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TITLE' . Microscopic Analysis, of Activated Sludge. Training

- Manual. IP 'INSTITUTION A Office of Viatr--4graik Operations(EPA)r, Cincin

L'Ohio. FatipAil Training and Operational Technology , . , '. - Center. :' '''

, * 3, FEPORT NO EPA-430/1-80-007 , , POP ODE Jun 80 NOTE 250p.; Contains,occasional light and broken type. -Pages'1-198 ',Field Key to Some Genera of ', and . ',Ciliated isiotoZoa removed due.to copyright % 'restrictions. H AVAILABLE FROMEPA Indtructional'Reiources Center, 1200 Chambers , Rd., 3rd,ylbor, Columbus, OH 432124$1.00 plus $0.:03 ...... per Otge). O

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EDRS PRICE MF01/PC10 Plus Postage. * . DESCRIPTORS .Biological Stiences; Data Analysis; *Laboratory

Equipment; *Laboratory Procedures; AIMicrobiology; .

. - *Microscopes:, Postsecondary Education; *Water , , 'Pollution; :water lesouices 'IDENTIFIERS Activated Sludge; Analytical Methods; *Waste Water Treatment

* a7tBSTRACT ---/-: 4 , This trIiniA4 manual pwents material on the useA Of a compound microscope to analyze microscope communities, present in wastewater tteatient procefses, for operational control. Course topics includei sampling techniques, sample ha moiling, lairdratory analysis, iderftificatiot, of organisms, data interpretation,. and use '4 heecomOcund microscope: This manual contains 26 chapters including reading material,- 'laboratory activities, 'and selected references. Prior expgriende ip-microscopy is not necessary. (Co) ..

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tr **********i***************************************4*************i.***** * Reproductions suppliedrbEDRS atethe best that can be made,. *.-- * fromIthe'original document.- **********,**************ts***********************Ig*******************

t° United States National Training EPA-430/1-80-007 Environmental Protection and Operational June 1980 , Agen Cy Technology Center Cincinnati OH 45268 Water aEPA Microscopic Analysis of N r-4 Activated Sludge A o rJ Training Manual

rA U.S DEPARTMENