg.media. Limnol. Oceanogr. 15:486-488. .-, * Jackson, H. W.; and W. A. Brungs. 1966. Biomonitorin4, of industriaTeffluents. Proc. 21st Ind. WasteConf., Purdue Univ., Eng. Ext. Bull. No. 121. pp. 117. , Kester,'D. R., I. W. Duedall, D. N. Connors, and R. M. Pytkowicz. 1967. Prepation of artificial seawater. Limnol.Oceanogr. 12W:176-179. LaRoche, G., R. Eisler, and C. M. Tarzwell. 1970. Bioassay pr cedures/ forOil and oil dispersant toxicity evaluation. JWPCF, '42:1982-1989. 4 r'-'1 Mount, D. I., and R. E. Warner. 1965. A,serial-diluter apparatus for continuous delivery of variousconcenttations in,water. PHS Publ. No. 99-WP-23. 16 pp. .. - . ., r Mount, D. I., and W. A. lirungs. 1967. A ilDtplified dosing apparatus for fish toxicology studies. WaterRes.'1:21-29. National Technical Advisory Committee. 1968. Water quality criteria. Report of the National TechnicalAdvisory Committee on Water Quality Criteria to the Secretary of the Interior. USDI, FWPCA,..Washington, D. C. 234 pp. - Symons, J.M. 1963. Simple continuous.flow, low and variable rate pump. JWPCF,35:1480-1485. Zaroogian, G. E., G. Pesch, and G. Morrison. 1969. Formulation of an 5-rfificial mediumsuitable for oyster larvae development. Amer. Zool. 9:1144. Zillich, J: 1969. The simultaneous use of continuous flow bioassays and automatic waterquality monitoring elluipment to evaluate the toxicity of waste water discharges. Presented at: 44th Annual Conf. of Mich. WaterPoll. Cont. Assoc., Boyne Falls, Mich.,Iiiite,16, 1969. 3 pp. 8 1 AI4 O. BIOASSAY REFERENCES 6.2 PhytoplAnktonAlgal Asay Beige,G. 1969. Predicted effects of fertilizers upon the algae production in Fern Lake. FiskDiv, Skr. Ser. HavUnders. 15:339-355. Davis, C. C..1964: Evidence for the eutrophication of Lake Eric frOm phytoplankton records. Limnol. Oceanogr. 9:275-283. 1 Edmondson, W. T., and G. C. Anderson: 1956. Artificial eutrophication of Lake Washington. Limnol. Occanogr. 1(1):47-53. Francisco, D. E. and C. M. Weiss. 1973. Algal response to detergent phosphate levels. JWPCF, 45(3);480.489. Fruh, E. G., K. M. Stewart, G. F. Lee, and G. A. Rohlich.1966. Measurements of Eutrophication and Trends. JWPCF, 38(8):1237 -1258. n Goldman, C. R., and R. C. Carter.19615.An investigation by rapid CI 4bioassay of factors affecting the cultural eutrophication of Luke Tahoc,'California. JWPCF, 37:1044-1063. Hasler, A. D. 1947. Eutrophication of lakes by domestic drainage. Ecology, 28(4):383-395. Johnson, J. M., T. 0. Odlaug, T. A. 01.1on and 0. leRuschrneyer. 1970. The potential productivity of fresh water environments as determined by an algal bioassay technique. Water Resc. Res. Ctr., Univ. Minn. Bull. No. 20. 77 pp. Joint Industry/Government Task toree on Eutrophication. 1969. Provisional Algal Assay Procedure. P. 0. Box 3011, Grand Central Statiop, New York, N.Y., 10017. 62 pp. Lake, Tahoe Area Council. 1970. Eutrophication of Surface WatersIndian Creek Reservoir, Fiist Progress Report, FWQA Grant No. 16010 DNY. A Maloney, T. E., W. E. Miller, and T. Shiroyama. 1972. Algal Responses to Nutrient Additions in Naturil Waters. I. Laboratory Assays. In: Nutrients and Eutrophication, Special Symposia, Vol. I, Amer. Soc. Limnol. Oceanogr., Lawrence, Kansas, p 134-140. '- Maloney, T. E., W. E. Miller, and N. L. Blind. 1973. Use of Algal Assay in Studying Eutrophication Problems. Proc. Internat. Assoc. Water Poll. Ref., Sixth Conference, Jerusalem, 1972.Pergamon Press. Middlebrooks, E. J., E. A. Pearson, M. Tunzi, A.Adirrarayana, P. H. McGauhey, and G. A. Rohlich. 1971. Eutrophication of surface water - Lake Tahoe. JWPCF, 43:242-251. -Miller, W. E. and T. E. Maloney. 1971. Effects of Secondary and Tertiary Waste Effluents on Algal Growth in a Lake River System. JWPCF,43(12):2361 - 2365. Murray, S., J. Schertig, and P. S. Dixon. 1971. Evaluaten I:If algal assay procedures - PAAP batch test. JWPCF, 43(10):1991-2003. Oglesby, R. T., and W. T. Edmondson, 1966: Control of Eutrophication. JWPCF, 38(9):1452-1460, Potash, M. 1956. A biological test for determining the potential productivity of water. Ecology, 37(4):631-639. Rawson, D. S. 1956. Algal indicators of lake types. Limnol. Oceanogr. 1:18-25. , Schreiber, W. 1927. Der Reinkultur von marinem Phytoplankton and deren Bedeutung fur die Erforschung der ProdUktionKtahigkeit des Meerwassers. Wissensch. Meeresunters. N.F., 16:1734. Shapiro, J. and R. Ribeiro. 1965. Algal growth and sewage effluent in the Potontac estuary. JWPCF, 37(7):1034-1043. Shelef, G., and R. Halperin. 1970. Wastewater nutrients and algae growth potential. In: H. I. Shuval, ed., Developments in Water Qty Research, Proc. Jerusalem Internael. Conf. on Water Quality and Poll. Res. June, 1969. Ann Arbor-Humphrey Science Publ. p. 2/1.-228. I', , . , Skulberg, 0. M. 1964. Algal problems related to the eutrophication of European water supplies, and a bioassay method toassess fertilizing influences of pollution on ikiland waters. In: D. F. Jackson, ed., Algae and Man, Plenum Press, N.Y. p. 262-299. Skukberg, 0. M. 1967. Algal cultures, as a means to assess the fertilizing influence of pollution. In: Advances in, Water Pollution Re Search, Vol. 1, Pcrgainon Press, Washington, D. C. Strom, K. M. 1933. Nutrition of algae. Experiments uponthe feasibility of the Schreiber method in fresh waters; the relative importance of iron and manganese in the nutritive medium; the nutritive substance given off by lake bottom muds. Arch. Hyditbiol. 25:38:47. Toerien, D. F., C. H. Huang, J. Radimsky, E. A. Pearson, and J. Scherfig. 1971. Final report.provisional algal assay procedures. Report No. 71-6, Sanit. Eng. Res. Lab., Coll. Eng. Sch. Pub. Hlth., Univ. Calif., Berkeley. 211 pp. U. S. Environmental Protection Agency. 1971. Algal assay procedure: bottle test. National Eutrophication Research Program, USEPA, Corvallis, Oregon. Wang, W., W. T. Sullivan, and R. L. Evans. 1973. A technique for evaluating algal growth potential in Illinois surface waters.111. St. Water Sur., Urbana, Rcpt. of Investigation 72, 16 pp. Weiss, C. M. and R. W. Helms. 1971. Interlaboratory precision test - Aneight-lallatoryevaluation of the Provisional Algal Assay Procedure: Bottle Test. National Eutrophication Reseal:eh Program, U. S. Environmental Protection Agency. Corvallis, Oregon. 70 PP:" BIOLOGICAL METHODS 6.3Periphyton 'Burbank, W. D., and D. M. Spoon. 1967. The use of fisile ciliates collected in plastic petri dishes for rapid assessment of water pollution. J. Proteozool. 14(4):739 -744. Cairns, J., Jr. 1968. The effects of dieldrin on diatoms. Mosq. News,28(2):177-179. Cairns, J., Jr. 1969. Rate of species diversity restoration following stress in freshwater protozoan communities. Univ. Kansas, Sci. Bull. 48:209-224. 11 Cairns, J., Jr., and K. L. Dicky. 1970. Reduction and restoration of the number of fresh-water protoioan species following acute exposure to copper and zinc. Trans. Kansas Acad. Sci. 73(1):1-10. Cairns, J., Jr., A, Scheier, and N. E. Hess. 1964. The effects of alkyl benzene sulfonate on aquatic organisms. Ind. Water Wastes, 1(9): 1-7,- Fitzgerald, G. P. 1964. Factors in the testing add application of algicides. Appl. Microbiol. 12(3):247-253. Jackson, H. W., and W. A. Brungs. 1966. Biomonitoring industriateffluents. Ind. Water Eng. 45:14.18. McIntire, C. D., R. L. Garrison, H. K. Phinney, and C. E. Warren. 1964. Primary production in laboratory streams.Litrufo4ceanogr. 9(11:92-102. McIntire, C. D., and H. K. Phinney. 1965. Laboratory sttis of periphyton production and community metabolism in lotic environ- ments. Ecol. Monogr. 35:237-258. McIntire, C. D. 1966a. Some effects of current velocity on periphyton communities in labOratory streams. lirdrobiol. 27:559-570. McIntire, C. D. 1966b. Some factors affecting respiration of periphyton communities in lotil environments. Ecology, 47:918-930. McIntire, C. D. 1968a. Structural characteristics of benthic algal communities in laboratory streams. Ecology, 493):520-537. McIntire, C. D. 1968b. Physiological-ecological studies of benthic algae in laboratory streams. JWPCF, 40(11) Part 1:1940-1952. Otto, N. E. 1968. Algaecidal evaluation methods using the filamentous green alga, Cladophora. Rep. No. WC-40, Div. Res.,Bur, 7. Reclam., USD1, Denver. Patrick, R. 1964. Tentative method of test for ev uating inhibitory toxicitof industrial waste waters.AST4sStanditrils,Part 23, pp. 517.525, American Society for Testing and M?terials, Philadelphia, Pa. Patrick. R. 1966. The effect of varying amounts and ratios of nitrogen and phosphate on algae blooms. Proc. Ind. Waste Conf. (Purdue) 21:-51. Patrick,. 1968. The structure of diatom communities insimilar ecological conditions. Amer. Nat. 102(924):173-183. Patrick, R., 'J. Cairns, Jr., and A. Scheier. 1968a. The relative sensitivity of diatoms, snails, and fish to twenty commonconstituets of industrial, wastes. Prog. Fish-Cult. 30(3)i137-140. 4 Patrick, R., B. Crum, and J. Coles. 1969. Temperature and -manganese as determining factors in the presence of diatom or blue-green algal floras in streams. Proc. Nat:Acad. Sci. Phil. 64(2):472-478. Patrick, R., N. A. Roberts, and B. Davis. 1,968b. The effect of changes in pH on thetructure of diatom communities. Not. Natur. 416:1-16. Phaup, J. D., and J. Gannon. 1967. Ecology of Sphaerotihr's in an bxperimental outdoor channel. Water Res..1 :523-541. Phinney, H. K., and C. D. Mcfntire. 1965. Effect of temperature on metabolism of periphyton communities developed in laboratory streams. Limnol. Oceanogr.0(3): 341 -344. t effect and growth of fresh-water algae. Trans. Amer. Microsc. Soc. 79(3):302-309. Whitford, L. A. 1,960. The eurn, . Whitford, L. A,, G. E. Dillard, anF. J. Schumacher. 1964. An artificial stream apparatus. for the study of lotic organisms. Limnol. Oceanogr. 9(4):598 -600. Whitton, B. A. 1967. Studies on the growth o erain Cladophora in culture. Arch. Milcrobiol. 58:21-29. Whitton, B. A. 1970. Toxicity of zinc, copper, and lead to Chlorophyta from flowing waters. Arch. Mikrobiol. 72:353-360. Williams, L. G., and D. 1. Mount. 1965. Influence of zinc on periphytic communities. Amer. J. Bot.52(1):-26-341."). Wuhrmann, K. 1964. River bacteriology and the role of bacteria in self-purification of rivers. In: Principles andAliplicatidns in Aquatic Microbiology. John Wiley, NY. 4 Zimmermann. P.1961. Experimentelle Untersuchungen uber die okologische Wirkung der Stromungsgeschwindigkeit auf die Lebensgemeinschaft. :es fliessenden Wassers. Schweiz. z. Hydrol. 23:1-81. 6.4 Macroinvertebrates -,) Arthur, J. W., and E. N. Leonard. 1970. Effects of copper on Gammarus pseudolimnaeus, Physa integre, and Campeloma decisum in soft water. J. Fish. Res. Bd. Canada, 27:1277-1281. r-- Bell, H. L., and A. V. Nebeker. 1969. Preliminary studies on the tolerance of aquatic insects to low pH. J. Kansas Entomol. Soc. Ae. :230-236. Biesinger, K. E., and G. M. Christensen. 1971. Metal effects on survival, growth, and reproduction and, metabolism of Daphnia magna. National Water Quality Laboratory, Duluth, Minnesota. 43 pp. BIOLOGICAL METI-IODS Dimick,R. E., and W. P. Breese. 1965. Bay mussel embryo bioassay. Proc. 12th Poirific Northwest Ind. Conf., College of Engineering, Univ. of Wash, pp. 165-175. - .. \ - Gaufin, A.R. 1971. Water quality requirements of aquatic insects. of Biology, Universityof of Utah. Contract No. , . 14-12-438,USpI, FWPCA,National Water Quality Laboratory, Duluth. 65 pp. . , Woelke, C. E. 1967. MeasureMent of water quality with the Pacific oyster embryo bioassay. In: Water Quality Criteria, Amer. Soc. for Testing and Materials, Special Tech. Pub. No. 416. pp. 112-120. / 6.5Fish Brungs, W. A. 1969. Chronic toxicity of zinc of the fathead minnow, Pimephales promelas Rafinesque. Trans. Amer. Fish. Soc. 98:272-279. J, G. 1970. Chronic malathion toxicity to the blucgill (Lepomis macrochirus,Rafinesque). Water Res. 4:673-684. McKim, J. M., and D. A. Benoit. 1971. Effects of long-term exposures to copper on survival, growth, and reprOduction of brook trout Salvelinus fontinalis (Machin). J. Fish. Rcs. Bd, Canada, 28:655-662. Mount, D. I., and C. E. Stephan. 1969. Chronic toxicity of copper to the fathead minnow (Phnephales promelas, Rafinesque).in soft 4 ' water. J. Fish. Res. Bd. Ganada, 26:2449-2457. a Sprague, J. B. 1969. Piteasurciftent of pollutant toxicity to fish. I. Bioassay methods for acute toxicity. Water Res. 3:793 -821. : Sprague, J. B. 1970. Measurement of pollutant toxicity to fish. II. Utilizing and applying bioassay results. Water Res. 4:3-32...` r I I A 4 RECOMMENDED BIOASSAY PROCEDURES NATIONAL WATER QUALITY LABORATORY DU LUTliNINNESOTA 1 4+ Recommended Bioassay 'Procedures are estab- Laboratory bioassays. An attempt has been made lished by the approval of the Committee to adopt the best acceptable procedures based on Aquatic Bioassays andand the Director of the on current evidence and opinion; although it; is National Water Quality Laboratory. The main recognized that altemalive procedures may be reasons, for establishing them are: (1) to permit -dal-five. Improvements (iii the proceduresare direct ,comparison Of test results, (2) to en-being bonsidered and tested, and revisions will courage the use of the best procedures available, be made when necessary. Comments and and (3) to encourage uniformity. These proce-suggestions are 'encouraged. du es should be used by National Water Quality La oratory personnel whenever iibssible, unless th reis a good, reason for using some other procedure. Recommended Bioassay Procedures consider the Director, National Water Quality Lab (NWQL) basic elements that are. believed to be importarit in obtaining reli4ble and reproducible results in Committee on Aquatic Bioassays,-NWQL 1 . r .1150 13 *Fathead Minnow Pirtiephalespromelas Rafinesque Chronic Tests April, 1971 (Revised January, 1972) 't 1.0 PHYSICAL SYSTEM and divided to form two separate larval 1/1.1Diluter chambers withseparatestandpipes,or separate 1/2 sq. ft. tanks my bused. Test Proportionaldiluters (Mount ,andBrungs, water to be supplied by deli '(ery tubes 1967) should be employed for all long-term from the mixing cells described in Step 2 exposures; Check the operation of the diluter above. '4, daily, either directly or throughmeasurement of Tess water depth in tanks and 'chambers for toxicant concentrations. A minimum of -five both a and b above should be 6 inches: toxicant concentrations andone contn4 should be used for each test witha dilution factor of, 1.4Flciw Rate not letghan, 0.30. An automatically triggered The flow Tate to each chamber (larvalor emergey aeration and alarm system must be a dUlt) should be equalto6 to '10 tank installed to alert staff in case of diluter,terhpera- volumes/24 hr. ture control or water supply failure. . 1.5Aeration 1.2 Toxicant Mixing. Total dissolved oxygen levels shouldnever be A container to promote mixing of toxicant-allowed to drop below 60% of, saturation, and bearing and w-cell water should be used between flow rates must(be increased ifoxygen levels do diluter and tanks for each concentration. drop below 60%. As a first alternative,Tflow rates Separate delivery tubes shouldrun from this can be increased above those specified in1.4. container to each duplicate tank. Check at least Only aerate (with oil free air) if testinga non - once every month to see that the intended volatile toxic agent, and thenas a last resort to amounts of water are going to each duplicate maintain dissolved oxygen at 60% of saturation. tank or chainber. 1.6Cleaning 1.3 Tank 7 All adult tanks, and larvae tanks and chambers Two arrangements of test tanks (glass,or after larvae swim-up, must be siphoneda mini- stainless steel with glass ends) can be utilized: mum of 2 times weekly and brushed or scraped when algal or fungus growth becomes excessive. a. Duplicate spawning tanks measuring 1 X 1 X 3 ft. long with a one sq. 'ft. portion at 1.7 Spawning Substrate one end screened off and divided in half for Use spawning substrates made from inverted the progeny. Test water is to be delivered cement and asbestos halved, 3-inch ID drain tile, separatelytotl larval and spawning or the equivalent, each of these being 3 inches chambers of each tank, with aboutone- long. third the water volume going to the former chamber as to the latter.' 1.8 'Egg Cup b. Duplicate spawnirig tanks measuring 1 X 1 Egg incubation cups are made from either X 2ft.long with a separate duplicate 3-inch sections of 2-inch OD (11/2-inch ID) progeny tank for each spawrii6g tank. The polyethylene water hose or 4-oz., 2-inch OD" larval tank for each spawning tank should round glass jars with the bottoms cut off. One be a minimum of 1cu. ft. dimensionally end of the jar or hose sections is covered with 15. 5 1. 4., BIOLOGICAL METHODS stainless steel orlylonscreen (with a. minimum 1.11Tempprature of 40 meshes per inch). Cups are oscillatedin Temperature should not deviateinstanta- the test water *X means of a rocker arm*appara- neously from 25°C by more than 2°C and tus driven by afr.pan. eleCtric motor (Mount,-should not remain outside the rangeof 24 to 1968). The vertical-travel distance of the cups 26°C for more than hours at a time. Temper- should be 1 to 1 1/2 inches. ature should be recor7d continuously. dr- Aig pi:1.9tight 1.12Disturbance. la' he lights ,ufied should simulate sunlight as Adults and larvae should be shieldedfrom nea 1y as possible. Acombination of Duro-Testjlisturbances such as people continuallywalking r: ',(Optima FS)' '2 and wide spectrum.Grow-lui0 past the chambers, or fromextraneous lights fluorescent tubes has proved satisfactory atthe that might alter the intended photoperiod. 1.13Construction Materials Construction materialswhichcontactthe .10 Photoperiod diluent water should not contain leachablesub- The photoperiods to be used (AppendixA) stances and should not sorbsignificant amounts simulate the dawn to dusk times of Evansville,of substances from the water. Stainlesssteel is 11 Indiana. Adjustments in day-length are tob probably the preferred constructionmaterial. ---irriaite on the first and fifteenth day of eve Glass absorb4s some trace. organics significantly. Evansville test month. The table isarranged soRubber shbied not bckosed. Plastic containing dusk that adjustments need be made only in the addiffves,stabilizers,plasticizers,etc., times.Regardless of the actual date that theshould not be used. Teflon, nylon,and their experiment is started, theEvansville test photo- equivalents should not containleachable period slidtld be adjusted so thatthe mean or should not sorbsignificant start ma terials and estimated hatching daie of the fish used to -limounts o substances. Unplasticized poly- 'the experiment' correspondso the Evansville ethylene polypropylene should not contain test day-length forDecembefirst. Also, the leachable substances, but may sorb yerysignifi- dawn and dusk times listed in r e table need not cant amounts of trace organiccompounds. Correspond to/ the actual time where theexperi- ment is1:Milig conducted. o illustrate these 1.14 Water points, an experiment started with5-day-old The fttater used should be from a-well or larvae in Duluth, Minneso at onAugust. 28 from a 5 spring if at all possible, or alternatively (actual date), would requiree of a December surface water source. Only as a last resortshould Evansville test photoperiod, nd the lightscould water supply St so long as they water from a chlorinated municipal go on anytime onThat day j be used. If it is thought that the watersupply remained on for 10 hours a 45 minutes. Ten with fish 15 Evans could be conceivably contaminated days later (Sept. 7 actual d' te, Dec. pathogens, the water should be passedthrough ille date) the day-lengtl would bethawed sterilizer immediately changes in an ultraviolet or similar to 10 hours and 30 minutes Gradual before it enters the test system. light intensity at dawn. asdusk (Drulnmond and Dawson, ,4970), if .esired,should be in- cluded within the day -l=gth showii, and should 2.0 BIOLOGICAL SYSTEM not last for more tha1/2 hour from full on to full off and vice ver a. 2.1 st Animals Ifossible, use'stoctsof fathead minnows Me ion of tra, ames does not constitute endorsement. fro=the National Water fuality Laboratoryin 2 Duro-Test, inc., Hainmond, Ind. Fish Toxicology 3 Sylvania, Inc., New York, N. Y. D uth,Minnesotaor the ' 16 FATHEAD MINNOW BIOAS§AY La boratory in NewtownOhio. Groups ofof them or not. The frequency oftreatment startingfish- Ciotti ntainamixtureof shoidd be held to a minimum. approximately °equal number ofeggs or larvae I from at least three different females. Setaside 2.5 Measuring Fish enough eggs or larvae at the start of the test to Measure total, lengths of 'all starting fish at 30 supply an adequate numtttr, of fish for theacute and 60 days by the photographic method used mortality bioassays used in determiningappli- cation factors. by McKim and Benoit' (1971). Larvaeor juve- niles are transferred to a glass box containing 1 62.2 Begjining Test inch of test water. Fish should be movd to and from this box in a .water-filled containe, rather In beginning the test, distribute 40to 50 eggs. than Whetting them. The glass box is placedon or 1- to 5-6y-old larvae per duplicate tank using a translucent millibeter grid over a fluorescent a stratified random assignment (see 4.3).All lightplatform to 'provide background illumi- acute mortality tuts should be conducted when nation. Photos are' then taken of the fishover the fish are 2 to 3 nionths old. Ifeggs-or 1-tothe millimeter grid and ttre enlarged into 8 by 10 5-daY>).1d Idrvaeare not available, fishup to 305,inchprints. The length of each fishis sub- days of age may be used to start thetesta" fish -sequently determined bl comparing it to the between ,20 and 60 days oldare used; , thegrid. Keep lengths of discarded fishseparate exposure should be designated a partial chronic from those of fish that are. to be kept. test, test animals may be added at the beginning so that fish can be removedperiodi- 2.6 Thinning cally for special examinations (see 2.12.) or for When the st6rting fish are sixty, (-± 1 or 2) days residue analysis-(see 3.4). . old, imparti4lly reduce the number of surviving 2.3Food fish in each tank to 15. Obviously injuredor crippled individuals may be discarded before the Feed the fl)h a frozen trout food (e.g., Oregon selection so long as the number is not reduced Moist). A minimum of once daily, fishshould be below 15; be sure to record the number of fed ad libitum the largest pellet they will take. deformed fish discarded from each tank. Asa Diets should be supplementedweekly with live last resort in obtaining 15 fish per tank, 1or 2 Orfrozen-live food (e.g., Daphnia, chopped fish may be selected for transfer fromone earthworms, fres h or frozen brine shrimp, etc.). duplicate to the other. Place five spawning. tiles Larvae should be fed a fine troutstarter a in each duplicate tank, separated fairly widely to minimum of 2 times daily; ad libitum;one reduce interactions between male fish guarding feeding each day of liveyoung zooplankton them. One should also be able to look under from mixed cultures of small copepods, rotifers, tiles from the end of the tanks. During the and prOtOzoans is highly recommended. Live spawning period, sexually maturing Males must food is especially important when larvaeare just be removed at weekly intervals,so thereare no beginning to feed, or about 8 to 10, days aftermore than four per tank. An effort should be egg deposition. Each batch of should be made not to remove' those males having well checked for pesticides (including DDT,TDE, established territories under tiles where recent dieldrin, lindane, methoxychlor, endrin, aldrin, spawnings have occurred. BHC, ,chlordane, toxaphene, 2,4-D, and PCBs), and the kinds-and amounts should be reported 2.7 Roving Eggs to the project officer or recorded. Rotve- eggs'from sprawning tiles starting at 2.4iDisease 12:00 noon Evansville test time (Appendix A) edel. day.. As indicated, in Step 1.10, the test (Handle diseaie 'outbreaks according to their time need hot corresp6nd to the actual time nature, with all tanks receiving the same treat- where the test is being conducted. Eggsare ment whether there6eems to be sick fish ill all loosened from the spawning tiles and at the. 1 0o BIOIQGICAL METtIODS t. same time separated from one anotherby lightly 2.9 Progeny 'Transfer placing a finger on the egg mass and moving it in Additional important information on hatch- a circular pattern with increasing pressure'until ability and larval survival is to begained by. the eggs begin to roll. The groups of eggs should'transferring controleggsimmediately, after then bei washed into separate, appropriately spawning to concentrations wherespawning is marked containers and subsequently handledreduced or absent, or to where anaffect is seen (counted, selcted for incubation, or'discarded) on, survival of eggsot/larvae, and by transferring as soon as possible after all eggshave been re-eggs from theseconcentrations to the control moved and the spawning tiles put back into the tanks. One larval chamber in, orcorresponding test tanks. All ,egg batches must bechecked to, each adult tank shouldalways be reserved for initially for different stages'S, f development. If it eggs produced in thattank. is determined that thereis more than one distinct stage of developMent present, then each 2.10 Larval ExpoSure and stage-must be considered as one spawning tank, handled separately as described in Step 2,8. From early spaWnings in each duplicate , use the larvaehatched in the egg incubator cups (Step 2.8. above) for 30 or 60 day growthand 2.8 Egg Inculmtion and Larval Selection survival exposures in e larval chambers.Plan for hatchability so that Impartially select 50 unbroken 'eggs from ahead in setting up e them in a n,ew groupof larvae is ready to be tested for spawnings of 50 eggs or more and place possible after the an egg incubatdr cup fordetermining viability 30 or 60 days as soon as and previously tested,group comes outof the larval and hatchability. Count the remaining eggs mortalities, and measure total discard then, yipNity_and hatchability deter- chambers. Record (>49 lengths.of larvaeat 30 and, if they arekept, 60, =nations must be made on each spawning days posthatch. At the timethe larval test is eggs) until the number of spawnings(>49 eggs) No fish equals the). number of terminated they should also be weighed. in each duplicate tank (larvae, juveniles, or adults) should befed within females in that tank. Subsequently, only eggs weighed. from every third spawning (>49 eggs)and none 24 hr's. of when they are to be be set up to of those obtained on weekends need Parental Termination determinehatchability; however, weekend 2.11 from tiles andthe Parental fish testing should be terminated spawns must still be removed day-length photo- eggs counted. If unforeseenproblems are -edcounL when, during the receding hatch- period, a one week period passesin which no tered in determining egg (viability and Measure ability, additional spawnings should besampled spawning occurs in any of the tanks. before switching to the setting up of eggsfrom total lengths and weights of parentalfish; check Every day, record the live sex/ and conditionof gonads. The gonads of every 'giircl,spawning. 'begun to regress and 'dead eggs in the incubator cups, removethe most parental fish will have dead ones, and clean the cup screens.Total from the spawning condition,and thus the dif- less distinct rUmbers of eggs- accounted for shouldalways ferences between the sexes will be add up to within two of 50 or theentire; batch is now than previously.Males and females that are because tb be discarded. When larvae beginto hatch, readily distinguishable from one another generally-after 4 to 6 days, they should not be of their externalcharacteristics should be the egg-cups selectedinitiallyfbr determining how to handled agaip\or removed from ovaries. One of until all have hatched.then,. if enough are still differentiate between testes and alive, 40 of these are eligible tobe transferred the more obvious e`xternalcharacteristics of immediately to a larvaltest chamber.Those females that have spawned is anextended, trans- the' number parent analcanal(urogenitalpapilla). The individuals selected out to bringv, located just ventral kept to 40 should be chosenimpartially. Entire gonads of both sexes will be survival and growth to the kidneys. Theovaries of the females at this egg-cup-groups not used for but perhaps con- studies should be counted and discarded: time will appear transparent, .18 FATHEAD MINNOW BIOASSAY t-J 'Wining some yellow pigment, coarsely granular, they have been proven. tobe necessary in the , and larger than testes. The testes of males will actual test system. The suggested surfactant is appear as slender, slightly milky, and very finely p-tert-octylphenoxynonadthoxy-ethanol ^(prl, 1, granular strands. Fish must not be,frozen before 3-tetramethylbutylphenoxynonaethbxy- making these examinations. ethanol, °PEI 0)- (Triton X-100,a product of 2.12 Special Examination's the Rohm and Haas Company, or equivalent). The use of solvents, surfactants,or other Fish and ,eggs obtained from the test should additives should be avoided whenever possible. be considered. for physiological, biochemical, If an additive is necessary, reagent gradeor histological and other examinations which may bettershpuldbeused. The amount of an indicate certain toxicant-related effects. additive used should be kept to a minimum, but 2.13NecessaryData the calculated concentra'tion ,:of a' solvent to which any test' organisms are exposed mustA Data that must be reported for each tank ofa never exceed one one-thousandth of the 96-hr. chronic test are: LC50 for test species under the test conditions and must never exceed one gram.per liter of a. NumDer and individual total length of water.The calculated concentration of sur- no(mal and deformed fish at 30 and 60 factant or other additive to whichany test days; total length, weight and number of organisms are exposed must never exceed one-, either sex, both normal and deformed, at twentieth of the concentration ofjthe toxicant end of test. and must never exceed one-tenth gramper liter b. Mortality during the test. of water. If any additive is used, two sets of c. Number of spawns and eggs. controls Must be used, one exposed to no addi- d. Hatchability. tives and one exposed to the highest level of e. Fry survival, growth, and deformities. additives to which any other organisms in the test are exposed. 3.0 CHEMICAL SYSTEM 3.2 IViasurement of Toxicant Concentytion 3.1Preparing a Stock Solution As .a minimum, the concentration of toxicant If a toxicant cannot- be introduced into the must be measured in one tank at each toxicant test watereas is, a stock solution should be pre- concentration every week for each set of dupli- * pared by dissolving the toxicant in water or an cate tanks, alternating tanks tit each concen- organic solvent. Acetone has been the most trationfrom week to week. Water samples, widelyused solvent, but dimethylformanide should be taken about midway between the top (DMF) and triethylene glycol may, be preferred and bottom and the sides of the tank and should in many cases.If none of these solvents are not include any surface scum cr material stirred-' acceptable, other water-miscibleolvents such as up from the bottom or sides of the methanol, ethanol,isopropanoi,acetonitrile, Equivolutne daily grab samplescan be coat,- dimethylacetamide (DMAC), 24thoxyethanol, posited for a week if it has been shown that the glyme (dimethylether of ethylefiegl col, .results the analysis are not affected by storage diglyme (dimethyl ether of diethylene ycol) of t e sami)le. and propylene glycol should be con idered.- nough grouped grab samples shouldbe However, dimethyl sulfoxide (DMSO) analyzed periodically throughout the test to not be used if atall possible because of itsdetermine whether or not the concentration of biological properties. toxicant is reasonably constant from day to da'y Problems of rate of solubilization of solubility in one tank and from one tank to its duplicate, limit should be solved by mechanical means if at Ifnot,enough samples mustbeanalyzed all -possible. Solvents, or as 'a last resort, sur- weekly throughout the test to show the vari- factants, can be used for this purpose, only after ability of the tOxicant concentration. e" 19 1 BIOLOGICAL METHODS . 3.3Measurement of Other Variables icants. If available,ieferencesamples should be analyzed periodicallyfor eachanaly.tical Temperature must be recorded continuously method. (see 1.11.). / . Dissolved oxygen must be measured inthe 4.0 STATISTICS ,te tanks daily, at least five clays a week on an alter- nating basis, so that each tank is analyzed once 4.1Duplicates each week. HowAr, if the toxicant or an 4/0 1 additive causes a depression in dissolved oxygen, Use true duplicates for level of toxic the toxicant concentration with the lowest dis- agent, i.e., no water connections betweendupli- solved oxygen concentration must be analyzed cate tanks. daily irivaddition to the above requirement. A control and one test concentration must be 41 Distribution of Tanks analyzed weekly for pH, alkalinity, hardness, The tanks, should be assigned to locations by \ ticidity, and conductance, or moreoften,if stratified random assignment (random assign- necessary, to show the variability in the test ment of one tank for each level of toxic agentin water. However, if any of these characteristicsa row followed by randomassignment of the are affected by the toxicant,the tanks mustbe second tank fOr each level of .toxic agent in analyzed for that characteristic daily, at least another or an extension of the same row). five days a week, on an alternating basis so that each tank is analyzed once every other week: 4.3Distribution of Test Organisms At a minimum, t e test water must be ana- -ng and near the middle of The test organismeould be assigned tetanks lyzed at thebeginning by stratified randoassignment (random assign- the test for calcium, magnesium,. sodium, po- ment of one test organism to each tank, random tassium, chloride, sulfate, total solids, and total assignment of, a second test organism to each dissolved solids. tank, etc.). 3.4Residue Analysis 5.0 MISCELLANEOUS When possible and deemed necessary, mature fish, and possibly eggs, larvae, and juveniles, 5.1Additionalihtormation obtained from the test, should be analyzed for toxicant residues. For fiqh, muscle should be All routine bioassay flow-thrbugh Methods analyzed, and gill, blood, brain, liver, bone, not covered in this procedure (e-,g., physical and kidney, GI tract, gonad, and skin should be con- chemical determinations,handli of fish) sidered for analysis. Analyses of whole organ- should be followed as described IR Standard isms may be done in addition to, but should not Methods for the Examination of Water and place of, analyses of individual Wastewater,' (American Public Health Associ- be done in requested' from tissues, especially muscle. ation, 1971), or information appropriate persons at Duluth or Newtown. . 3.5 Methods When they will provide the desired infor- 5.2 Acicnowledgments mation with acceptable precision and accuracy, These procedures for the fathead minnow methods described in Methods for Chemical were compiled by JohnEaton for the Commit- Analysts of Water and Wastes (EPA, 1971)tee on Aquatic Bioassays. Theparticipating should be used unless there is 'another method members of this committee are: Robert Andrew, which requires much less time and can provide John Arthur, Duane ,Benoit, Gerald Bouck, the desired information with the same or better William Brungs, Gary Chapman, John Eaton, -.precision and accuracy. At a minimum, accuracy John Hale, Kenneth Hokanson, James McKim, should be measured using the method of known QuentinPickering, Wesley Smith,Charles additions for allanalytical methods for tox- Stephan, and James Tucker. - FATHEAD MINNOW BIOASSAY -11114 6.0 REFERENCES For additional information concerning flow through bioassays with fatheadminnows, the foil ences are listed: . American Public Health Association. 1971. Standard methods for the examination ofWater artcl was ater. 13th ed. APHA. New York. , ',. . * ../ . 0 Brungs, William A.:1969. Chronic toxicity of zinc to the fathead minnow, Pimephlsprortittas Rafinesque. Trans. Abler. Fish. Soc. 9812).272 -279. . . , \ Btungs, William A. 1971. Chronic effects . of low dissolvedoxygen conicentrations on the fathead minnow (Pimephales promelas). J. Fish. kes. Bd. Canada, 28(8): 1119-1123. Brungs, William A...1971. Chronic effects of constant elevated temperatureon the fathead minnow (Pimephales promelas ). Trans. Amer. Fish. Soc. 100(4: 659-664. '> Carlson, Dale R. 1967. Fathead minnow, Pimephales promelas Rafinesque the Des Moines River, Boone County, Iowa, and the Skunk River drainage Hafnilton and Story Counties, Iowa. Iowa State J.i., 41(3): 363-374. Drummond, Robert A., and Walter F. Dawstk 1970. An ine7if5ensivenneod for simulating Diel patterns of lighting in the Ikboratork- Trans. Amer. Fish. Soc. 99(2):434-435. ,1 lsaak, Daniel. 1961. The ecological life history of the fathead minnow, Pimephafes promelas(Rafinesque ).Ph.D. Thesis, Library, kiniv. of Minnesota. Markus, Henry C. 1934. Life history of the fathead minnow (Pimephales promelas J. Copcia, (3):116-122. McKim, J. M., and D. A. Benoit. 1971. Effect of long-termexposures to copper on survival, reproduction, and growth of brook trout Solvelinuslontinalis (Mitchill). J. Fish. Res. Bd: Canada, 28: 655-662. Mount, Donald I. 1968. Chronic toxicity of copper to fathead minnows (Pimephalespromelas, Rafinesque). Water Res. 2: 215-223. Mount, Donald I., and William Brungs. 1967. A simplified dosingapparatu for fish toxicdlogy sfudies. Water Res. 1: 21-29. Mount, Donald I., and Charles E. Stephan. 1967. A method for establis acct le toxicant limits for fish - hion d the butoxyethanol ester of 2,4-D. Trans. Amer. Fish..Soc. 96(2): 185-193. Mount, Donald I., and Charles E. Stephan. 1969. Chronic toxicity ofcopper to the fathead minnow (Pimephales promelas) in soft water. J. Fish. Res. Bd. Canada, 26(9): 2449-2457. Mount, Donald' I., and Richard E. Warner. 1965. A serial-dilution apparatus for, continuous delivery of variousconcentrations of materials in water. PHS Publ. No. 999-WP-23. 16 pp., Pickering, Quentin H., and Thomas 0. Thatcher. 1970. The chronic toxicity of linear alkylate sulfonate(LAS) to Pimephales promelas Rafinesque. JWPCF, 42(2): 243-254. Pickering, Quentin H., and' William N. Vigor. 1965. The-acute toxicity of zinc to eggs and fry of the fathead minnow. Prpgr.Fish-Cult. 427(3):.153 -157. "Air Verma, Prabha. 1969. NOrmal stages in the development of Cyprinus.carpiovar. communis L. Acta biol. Acad. Sci. Hung.-21(2): 207-218. 21 FATHEAD MINNOW BIOASSAY , A Appendix A fi Test (Evafitsville, Indianaqhotoperiod For Fathead Minnow' Chronic Dawn to Dusk , Time Date Day-length (houriand minute) 6:00 - 4:45) DEC. 1 .10:45) 6:00 -4:30) 15.10:30) 6:00 - 4:30) JAN. 1 10:30) 6:00 - 4:45) 15 10:45) ) 6:00 - 5:15) FEB. 1 11:15) 5-mfonth pre-spawning 6:00 - 5:45) 15 11:45) growth ffieriod ) 6:00 - 6:5) MAR. 1 12:15) 6:00 - r00) 15 13_:00) 6:00 - 7:30) APR. 1 13:30) 6:00 - 8:15) 15 414:15) 6:00 - 8:45) MAY 1 14:45) 6:00 - 9:15) 15 15:15) 6:00 - 9:30)'JUNE 1 15:30) 6:00 - 9:45) 15 15.A5) 4-month sPawnin ) period 6:00- 9:45) JULY 1 15:45) 6:00 - 9:30) 15 15:30) ) 6:00 : 9:00) AUG. 1 15:00) 6300 - 8:30) , 15 14:30) 6:00 - 8:00) SEPT-. .1 14:00) 6:00 - 7:30) 15 13:30) ) 6:00 - 6:45) OCT. I 12:45Y post spawninig period 6:00 - 6.:15) 15 12:15) . . / 6:00 - 5:30) NOV. 1 11:30). 6:60 5:00) 15 11:00) A . a e 4 ' Brodk Trout SahA litlysforin,aies (Mitchill).Partialpronic Tests April, 1;971 (Revised Jan' al-y0972) 1.0 PHYSICAL SYSTEM 1.44 glow Rate . 1.1Diluter Flow rates for each duplicate spawning. tank Proportional and "growth chamber shouldbe- 6-1Q.: tank diluters (Mount an?' Brung's, volumes/24 1967) should be employed,for -long-term L. . Q, exposures.. Check theoperation of the diluter L 1.5'Aeration daily, either directly or through _the.Measure,- ok trout tanks and.growth chambersmust. ment oftolxi9litconcentrations.A niinirpum of .he aerated With .oil freeair unless there are no five toxicant concentrations'and one control flow limitations and '60% of saturation should 'be used. fo'r.' each can be test with a,. dilution . aintaine\d. Total dissolved'oxygen levels should factor of not legs than 0.30.An autOmaticallf*yer be allowed, to drop below60% of satu-- triggered emergency aeration andalarm system- '''. must be installed to alert staff:incase of diluter, a . temperature controlor Water supply,failure. Cleaning All tanks :and Chanibers must be siphOpcd*, - 1.2 toxicqnt Mixing . . o daily. and brushed at leastonce per week. When A container 'to promoteaxing Of takicant-apawning commences, 'gravel baskets must be re- hearing and w-,Cell water shoube used between inoved agd cleaned dailY. 1..idiluter and tanks fore ch' c,oncentration. Spawning. Substrates' -.Separate delivery tubes sh uld run from this . Use MD spawning substrates! per duplicate container to each duplicate, tank. Checkto see' :that. same amount of water hoes to duplicate,'fade of plastic or stainless steel Whichmeasure ; tanks and that the toxicant,doncentratiois the at leaste X 10 X 12 in with 2 inches Of .25 to same,in both. 'z,.,50 inchstream gt'avel covering the bottom and, a. 20 mesh stainless'steelor nylon screen attached 17 t. , ';.1.3Tank to the,ends for circulation of water. °' Each duppcate spawning 'tank(preferably', t.8 -Egg Cup 7% stainless' steel). should measure X 3 ' ft.- Egg incubation cups are made Jrprn4-oz.,a wide witha water depth of .,,l';fo.ot and alevin- rourid glass jars with he bottoms cut- ' TUv,enile growth chambers.(glasgor stainlesssteel off and replaced 'with stainlesssteel or nylOn with glass obttorn) -7 X 1.5 X 5 in. wide'with screen (40 meshes per.inch)i Cups are oscillated t water depth 'of'S inches: Growth chambersccan in the test water' by means-ofa rocket, arm be'supplieclitet ;waterbyith' separate delivery apparatus driVen by .2 r.p,m. electr4 ,motor ° tubes fro! the, mixing cells deseribeclin. Step 2 (Mount; 196,8). abbN)e or from ° test water delivered from' the 1;.9Light eel) to each duplicate pawning..tank.In the second chdice, testwater must always flow The lights :.used shdUld sfiii`ulate'sunlight as through growth :chambers:befo're,entering the nearly, us posSible. -A combination of:Durci,Test spawning tank. Eabh growth chambershould be (Optima. F-S)l 'and - "wide spectrum GrOliix3 designed so that the Mastater can be &mine' fluorescent' tubes has proved Satisfactofylat 'theme- down to ,1inch and -the chamber NWOL. -, transferred . over a fluorescent light boi for photographing 'Mention of trade names doei not coniktute endorsement; the fish (see 2.1A0). 2 tiuni-Test, Iv Hammond, Ind. . 7'.. ?Sylvania, Inc., New York, N. Y. tt BIOLOGICAL METHODS material. 1.10 Photoperiod probably the preferred construction Glass absorbs some trace organicssignificantly. Thy photoperiods to be used(Appendix A) Rubber should not be used.- Plasticcontaining of Evansville, ,,mulate the daWn to dusk times fillers,additives, stabilizers,plasticizers,etc., 'Indiana.Evansville dates Must correspond to should not be' used. Teflon, nylon,and their Ictual dates jn order to avoid putting naturalequivalents should notcontainleachable reproductive cycles out of phase.. Adjustmentsin materials and should not sorbsignificant" photoperiod are to be Made on thefirst and amounts of most substances.Unplasticized pot; fifteenth of every' Evansville test month.The yethylene and polyprOpylene should notcontain tableis arranged so that adjustmentsneed be leachable substances, but may sorb verysignify dawn and made only in the dusk times. The cant amounts of trace organiccompounds. dusk times listed in the table(Evansville test actual test time) need not correspond to the 1.14 Water times where thetestis being condticted. To_ .from a well o -illustrate this _point, a test started onMarch first The water used should be photoperiod for spring if at all possible,'or alternativelyfront a would require.' the use of the ghould * Evansville test ,ate March first: and thelights surface water source. Only as a last re,sort just so long as water from a chlorinat'dmunicipal water supply could go ohanx., time on that day the water supply they 'remained on for twelvehours and fifteen be used. If it is thought that minutes. ,Fifteen days later. thephotoperiod, conceivablycontaminatedontaminated witfist pathogens, the water sh .uld" be passedthrough ) wo be changed .to thirteen> hotirs.Gradual light intensity 1:atdawn and dusk an ultravioletor similar;stesilizer immediately ../ in- before it enters(the test system: rl-ror4d-and. It6,vs.ohl7 701: may' .e...... , , 41'wit'hinthe.' photoperiods shown; and should ncA Iasi for more than 1 / 2 hourfrom full 2.0 BIOLOGICALSYSTEM -c-- on toli.L11.,_oTfand vice versa. 4 Test'Animals C/: 1.11Ternperatufeiz. - Yearling fish should be collected no laterthan Utilize the attached temperature regime(see Mara l' and acclimated in thelahOratory to:tesq Appendix B). Temperatures shouldnot deviate temperature and water qualityfor at least one instantaneously from the specified testtempera- month before the test is initiated..Suitability of ture by more than 2°C andshould not remain fish f6- testing should be judged onthe basis of. outside the specified temperatiire ±1°C for more acceptance of food, apparentlack of diseaks, than 4,8 hours at a time. and 2% or .less mot-- lity duringaccliniation with nomortality two weeks prior to test. Set aside 1-.12Disturbance enough fish to supply an adequate numberfor Spawning tanks and growth chambers mustbe short-term bioassay exposuresUsed 'in deter- covered with a screen to -confinethe fish .and Mining ' . .Record inortalities,daily, and ,measure fish Additional important infornption on hatch- directly' at initiation of test, afterAhree months ability and alevin survival can be gained by trans- and atthinning (see 2.6) .(tbtal length and ferring control eggs- immediately after spawning weight). Fish should not be fed 24 hours before to concentrations whefe spawning is reduced or weighing 'and anesthetized with MS-222.absent, or to where In affecris seen on survival tofacilitate measuring..(100, mg MS-222/liter,of eggs or alevin, aila by t ansferringleggs from water). these concentrations to thecontrol tanks. Two growth chambers for each 441icate spawning 2.6 "Thinning tank should always -be' reserved for eggs pro - When secondary sexual characteristics are.well du -n thattank. developed (approximately two weeks prior to expected spawning), separate males; females and 2.10 :Hatch anci Alevin'Thinning . 9 . undeveloped .fishin .each -, duplicate and ran- Remove dead eggs daily from tlietctchability domly reduce sexually mature fish (see 4.4) to cups .described in Step 2.8 abovec, When Hatching the desired number of.2 males and 4 ,females, commences, record the number hatched daily in anddiscard undevelopedfishafter exami- each cup. Upon completion of hatch in any cup, nation. Place two spawning substrates (described ,randomly (see 4.4) select 25 alevins-from that earlier) in each dtipl?cate. Record the number of c tip. Dead. or deformed,alevins must not be in- mature, .immature, deformed and injured males '-'Tr uded in the -randoVselection but 'should 'be and females in each tank and the number from counted as being dead or defgrmed upon hatch; each category ,discarded. Measure total- length Measure total lengths of the 25 selected and 'f'and weight of all 'fish in each.cate.tory ,before discarded alevins. Total lengths are measured V 'any are. discarded and note which ones were did the photographic method used, by McKim. and carded. Benoit (1971). The fish are trtnsferred to a grass box containing 1 inch of tcs water. They sh, 2.7Removing Eggs be moved to and from this box in a water RernOve eggs from the retld at a fixed time container, rather than by netting them. The glass each clay .(preferablyge,c1 :00 p.m. Evansville is placed on a translucelnt eter grid 27 ! C. BIOLOGICAL METHODS ; over afluorescent light box which provides2.12 Parental Termination 4a 4 _ background illumination.- Photos are then taken All parental fish should be terminated when-ao of the fish over the millimeter grid and arethree-week period passes in which no spawning enlarged into 4,X 10 inch prints-r-The length Ofoccurs in any of the spawning tanks.Record each fish is sAbsequently determined 1:0_3:,, conk mortality and weigh and measure total length of_ paring it to the grid. Keep lengths of discardedparental fish, check sex and condition of:gonads' alevins separate from those which are kept. Place (e.g., reabsorption, degree of maturation, spent the 25 selectealevins back into the incubatorovaries, etc.) (see 3.4). ...cupandpres e the discarded ones for initial weights. 2.13Special Examinatipas Fish and eggs obtained from the test should 2.11Alevin-Juvenile Exposure be considered for physiological, biochemical, and ihistologicalinvestigations which may Randomly (see 4.4) select from the incuba-indicate certain toxicant-related effects. tion cups two groups of 25 alevins each per duplicate for ,90-day grOwth_ and survival expo- 2.14 Necessary Data sures the,.growth chambers. Hatching -from one 4)awh may be spread out over ,a 3-to6-day Data that must be reported for each tank of a period; therefore, the median-hatch date shouldchronic test are: beused to establVL the 90-day growth and sur- vival period for ea cir of the two group of alevins. a. Number and individual weights and'total If it is determined that the median-hatch dates lengths of normal, deformed, and injured for the five groups per duplicate will be mare rtature and immature males and females at than three weeks1apart, then the two grodps of initiation of test, three months after test 25 alevins must be selected from those which are commences, at thinning and at the endof less than three weeks old. The remaining groups test. in the duplicate which do not hatch during,the b. Mortality during the test. three-week period are used only for hatchability c. Number of spawns and eggs. Amean,.:', results and 'then photographed for lengths and incubation time should be calculated using preserved for initial weights. In order to equalize date of spawning and the median-hatch the effects of the incubation cups on groyth, all dates. groups selected for the 90-day exposure must' d. Hatchability. remainin the incubation cups three weeks e. Fry survival, growth and deformities. before theyarereleased \intothegrowth chambers. Each of the two groups selected per 3.0 CHEMICALSYSTEM dOplicate must be kept separate during (the 9 day period. Record mortalities daily, along 3.1Preparing a Stock Solution wig total lengtIA 30 and 60 days post-hatch,and If a toxicant cannot be introduced into theI 3.5 Methods 4:3Distribution of Test Organisms The test organisms should be assigned to tanks e desired infor- . When they will p by stratified random assignment (random assign- mation with acceptable eision and accuracy, ment of one test organism to each tank, random methods described in M thods-for Chemical assignment of a second test organism to each Analysis of Water d Wastes (EPA, 1971) tank, etc.). should be used unlethere is another method ,which requires m ch less time andcan provide 4.4Selection and Thinning Test Organisms the desired information, with the same or better At time of selection or thinning of test precision and accuracy. At a minimum, accuracy organidns the choice must be random (random, should be measured using the method of known as defined statistically). additions forallanalytical methods for toxicants. If available, reference sample should 5.0 MISCELLANEOUS 44' be analyzed periodically- kir each analytical method. 5.1Additional Information All routine bioassay flow-through' Methods ,4.0 STATISTICS not covered in this procedure (e.g., physical and chemical determinations, handling offish) should be followed as described in Standard 4.1Duplicates Methods for the Examination of Water and Use true duplicates for each level of the toxic Wastewater (American Public Health Associ- agent, i.e., no water connections between dupli- ation, 1971). cate tanks. 5.2 Acknowledgments -4.2Dish4.tition of Tanks C- These procedures for the brook trout were compiled by J. M. McKim and D. A. Benoit for The' tanks should be assigned to locations by the Committee on. AquaticBioassays.The stratified random assignment (random assign- participating members of this committee are: ment of one tank for each level of the toxic Robert Andrew, John Arthur, Duane Benoit, agent in a row, followed by random-assignment Gerald Bouck, Wil1ia3x1. Brungs, Gary Chapman, of the second tank for each levik of the toxicJohn Eaton, John ,Hale, KennethHokanton, agent in another or an extension the same.James McKim, Quentin Picketing, We eSmith, row). Charles Stephan, andJames Tucker. '6.0 REFERENCES For additional information concerning flow-through bioassay tests with brook trout, the fqllowing references are listed: "`- 1 Allison, L. N. 1951. Delay of spawning in eatern brook trout by means of artificially prolonged light intervals. Prog. Fish-Cult. 13: 111-116. American Public Health Association. 197J. Standard methods for the examination of water and wastewater. 13th ed. APHA, New York. 0 Carson, B. W. 1955. Four years progress in the use- of -artificially'controlled light to induce early -spawning of brook trcut.Prog. Fish-Cult. 17: 99-102. Drummond, Robert A., and Walter F. Davison. 1970. An inTpensive method for simulating Diel patterns of lighting in thelaboratory. Trans. Amer. Fish. Soc. 99(2): 434-435. Fabricius, E. 1953. Aquarium observations on the spawning behaviOr of the char,Salmo alpinus. Rep. Inst.Freshwater Res., Droning- holm, 34: 14-48. Hale, J. G. 19633. Observations on brook trout, Splvelinus fontinalis spawning in 10-galion aquaria. Trans. Amer. Fish. Soc. 97:7, 299-301. Henderson, N. E. 1962. The annual cycle in the testis of the eastern brook tout, Salvelinus jontinalis (Mitchill).Can. J.ZoOl..40: 631-645. 30 BROOK TROUT BIOASSAY -Henderson, N. E. 1963. Influence of light and temperature on the reproductive cycle of the eastefn brook trout Salvelinus joittinalis v (Machin). J. Fish. Res. Bd. Canada, 20(4): 859-897. , Hoover, E. E., and H. E. Hubbard, 1937. Modification of the sexual cycle in trout by control of light. Copeia, 4: 206-210. MacFadden,1 1961. A population study of the brook troufSalvelinus fontinalis (Mitchill). Wildlife Soc. Pub. No. 7. McKim, J. M., and*D. A. Benoit. 1971. Effect of long-term exposures to copper on survival, reproduction, and growth of brook trout Salvelinus fontinalis (Mitchill). J. Fish. Res. Bd. Canada, 28: 655662. Mouni, Donald I. 1968. Chronic toxicity ofcopper to fathead minnows (4'imephales promelas,,liafinesque). Water Res: 2: 215-223. ,...i Mount, Donald I., and William Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Res., 1: 21-29. Pyle, E. A.A969. The effect of constant light or constant darkness on the growth and sexual maturity of brook trout. Fish. Res. Bull. No. 31. The nutrition of trout, Cortland Hatchery Report No. 36, p 13-1.9. ) U. S. Environmental Protection Agency, 1971. Methods for Chemical Analysis of Water andWastes. Analytical Quality Ccintrol Laboratory, Cincinnati, Ohio. Wydoski, R. S., and E. L. Cooper. 1966. Maturatien and fedundity of brook trout from infertile'streams. J. Fish. Re's. Bd. Canada, 23(5): 623-649. .1 tf% r 1 . , BIOLOGICAL MET,' Appendix A Test (Evansville, Indiana) Photoperiod For Brook Trout Partial Chronic Dawn to Dusk Time Date -Day-length (hour and minute) 6:00- 6:15) MAR. 1 12:15) 6:00( 7:00) 15 13:00). 6:00 - 7:30) APR.' 1 13:30) 6:00 - 8:15). 15 14...15) , . 6:00 - 8:45) MAY 1 14:45) 6:00 - 9:15) 15.15:15) 6:00 - 9:30) JUNE '1 15:30) Juvenile-adult eXposiire 6:00 - 9:45) 15 1,5:45) ye, -9:451 JULY 1 15:45) VA tj- 6:00 - 9:30) 15 15:30) 6100 - 9:00) AUG. 1 15:00) 6:00 - 8:30) , 15 14:30) ) 6:00 - 8:00) SEPT. 14:00) 6:00 - '7:30) 15 13 30) 6:00 - 6:45) OCT. 1 .12:45) _ ,t 6:00 - 6:15) ...- c15 12:15)Spawning and egg incubation. ) 6:00-.5:30) NOV..1 11:30) 6:00 - 5:00) 15 11:00) 6:00 - 4:45). DEC. 1. 10:45) 6:00 - 4:30) ,15.10:3e) t . 6:00 - 4:30) JAN. 1 10:30) Alevin-juvenile exposure:- 6:00 - 15 10:45) ) 6:005:15) FEB. 1 1i:15) 6:00 - 5:45) 15,11:45) 44 9o ti 4 e ,T 32, BROOK TROUT BIOASSAY Appendix B Tetnperature Regime for. Brook Trout Partial Ironic Months Temperature ° C Mar. 9 Apr. May 14 Juvenile-adult exposure June 15° July '15 'Mg. 15 Sept. 12 Oct. 9 Spawning and egg incubatiori constant temperature Nov. 9 niust be established just Dec. 9 prior to spawning and egg incubation, and maintained Jan. Alevin-juvenile exposure throughout the 3-month Feb. r 9 alevin-juvenile exposure. Mar. 9 1* 33 F) la APPENDIX Page 1.0 BENCH SHEETS 1: 1.1 Phytoplankton Sedgwick-Rafter Count i. 1 1.2 Zooplankton Count 2, 1.3 Plankton and Periphyton Diatom Analysis 3 :1.4' Periphyton Sedgwick-Rafter Count , ( 4 1.5 Plankton and Periphyton Pigment and Biomags 5 1.6 Macroibvertebrates .6 2.0 EQUIPMENT. AND SUPPLIES 7 , 2:1 Plankton: and Periphyton 7' 2.2 Ivracroblvertebrates 8. 2.3 Fish ...... r ...... ,f . 11. 3.0 ,UNITS OF MEASUREMENT CONVERSION FACTORS 13 zit c. 10, f. 1' 1 .110. " BENCH SHEETS 1.1 :1iyibp1ankton. Sedgyvick-Itafter Count P;y10planktonSedgsick-Rofter Count River or Lake 4 ,Date, Analyzed Station No. v. Station, Analyzed by ra te Collected, 'State , co ES ORGANISM 1ALLY CAL TOTALS Tote_ coocoid blue- enal Totalfilamentous blue- en algae{ . 01, Total coccoidgreen algae1: "renal filamentous greenlagae 6.! -Total en fl 1111111111.111111 111111111MMAIIIIIIMINIMMIIIME1111111111111111111112=511111111 _ atea< Most Diatom. Abundant Centr4c Melcm. OthersiTotal c/ml Al e' Shells Live , I To live centric diatoms- s 1 c /al. Pennare Shells Pennates , I -4 I Total liva ;ennate diatass.c S-p Factor: TOTAL LIVE ALGAE' C/m1 ) 4. Remarks: First check Wash. sheet Recorded Wash. sheet checked 1 vs4 47 1.2Zooplanktori Count \ oR , Znoplo.nkton Count \ COM404ANDNK TALLY CjITTER aarttaaA Karsten& Brachionur Syncyncbaeta T7ichocers Total/Rotifers per liter-< mitsocamA, Ceriodaphnia riOpsponA Nauplii CyclopsA related genera -Diaromils Total Crustacea r liter, . NEMATODES(per liter) = OTHER i/VilipTEBRATES: per liter)/ Most Factor Abundant Rotifers ' 1.3Plankton and. Periphyton DLit Om Analysis. .PLANKTbN AND PERIPRYTON D.IATON ANALYSIS Rivbr Station State Station Nusear Live Centiice Dead Centrics t. Date C011ected Live Pennates Dead Pennates Analyzed by Total Live Total Dead Date Analyzed S-R Count Counting Time' Species Total _ Species Coscimodiscua Ah. 'Pragilaria crotonensie cyclotella , 4construens Veneghiniana Frustulia 17lie °airs ambigua Gompboneie granulata kiyrosiges - diets:1s tiferion circulare a' Ilavicula' C's (Rbdzosolenia . StephanodIscus hantzechii invisitatus aati-ea ndnutula 5kitischia r. Other centric' Achnanthes dbaphiprora Pinnularla Aapbora l Pleurosigas . . 4 ,Aaterionella torsos& ihoicos curvate .144-j Caloneia.. RhoPelodia Coccopels Surirella CyzetoRleu.ra Cymbella Synedra 41..7., 4, Dia vul1az4 3. Diploneis omit/lid bellaria Epithemia feneatreta Eunotia) flocculase FIRST SECOND THIRD FOURTH Percent ode others 4 '.No. species R 4?° )/' 1.4Periphyton Sedpkick-Rafter Count . . - 'Q',,=. A. PER HYMNsi,xc4icl-kATTE2 cOODP , -... -,-,', .-. -River or Lake . Inclusive Dates 4,,,, - "."' Station , ° Date Antlzed ,..- ; .,1, State Analyzed -by "- . 2 ,'cODE ORGANISM .Tally C mm .4+ Total coccoid blue-green algae 'c a Total filamentolss blue-green algae < . -41 /Total , coccoid green algae < Total filamentous green algae. a reen ellates Otheroccoid al e I " "-Other < . Filanientnus.bacteria a'nd fungi < GP . Protozoa:< -; /min . 41. Diatoms Centi-ic Shells. LiVe Centrics Arrirn Total live centric diatoms . , Pennate Shells --':Liv_e'Pennates v Total live pennate clgatans . # Rreservatrive S-R Factor. 'TOTAL No. Slidei . (Geils/mm4) ti -Area S-craped Remarks): Scrapings diluted to ml s. ;First check* Recorded ° 1.5Plankton and Petiphyton Pigment and Biomass , PLANKTON AND PERIPH CHLOROPHYLL AND BIOMASS IDENTIFYING INFORMATION: 2 X. Station: B. Date: CI Method of Sample Colifetion and Handling: r.4 II:. SFECTROPHCTOMETER DATA: A. OTICALMITyetaAaMENTS: Extract Dilution OptiOnl Denaityligadirngs--1663 Volume khetor 0 6636*645 630,. 663a* b/a 'Hop. I. 2. 3. *(b -"before acidification; a afteracidification)' B. CHL6ROPiiYLL.CALCULAXIONS: Concentration of .4-, Sample area Chlotophyll content Chlorophyll in Extract or volume of sample (mg/1). ' (liters; m2) ( ug/1; mg/m2) .\,... , Chi. a Chl b ..Chl c i Chl a Chl b Chl c i. ' N,s .Rep. 1. 2. ,1 . 3. 4. III. FLDOROMFfFir Instrument Used: Weadi kore,(6) Rddding After(a) 1 ,A0dification Acidification ' DilutiOn -Reading - Setts. Factor Et4r. Level (54 Re (3) Rb/ita. Rep. 0 1. 4. N. ORGANIC MATTER (ASII=FREE WEICIL.`) A. -Empty tI Weight Weight E;iipae A34. ' Organic' Cruc. ible with Dry After Dryry -F ' Matter No. Sample Firing Welight Weight (EF/m9 (11),, (C.) '- (PA). (B-C) a 1.6. Macroinvertebrates 49, 414# MACROINVERTEBRATE LAB BENCH, SHEET Name f water body Lot.ft. Collec ed by imStation No. Sorted IFDate collected L(N)1 P1 TOTAL f, DRY, WGT TOTAL DRY WGT DI PTE RA7 CRUSTACEA # (mg) ft ("A ft T RI CHOPTE RA HIR1JDINEA PLECOPTERA NEMATODA 2 EP HEMEROPTERA - rYBIVALVIA . TOCIATA GAST ROFODA e -1. NEU ROPTE OVAER HEMiPTE RA COLEOOTERA Total lk of organism Tofal dry weight Total ft of taxa Ash-,free ,weight N = nymph, P = pupae ** Initials of taxonomists in -this' Column d. Y - - , 2.0 QUI MENT AND SUPPLIES 41. This section contains an abbreviated list of 'equipment andsupplies used for the collection and analysis of biological samples. The companies andaddresses are listed alphabetically at the end of the table. Mention of commercial sources, or prociucts in this,section does not constitute endorsement by the U. 5. Envirowental Protection Agency. . ppros,. Item ,Source* Cat. No. Unit cOSt (1973) . I. 2.1Plankton and Periphytion - * ; . Sampltng an4 field equipment 1 Water sampler,alpha, hot tle, nonmetallic, transparent,.6 liter 'N.,. (30) 1160TT $ 150.00 Plankton saMpft.ir'ftlarr Gr 1 *See list of suppliersiit the end of this table: Item Cat. No. Unit Approx. Source Cost (1973) Light meter - (29) Model 756 100.00 Muffle furnace, 1635 Temco, Thermolyne, 240 -volts . . 180.00 Temperature control for muffle,furnacc, Amplitrol Proportioning Controller, 0-2400°F, for 240 volt furnaceteciimmended for use with Temco 163').1 230.00 Oven, Thermozone, forced draft, double walledahree shelves, 230°C. '350.00 Pipetting machine automatic, large, BBL. (for dispensing preServalive)., 7750-M10 320.00. "Spectrophotometer, double-beam, recording, resolution 2 `or better at 663.nm; Gileman-124 or equivalent. Washer, mechanical, glassware, variable speed, Southern cross, Model, 300-B-2, , - IA Complete. 330.00 , . . Supplies Cubitainer, 1 qt (approx IJiter) 1 doz. 7.00 .ub Rainer shipping carton, 1 qt ...... 1 doz. gr 4.00 ittles, pill, square, DURAGLAS, 3 ounce for periphyton samples. Do notuse . c pS supplied with bottles. t clear glass 1/2 gross" '8.00 , amber glass 1/2 gross 1§.00 Caps, Polysial, black, size38,G. CAIthreadNo. 400. Use on Duraglas bottles - above, (16) 1/2 gross Crucible's, Co'ors,,Itigh form, porcelfin. size 1, capacity 3Q ml (24) 3319-055'Case (36) 11125.0 Crticible Covers for above, Size G . , . '(24) 33I9-D47 Case (72) Desiccator, aluminun, with shelf (24) 3747-C10 22.00 Merthiolate, poWder No. 20, (Thimerosal, NJ') (13) '/4 ounce 2.00 - . 1 ounce 7.00 1 pound . 95.00 ' -1 1 Metal plate, 5,X 10 X 1/8jnches,'steel (to transfer cover glasses betWeen . 0 (iot-plates). . Micrometer, eye-piece, whipple , p, .64,st"* ', (J6) . 18:00 Micrometer, stage (American Optical) ' (36) ,, 400 31:00 1' Mounting medium, HYRAX s,' .,... (3)-. 1 ounce 10.00 Pipettes, disposable, Pasteur type, 5-3/4 inches (23)., .N205-2, 21/2 gross 8.0(1 Sedgwick-Rafter CountireChamber, as preset ibed by "Standard Methods for the .e examination ()M ater and Wastes." . . . (30) 1801 9.00 issue grinder, glass, Dualt, complete , . (12) , size C 10.00 VialS,'Opticlear, Owens-Illinois, 3,drams, snap caps, for diatom prreparation. (-21) SK-3 a Gross 11'.06 ,2.2Macro inver tebta t ) Boat, flat botte:m, 14-lb teet, ArkansaS Traveler or Boston Whaler with winch sitch-block meter wheel, and ttailer, 18 hp Outboard motor, Life RI., other accessories ... (17) ." 'v 3;000.00 ' N hastening tools: /4 (20) able clamps, 1/8 inch 2&.!'*; 3.00 Nicro-press 'sleeves, 1/8 inch Nu.. 6.00 Nicro-press tool; 1/8 inch' .32.0() Wire cutter, Fele() 7-' 7.00 Wire thimPles,'1/8 inch 2.00 Cable, 1/8%inch, galvanized steel '89.00 Large capacity, metal wash tubs . -3.00 Cote' sampler, K. B., multiple, and gravity corers (30) )225.00 ,kirdboard multiplate sampler (30) 7,50 Trawl net 1 (30) 100.00 'Drift net, stream (30), 15 1.00 Grabs Ponar s. 1725' 290.00 Ekman, 6 X 6 inch ,19613 78.do Petersen, 100 square inch r750 200.00 Fights for Petersen 1 \751;..` 2,5.00 1!"c14 k Approx. Item Sdurce Cat. No. Unit cost (1973) Basket, Bar-B-Q, (R13 -75) Tumbler (22) 12 25.00 Sieve, US standard No. 30 (0.59 In opening) and others as needed (26) V 73250 L 1 each 10.00 ArtIow meter, TSK, (propeller t (10) 313 TS: 2op:QO Flow meter, electromagneticwo-axis (15) 2,600.00 Mounting media, CMC-94F (6) 4 ounce 2.00 Mount ing Media,CMC-S (6) 4 ounce 2:00 (31) 94370 1 500.00 . Low-temp bath Water pump, PO.Ky-encapsulated, submersible and open (14) 1A-MD 2 50.00 Sounding equipment and specialized gear (7,9,11) . Large, constant temperature holding tanks with 1/3 hp waterchiller, charcoal 15) MT -700 540.00 !r*:Polyeth3,1ene bottle's, dark bottles, tubing J' (18) tahn electrobalance (27) DTL 1,00'0.00 tinlapped 1 pOund 0.30 Porcelain balls for baskets (2-inch diameter) 0 st; Porcelain intatipla(es s 41 / 7.50 Offerential, 9 unit, Clay-Adams B412 . ) 105.00 -*Counter,Counter, hand .taliy 3297-1410 11,00 Magnifier, Dazor, 2X, floating, with illuminator and base. 6) N A 95 50.00 Microscope, compound, trinocular, equipped tor bright -belt! a41 Pita3e microscopy I. . N1.4 with 10X and 15X Wide-field oculars, 4,0 X, 10X, 20X, 454ant1190X bright:- field objectives, and 45X and 160X phase objectives. 2,000.00 Stereoscopic dissecting Microscope 2) L 1,000.00 Tesso.var photomacrogaphic Zoom System 49-65-01 1 1,77-9.00 h Camera body, 35 mm Zeiss Contarex, for Tessovar (32) .10-2611 '1 600.00 (6) 375AA4514 42.50 Stirrer, magnetic --_:. Aquaria (of various sizes) (6) Aquatic dip nets le (6) 10 gross Microscope Slides and Cover slips, Standarif square, 15 'min, , (6) 320A io 31.00, 320A210 '1 ounce., 3.50 Vials, specimen, glass,;l dram, 15 mm X 45 min e6) 315A 57 10 poss 78.00 4)stri dish, ruled grid, 150 mm X 15 mm (2)' 315AA4094 '12 Freeze dryerwith freiriing shelf (28) 10-800 1 loo 4,00b :00 Vacuum oven (19) 5831 300.00 0 0 Sources of equipment and supplies for plankton, periphyton, and macroinvertebrates I. Coleman Instruments 17. MonArk Boat Company '42 Madison St. Monticello, AK 71655 Maywood, IL 60153 18. Nalge Corporation 2. Corning Glass Work& Rochester, NY 14602 11470 Merchandise Mart Chicago ,1L 60654 19. Nation;\Appliance Cont P. O. Box 23008 Custom Research and Development Company, Inc. Portland, OR 97223 Mt. Vernon Rd., Route 1, Box 1586 Auburn, CA 95603 20. National Telephone Supply Company 3100 Superior 4.. Ferro Corporation Cleveland OH P. 0. Box 20 East Liverpool, OH 43920 21. Owens-Illinois . P. 0. Box 1035 5.Frigid Unitl, Inc. Toledo, OH 43666 4 3214 Sylvania Ave. Toledo, 011.43613 22. Paramont Wire, Inc. 6. General Biological Inc.. 1035 Westminster Ave. 8200 S. Hoye Ave. Alhambra, CA 91803 Chicano; 60620 23. Scientific Products .7. -6-M Man ufacturing & Instrument Company 1210 Leon Place 2417 Thh-p Ave. EvanstonIL 60201 New York\ NY 10451 24. Arthur H. Thomas Company 8. Hedviin Corporation Vine Street at Third 1209 E. LincOlnWay P. O. Box 779 Laporte, IN 46350 Philadelphia, PA 1.9105' 9. Hydro Prjoducvs, 25. G. K. Turner, Assoc. 11777 SeTrrento Valley Rd: .2524 Pulgas Ave. San Diego, CA1:921m2.1 Palo Alto, CA 943Q3 10. Inter Ocean, Inc. 26. W. S. Tyler Company 3446 Kurtz St. Mentor, OH 44060 San Diego, CA 92110 27. Ventron Instrument Corporation ik. Kattt Scie ptific Instruments .7500 Jefferson St. **°- PAX Box 1166 Paramont, CA 90723 El Cajon, CA 92022 28. Virtis Company 124Kontes Glass Company Gardiny, NY 12525 ''.3/inelind, NJ 08360 29. West441a-avrments, Inc. 13. EliLilly Company 614 Freiinghuysen Ave. 307 E. McCarty St. Newark, NJ 07114 Indianapolis, IN 46206 SO. Wildlife. Supply Company 14. March Manufacturing Company 301 Cass St. Glenview, -IL 60025 .11 Saginaw4MI 48602 15. Marsh-McBirney, Itic. 31.. Wilkens-Anderson Company 2281 Lewis Ave. 4525 W. Division St. Rockville, MD#20851 Chicago, IL 60651? 16. Matheson Scientific 32. Carl Zeiss, Inc. .4 -1850 Greenleaf AW. 444 Fifth Ave.- Elk Grove Village, IL 60007 New York,NY 10018 1 L. 10 2.3Fish Sources of information on fishery sampling equipment, ... American Association for the Advancement of Science. Annual guide to scientific instrumehts (Published inScience). American Societyf Limnology and-Odeanography, 1964. Sources of lininological ary:1 oceanographiE apparatus and supplies. Special Publ. No. 1.1 xii. Oceanology Int onal Ye.irbook/Directory. Sinha, E. Z., and C. L. Kuehhe. 1963.' Bibliography on oceanographic instruments. 1. General. II.Waves, currents, andott geophysical parameters. Meteorol. Geoasirophys. Abst. Amer. Meterol. Soc.14:1242-1298; 1589-1637. U.S. Fish and Wildlife service. 1959. Partial list Ofmagufacturers of fishing gear and accessories and vessel e pment. 'Fishery Leaflet . 195. 27 pp. Water Pollution Control Federation Yearbook. rr Cr 1 ML A UNITS,OFMEASVROVIEST STATES conversion -.Factors and SpecialTables IEPARTMENT OF ERIN UCATION c. October, 1172. REPRINTED`FROM Units ofWeightandMeasure, Internationat,(Metric) and U.S.Customary NBS Miscellaneobs;Publication 286- May,1967' -WITH THE COURT Y . TE,RtfiCAt INF, RMATIO"N OFF IC -NATIONAL itiSE4CH,CENTE11, Ci,NCINN 1± -113: ENVIRONMENTALgPROT*T01.0iVAilifNCY b . ,CONMENTS (Reprint includes only those_items asterisked) * INTRODUCTION THE INTERNATIONAL SYSTEM Prefixes HISTORICAL OUTLINE France The United States WEIGHTS AND MEASURES IN THE WORLD'S INDEPENDENT STATES., Metric NOnmetric 6 IMPORTANT DATES IN. U.S,METRIC HISTO 7 0 .'SELECTED BIBLIOGRAPHY *DEFINITIONS, *Definitions of Unita 9 *SPELLING AND SYMBOLS FOR UNITS 10 Some Units and Their Symbols 10 UNIT OF MEASUREMENTCONVERSION FACTORS ,, * 44Length _ - 11 *Maas 2- 12 *CapaCity, or Volume "13 *Area ,, ,t, 17 *SPECIAL TABLES ., 0 *Equivalents of Decimal and Binary Fractions of an Inch in Millimeters 18 *International Nautical Miles and Kilometers ,_ 19 UNITS OF MEASUREMENTTABLES OF EQUIVALENTS . .. Length -: 41 y Mass f 127 4 .Cap icity, or 161 Volume , d. Area 7:- 219 . ,Chisholm, L.J., Units of Weight and'Me sure. Intel'national (Metric) and U.S. Customary. (MS Mi cellanebUs Publication 286);, For S4e4IV Superintendent of -Do ents, U. S, -Governdent 'Printing Office, Washington, D.C. 20402. Price $2:25. ° . A ' t. *149 a a Units Jof. Weight and', Meagure International (Metric).and U.S. Gusto ark L. J: Chisholm"' 1 The primary purpose of thiii publication isto.maice available thd most oftelf-- . needed weights and measures conversiontablesconversions between the U. S. Customary System and Internatonal (Metric) System. A secondary purposeis', to present a brief historical outline of the International(Metric) System following it from its country of origin, France, through its progressin the United States. Key Words: Conversion, tables, Interns System (SI), Metric Systert-, U. S. CuStomary,,Systein; and measures, weights and . --. meavines;,ahbreviatioru :wei Pit and miasures systems, weights and measures ilriiisi. IntrOduct Two systemS of weights and measures exist side by side inthe United States today, with roughly equal but separate legislative sanction: the U. S.Customary System and the International .(Metric) System. Throughout U. S. hisioiy, theCustomary System- (inherited 4 frbm, but now different from, the ,BritishImperial System) has been, as its name implieit; 4, customarily used; a plethora of Federal and State legislationhas givertapth-fough impli4j; thin, standing as our primary weights and measures system.nowevel'," the Metric System (inCorporated in the scientists' new SI ,or Systeine International d'Unites) is theonly pys- . tern.that has ever received specific legislative sanction byCongress. The "Law of 1866" reach: It shall be lawful throughout the United Statesof America to employ the weights and measures of the metric system; and no contract ordealing,--Or pleading in any court, shall be deemed invalid or liable toobjection because the weights or measures expressed or referred tother4in are weights or measuresf the metric system9, Over the last 1061rears, the Metric System hasIken sloW,steadily increasing use in the United States and, today, is of importance nearlyequal to the. tomary System. The International System * For'uptodate information on the international metricsyqlem,,,,, See current edition of The InternationalSystem of Units (l)i Editors: Chester Page and Paul-VigoureUx(NBS Special Publication 330). For sale by ,Superintendent Of Documents, IS.Government Printing Office; Washington, D.C.H20402.-Price.* cents.- For .NBS policy on the usage of SI, see. NBS Techn±cal.NewsBulletin Vol. 55,11o. 1,. pp.. 18-20, Jantary 1971. k I Act of 28 July 1886 (14 stet. 339)--AnAct to authorise the use-ofethe Metric system of Weights and Measures. a 3 (' Slz untAft'mive been adopted to serve as thebasefor the International Sys' tem:' It"' - . ." ._meter -Vass ° kilogram _____ ----- = - L ------second Electric, current, _ _ - 27------", TherriipOyn ie, tetl7"perature , Light intensity ". 4:tcandela. .,/ the 'other More erequentry used units of the SI and their symbols'and, where ,applicsable, their derhrationsare listed below. SUPPLEMENTARY --UNITS uantiiy Unit Syinbo Plane angle radian rad Soli angle steradian sr DERIVED UNITS -Area square meter Volunie cubic meter m' 1 Frequ-ency hertz Hz (s-') Density kilogram' per cubic meter kg/n0 Velocity meter per second ''.m/s Angular velocity radian pet second i/q Acceleration meter per second squared. m/s, Angular acceleration radian per second squared rad /s'2 Force' newton N (kg- m/s2),, Pressure- newton per square meter ,N/m2 I{inem)ttic viscosity 'square meter per second - " ' m2/s Dynamic viscosity newton-second per sol, -.re meterN-s/m2 Work, energy, quantity of heatjoule J (Nm) Power watt W (Vs) Electric c1Ige coulomb C (A s) Voltage,potential;difference,volt V (W/A) -electroniOti*e:lOrea ° Electric field strength volt per meter V/m Electric resistance ohm ,42 (V/A) Electric capacitance - farad F (As/V) Magnetic flux weber Wb (V -s) Inductance ,henry H :(V-s/A) Magnetic flux density tesla T' (Wb/m2) Magnetic field strength ampere per meter A/m Magnetomotive -force ampere A Flux of light lumen im (cd Luminance candela per square meter cd/m2 Illumination lux lx (1m/m2) * Recent ,(1971-) ,additionorthe mole as the unit forvamount of snlostance bririgs the total tosO.EAunits. See asterisked footr note on,page.l. No PAGES .328 MISSING FROM DOCUMENT PRIOR TO BEING SHIPPED TO ,EDRS FOR FILMING. BESX COPY "AVAILABLE Definitions In its original conception, the, meVir *as the fundamental unit of the-MetricSystem, and all units of length and capacity were t,be derived directly from .the meter which was --,ti. intended to be equal to one ten - millionthf the earth's quadrant.'FurthermOre, it was'. originally planned that ths,,unit of mass, thkilogram, should be identical:With the mass of a cubic decitneter of waye at its maximum dehsity.The units ofetigt1CrInd mass are_now -,`, defined, independently of these conceptions. .; : iin'WoOts and Iri October 1960 the Eleventh °General (International) Conference , Measures redefined the meter as equal -to 1 650 763.73 waveleniths of the orange-redradia= tion in vacuum of krypton 86 correponding to theunperturbed; transitipr heiweerOhe 4't, 27910 and 5A levels. '4 ,, 7 -The kilogram is independently defined as the mass of a particular platinum-iridiuin standard, the International Prototype' Kilogram, whin is kept at theInternational Bureau o( Weights 4nd Measures in Sevres, France. . - -',-: The liter has- been defined, since October 1964, as .being equal toicubic. deeimeter. The meter is thus aUnit oh whicis based all,.4etric standards and'measureinents oflength, area,and, volume. ., Definitions 'of 'Units 4' ttl -V' Lenit11. - : . , . wavelengths in a vacItum of the orange- A meter is a unit of length equal-to. 1 650 763.73 d 'radiation of krypton 86. A yard is a unit of length equal to 0.914 4Meter.. - A kilogram unit of ss equal to the mass of theIntanatioiyil,Prototype An avoirdupois pound is aunit ofmass equal to 0.453 59237 kilogram,- Capacity, or Volume A cubic meter is a unit of volume equal to a cubethe edges of which are 1 meter. A lifer is a unit Of Volume equal to;a cubic decimeter. A cultic Yard is a unit of volume*equal to a±cube the,edges of ,which are 17yard; . - A gallon i4 a Unit of .volume *equalto 2,31 cubic' inches.It is used or measuring liquids > . ohly.. A busbel_is a unit of volUrne.equal to"2 1;50.42cribit;iriches. It i9 used 'for measuring ci,ry comrnodities only. Aria sq r is 'a unit of area equal oto the Area of a, square the, sides. ofWhich ate 14. ' in of\Which-are 1 yard, . sgUalle yar'd is a unit of area equal to the area of a square, the side Spelling arAd Symbols for Units , The spelling ,,o'r the nanues-611'Units as- ado)ted by theNational itteau of Sta.ndaKds, is that given,in:-the list lieluw. The "Erelling ofthe metric .1.Mitas: in accordance with that giveri_in, the lak"of ?g, 1866, legEliting the iNletrie System in tjie United StateS. Following the name of each unit in the list belowgiven the''symbolt at the Bureau has adopted: Attention i.particularly ,called to the.follfting principles: *No period.,is used With'SyrnbOls,for',units. Whenever " for inch m ht be,confused preposition 'tinch" should be spelled out. r, The exponents and ;1."' 'are Used to signify "squaie" and it respectively, instead of the..Symbolsl`k""or. o4`elf;':. .111,..tveveri,-kei4uently- used in technical lilittratureOi the U, S:. CiistOintaiy units. ,*? ' - r'The s9ri a synibOl is used for both`singulfrand plural. Some*Unifs and"Theii Symbols !Un Symbol Symbol I Unit ° 'Symbol. . acre' acre fathom Path millimeter him . are a foot minim ° minim +, laiirekh bbl furlong furlong ounces oz 'beardlOOt , i. .galtoriv° . "Otiice, ft voirddpol# oz avdp, buihel` bu ,grain:;: grain -.`,L ounce,tiquid oz car gram, ounce, troy, otr " . .Gelsslrdey3ree, "(;" hectare ha* peck peck cent re ca ,hect-ogritm hg . pennyweiglit dwt cetifigra-rn dg `hectoliter hl . o 4 pt centinter 9 cl `hectometer t. : pound . . . centimeter hogshead . ib pound, avoirdupois. lb avdp ". ,,, ," et; hundredweiiht cwt . pound; tro$, 1 cubic tEentimeter 3' --t- quart, //quid modliq qt. cubic`de Internathina .rod,* . cubio de eter. Nautue"al:ile A cubic ft' -," .square centimeter cm' r 4cubid hectoindter hm3 .kilograni dm - cubic inch, inl kiloliter . square dekaineter , 'dal-ns- cubic kilometer km' ilometer . square foot , meter'°-. in Pkc; square hectometer hms- .Pubicmiler liquid square inch:: v cubic Millimeter min' liter ,4* square kilometer. km12% cubic yard ".!4 yd' * Meter square mqtOr n13 de4. microgram square mile deciliter- dl 'rnicroiric4 4. - squire millimeter "; de'cirri;lter ' 'I r3m..Q miCrofitei square yard dekagram . deg.; stere ' dekaliter dal mide ctonAng slYotdeerigsr 4n dekameter ton, metric dram, avoirdupois.-- ,,s1-!_avdp ton,_Ishort hort, to41` yard .3'd . Units of MeasurementConversion Factors Units of,Length To C,onvert from to.Convert from Centimeters r Metepr . To Multiply by To Multiply by Inches 0.3937008 " 39.37008 Feet 0,03280840 Feet 3.280840 Yards 4_ 0.01093613 Yanhi 1.093613 Meters. ' f' 0.01 Miles. 0.00062137 'Millimeters 1000 Centimeters 100 Kilometers 0.001. To Convert from Feet To Convert from To Multiply by Inches To Multiply by Inches 12 Yards 0.3333333 Feet_ )7(4..% 0.08333333 Miles 0.00018939 Yards 0.02777778 Centimeters 30.48 Centimeters 1 2.54 Meters 0.3048 Meters 0.0254 Kilometers 0.0003048 All boldface figures are exact; the others generally are given to seven significant. figures.' In using conversittn factors, it is possible to perform division as well as the multiplication process shown here. Division may be particularly advantageous where more than the significant figures published here are required. Division may be performed in lieu of multiplication by using the reciprocal of any indicated mul-ad tiplier visor. For example, to convert from centimeters to inches by division, refet to the table headed To Con ert from Inches" and use the factor listed at ,"centimeters" (p.54) as divisor. To Convert from To Convert from Yards MIles To Multiply by To Multiply by Inches .36 Inches 63360 Feet 3 Feet 5280 Miles 0.00056818 Yards 1760 Centimeters 91.44 Centimeters 160934.4 Meters 0.9144 Meters 1609.344 %. Kilometers 1.609344 1 c) Units of Mass To Convert from To Convert from `- Grams )Cliggrams To Multiply bye To Multiply Grains 15.43236-\ Grains' 15432.36 Ayoirdupois Drams 0.564383 4 / Avoirdupois Drama 564.383 4 Avoirdupois Otthees 0.035273 9t1 Avoirdupois Ounce; 35.273 96 Troy ,Ounces 0.032150 75' Troy Ounces' 32,150 75 Troy Pounds 2.679 229 TroOlounds' 0.002..679, 23 Avoirdupois Pounds 2.204 623 Avoirdupois Pounds ' 0.002204 62 Milligrams . 1000 Grams - 1000 Kilograms 0.001 Short Hundredweights 0.022 04623 Short Tone 0.001 10231 Long Tons 0.000 9842 TO Convert from Metric Tons 0.001 ., Metric Tone To Multiply by Avoirdupois Pounds 2204.623 Short Hundredweights 22.04623 Short Tons 1.102311'3 Long Tons 0.984206 5 Kilograrns 1000 b To Convert from -! To Convert froth Grains Avoirdupois Ounces To Muliirlly by To Multiply by Avoirdupois Drams 0.03657143 Grains, 437.5 Avoirdupois Ounces 0.00228571 Avoirdupois Drama 16 Troy OunceS 0.00208333 Troy Ounces 4 , 0.911 Troy Pounds ! 0.00017361 Troy Pounds 0.075 486- Avoirdupois Pounds 0.00014286 Avoirdupois Pounds 0.0625 Milligrams 64.798'91 'Grams 28.349523125 Grams 0.06479891 Kilograms 0.028349523125 Kilograms 0.00006479891 To Convert from To Convert from Short Hundredweights ,,Pt;oirdupois Pounds To Multiply by To Multiply by Avoirdupois Pounds 100 b Grains 7000 Short Tons 0.05 Avoirdupois Drams_ _ _ _ 256 Long Tons 0.04464286 Avoirdupois Ounces: 16 Kilogram's r 45.359237 Troy Ounces 14.58333 Metric Ton's 0.045359237 'Troy Pounds 1.215278 Grams 453:59237 Kilograms 0.45359237 Short Hundredweights 0.01 Short Tons 0.0005 Long Tong 0.0004164286 Metric Tong 0.00045359237 b 12 TO Convert from To Convert from Short Tons Long Tons To 1. Multiply by To Multiply by. Avoirdupois Pourids 2 000 Avoirdupois Ounce:S.__ 35 Short Hundredweights 20 Avoirdupois Pounds___ Long Tons 0.892 857 1 Short Hundredweights_ Kilograms 907.184 74 Short Tons .12 Metric Tons 0.907 184 74 Kilograms 1 016.046 908 8 4 Metric.Tons ______1.016 046 908.8 I ToConvekit from Troy Out ices, To Convert from To Multiply by Troy Pounds To Multiply by Grains 480 Avoirdupois Drama 17.554 29 Grains 5 760 Avoirdupois Ounces 1.097 143 Avoirdupois Drama 210.651 4 Troy Pounds 0.083 333 3 Avoirdupois Ounces 13.165 71 Avoirdupois Pounds 0.068 571 43 Troy Ounces 12 Grams 31.103 476 8 Avoirdupois Pounds 0.822 857 1 Grams 373.241 721 6 Units of Capacity, or Volume, Liquid - Measure To Convert from To Convert from Milliliters Liters To Multipl; by Multiply by Minims 16.230 73 Liquid Ounces 33.814 .02 Liquid Ounces 0.033 814.02 Gills 8.453 506 Gills 0.008 453 5 Liquid Pints 2.113 376 Liquid Pints 0.002 113 4 Liquid Quarts 1.056 688 Gallons 0.264 17205. . Liquid. Quarts 0.0011056 7.1 4" Gallons 0.000 264 17 Cubic Inches .h, 61.023 74 Cubic Inches 0.061 023 74 Cubic Feet 0.035 314 67 Liters k 0.001 Milliliters 1000 Cubic Meters 0:001 Cubic Yards Ilan 307 95 To Convert from Cubic Meters To Multipby To Convert from Minims Gallons . 264.172 05 To Multiply by Cubic Inches 61 0 4 Cubic Feet 35.314 67 Liquid Ounces 0.002 083 33 Liters 1 000 Gills 0.000 520 83 Cubic Yards 1.307 950 6 Cubic Inches 0.003.759 77 Milliliters 0.061 611 52 13 .4" To Convert from To Convert from Liquid Pints 0 Multiply by To Multiply by iVlinini 1920 Minims 7 680 Liquid Ounces 4 iquid Ounces 16 Liquid Pints 0.25 ills 4 Liquid Quarts 0.125 Liquid Quarts 0.5 Gallons 0.03125 Canons 0.125 Cubic (Inches 7.21875 , Cubic Inches._/ 28.875 Cubic Feet 0.004177 51,7`,-1., 0.016 710 07 Milliliters 118.29411825 Milliliters 473.176 473 Liters . 6118294 1113 25 Liters 0.473 176 473 Y. To Convert from Liquid O'unces To Convert from To ' Multiply by Cub( Feet To ir Multiply by Minims 480 Gills 0.25 Liquid 0.unces_(__T_ ----'957.506 5 Liquid Pints 0.0625 Gills /39.376 6 Liquid Quarts 0.03125 Li uid Pints '59.84416 Gallons 0.0078125 Liquid Quarts 29.92208 Gallons 7.480519 Cubic Inches 1.8046875 Cubic Feet 0.00104438 Cubic Inches '1728 Milliliters 29.57353 ' Liters 28416846592 Liters 0.02957353 Cubic Meters 0.028316846592 Cubic Yards 0.03703704 To Convert from Cubic Inches To Convert from To Multiply by Cubic Yards To Multiply by Minims 20.974 0 Liquid Ounces 0.554'112 6 Gallons 201.974 0 gills 0.138 528 1 Cubic Inches 46 656 Liquid Pints 0.034 632,03 Cubic Feet 27 Liquid Quarts 0.017 316 02 Liters 764.354 857 984 Gallons 0.004 329 0 Collie Meters 0.764, 554 857 984 Cubic Feet 0.000 578\7 Milliliters 16.387 064 Liters 0.016 387 064 Cubic.Meters, 0.00.0 016 387064 Cubic Yards 0.000 021 43 To Convert 'hop Toonvert from LiquidQuaqts allon Tp Multiply by To Multiply by Mini Ms 15.360 Minims 61440 i Liquid Ounces 32 , Liquid Ounces...._ 128 . Gills._ 8 'bills 32 Liquid Pints 2 Liquid Pints 8 Gallons 0,25 Lictkiid Quarts,,. A Cubic Inches ',1 231 Cubic Inches '- 57.75 Cubic Feet 0,03342914 Cubic Feet p.183 6806 Milliliters 946.352946 killiliteits 3785.411 784 s . Liters 0.946352946 Liters A 3.785 411784 Cubic Meters 0.003.785411784 CulticYards 0.004,r9M13 3 Units of Capacity, or Voluel Dry Measure -.- To Convert from To Convert from Liters i3ekaliter To Multiply by To ultiply by Dry Pints 1,816 166' Dry Pints *Mr 66 Dry Quarts 0.908 '0198 Dry Quarts' 9.08082c,8 Pecks 0.113 5 4 Pecks -,-. 1..135104 BtAiels 0.028 377 59 Bushels 0.283g75 9 Dekalittrs 0 1. Cubic Inches ,610.237 4' Cubic Feet, 0.353146 7 Liters 7- 10 To Convert from. . Cubic Meters To Multiply by To Convert fr 17 Dry Pints Pecks 113.510 4 ;To Multiply by Bushels 28.377 59 ., Dry Quart* '. O. Pecks 0 0. 0 g 5 Bushels f - 0.015 625 Cubic Inches 33.600 312 5 CCubic Feet 0.019 444 63 Liters 0.550 610 "47. DekaliTem-.. 0.055 061 05 1 15 0 19 r) 4V To Convert from To Convert from Dry Quarts Pecks To Multiply by To Multiply by Dry Pints 2 Dry Pints 16 Pecks 0.125 . Dry,Quarts 8 Bushels 0.031 25 Bushels _ 0.25 Cubic Inches 67.200 625 Cubic Inches 7.605 Cubic Feet 0.038 889 25 Cubic Feet .0.311114 Liters f\ 1.101 221 Dekaliters 0.110 122 1 Uteri' 8.8097675 Dekaliters 0.880976.75 Cubic Meters 0.00880977 Cubic Yards 0.01152274 To Convert from To Overt from Busks Is Cultic Inches To Multiply by To / Multiply by Dry Pints 64 Dry Pints 0.029761.6 Dr Quarts 32 Dry Quarts 0.0148808 PWIcs 4 Peeks 0.00186010 Cubic Inches 2150.42 Bushels 0.000465025 Cubic Feet 1.244456 Liters "35.23907 Dekaliters 3.523907 Trl Convertfrom Cubic Meters 0.03523907 Cubic Yards Cubic Yards 0.04609096 To Multiply by Pecks 86.784 91 Bushels 21.696 227 Tp Convert from `Cubic Feet To Multiply by - Dry Pints 51.428 09 Dry Quarts 2 14 05 Pecks 3.214 256 Burhels 0.803 56b 95 s a r r. To Convert from To Convert from , Square Centimeters Square Meters o Multiply by To c J Multiply by Square .Inches 0:1550003 Square Incites 1,550.003 Square Feet_ '0.00107639 Square Feet 10.76391 Square Yards or 0.000119599 Square Yards 1.195990 Sqtiare Meters 0.0001 'Acres 0.000247105 Square Centimeters 10 000 Hectares 0.0001 1, To Convert from I Hectares To Multiply by To Convert from Square Inches Square Feet 107639.1 To Multiply by Square 11959.90 Acres 2,471054 Square Feet_ 2 0.006 944 44 Square Miles 0.00386102 Square Yards 0.000 771 605 Square Meters 10000 quaro Centimeters 6.451 6 bAuare Meters 0.000 645 16 To Convert from To Convert from Square Feet Square 'Yards To Multiply by 71 Multiply by Square Inches 1,44 Square _ 1;19-6 Square Yards 0.111111 1 Square Feet: Acres 0,000022957/ Acres 0.0002086116 Square Centimeters 929.0304 Square Miles 0.000000322830 6 Square Me ors 0.09290304 Square Centimeters_ 8 341.2736 Square Meters 0.83612736 Hectares 0.0000836127361 7 4 TO Convert from Acres To Multiply. by To Convert from Square Miles Square Feet 43560 .',- To Multiply by Square Yards 4840i Square Miles 0.0015625 Square Feet 274,878 400 Square Meters - 4046.856'4224 Square Yards - 3 097 600 0.404685 640p Hectaies . 64224 Acres__.__ 1 Square Meters 2,589 988.110 336 .Hectares_ _ 258.998 811 033 6 ,194 17 4 Special Tables " Length-Inches and Millimeters-Equivalents of Decimal and Binary Fractions of an Inch in Millimeters, From I/64 'to I Inch Decimals ci Decimals %'s8th-.16ths32ds.64ths Milli- of InchWes40Rho16ths32ds64ths Milli-, of meters an inch s meters an inch ,.- 1 0.3970.01562 33 13.0970.515SIM / sa 1 2 .794 .03125 ( - - 17 34 13.494 .53125 '3 1.191 .048875 35- 13.891 .546875 1 2 4 1.588 .0625 9 18 38 14.288 .5825 r- r 5 1.984 .078125 37. .\14:684 .578125 3 8 2.381 .09 19 38 15.081 .59375 . 7 2.778 .1093. 39 15.478 .609375 1 2. 4 Er"" -3..175 .1250 5 10" -,I 40 - 15.875 .825 I. \ 9 A., 3.572 0025 41 16.272 .640625 5 10 3.969. .151 5 21 42 16.669 .85825 11 -= 4.386 .171875 43 17.066 .871875 3 8, 12 4.78 .1875 I11 22 . 44 17,. 4g2 .6875 . 13 5.1 9 . .203125 45 17.859 .70312 7 14 5.556 .21875 23 48. .71875 .....,.. 18.256 15 5.953 .2.34375 _ 470' .18.653 .734375 1 2 8 -- 6 - 8.350 .2500 3 8 12 24 48 191050 .75' 17 6.747 .2656,251 . 49 =19.447 .785825 9 18 7.144 .28125 25 50 19.844 .78125 19 7.541 .296875 51 a20.241 .796875 10 20 7.938 .3125 13 28 52 ,c 20.638 .8125 . 40' 21 8.334 .328125 53 21.034 .828125 11 22 8.731 .3975 , 27 54 =21.431 .84375 55 =21.828 .859375 3 8 12 2324 Q....99.512258 .3375950375 7 14 28 56 ...22.225 .875 25 9.922' .390625 57 22.822 .890625 13 28 - 10.319", .40625 ,04' 29 58 23.019 .90625 .41 27' 10.716 .421875 - 59 -23.416 :921875 7 14 28 11 112 .4375 30 60 23.812 9375 n` , 29'11.509 .4535 81 .- 24.209 ,:953125 15 31L- 1?.'906 ..4687 31 82 .E 24.606,/ .96875 31 12.303 .484375 ki 63 =25.003 .9134375 2 4 8 18 32 12.700 .5 1 2 4 18 611 , 84 =25.400'1.000 , Length - International Nautical Miles and Kilometers MAW relation: International Nau-tical Mile 1.852 kilometer? / / 'Int. nautical Int. nautical . Int. nautical Int. nautical . I miles Kilimieters miles Kilometers iKilometers miles Ki brneters' 'les. - 0 50 92 . 600 0 26.9978 1 1.852 N1 94.452 1 . 0.5400, 1 27.5378 3.7Q4 2 96.304 2 1.0799 2 28.0778 . 3. $5.556 3a 98.156 3 '' 1.8199 3 28.8177 .4 7.408 I 4 100.008 4 : 2.1598 4 29.1577 5 'N 9.260 5 101.860 5, 2.8998 5 29.8978 8 .11.112 8 103.712 8 3.2397 ''''''""" 8 -"" 30.2376 7 964 7 105.584 7 6 , 3.7197 7 30.7775, 8 14.816 8 107.416- 8 4.3197 v8 -31.3175 9 16.668 S 109.288 - 9 4.8596 9 31.8575 ..- 6,a 10' 18.520 60 111.120 '10 5.3996 60 32.3974 '20.372 1 112.972 '1 p. 5.9795 1 32.9374 2: 22.224 2 114.824. '2 8.4795 V 2 33.4773 24.076 i 3 116.678 3 7.0, 194 3 34.0173 4 25.928 4 118.528 4 7. 94 4 34.5572 / . . / 5 27.780 5' 120.380 , 5 94 5 35.0972 /. 61 8 29.6321 6 122.232 6 .- 8.6393 6 35.6371 7 7 31.48 7 124.084 7 9.1793 7 36.1771 . -8 33 . $ . 125.936' 8 9.7192 8 36.7171 (..9 .188 9 127.788 9 10.2592 .9 37.257 / 20 37.040 70 129.640 20 104;991 70 37.7970 1 38.892 1 131.492 1 11:3391 1 2 t 40.744 ' 2 133.344 2 11.8790 2 38. 69 3 42.596 3, 135.196 ,3 12.4190 $ ? 3.1883 4 44.448 4 '. 137.048 4 12.9590 4 39/9568 5 46.300 ,5 . 138.900 5 13.4989 5 46.406 /0 4- (E- 48.152 6 140.752 6 14..0389 6 41.0367 50.004. .7 142.604 7 14.5788 7 & i\7 $0,6 8 51,856 8 144.456 * 8 , ' 15.1188N, 8 /42. 166 9 53.708 9 146.308 , - 9 15.66$7 ` 9 / 42566 1 / 30 55.560 80 148.160 30. 18.1987 80 i ,4,i.1965 1 57.412 1 150.012 1 16.7387 1 1 / 14.7365 2 59.264 2 151.864 '. 2 17.2786 N'1,: 2 '' j ,%2765 3 61.116 3 153.'716 3 17.8186 ' 3 441640 / 4 62.968 4 155.568 4 18.3585 .4 450564 / - ei . 5 8.820.820 . 5 157.420 5 18.8985, 5 , 45. 8963/ 8 651.672 6 159.272 6 19.4384 6 '/ 46.4363/ 7 6g.524 7 161.124 7 19%9754 7 7.1 .46.9462' . 8 -, 70.376 . 102.976 8 20.5184 8 / 47:5162 9 72.228 9 '164.828 9 .21.0583, 48.0562 .s., . . 40 74.08q 90 166.680 40 24.5983 48.5661 %I 75.9324 1 168.532 , 1 22.1382 1 2 77.784 170.384 2 22.8782 49. 760.i- 3 ., .. 79.636 : 3 ,172.236 . 3 23.218k /3 50 2160'4' 4 481.488' ' ,4 '' 174.088 4 23.7581`\ /4 50.7559 ,, 5 83.340 5 175.940 5 '' 24.2981 5 51.2959 0 85.192 , (,. 177. 6 24.8380 51.8359 7 87.044 7 179. 7 25.3780 52.3758 8 . 88.896 '8 181.496 8 . 25. 179 8 §2.9158 - 9 90.748 , 9 183.348 9' 26. 79. 9 3.4557 ipik 185.200' 1 00 ..\ '53'97.2 *U.S. GOvERUMENT) pRIFING lFjCE:.1974 1 70-58175309 , 19 1 t)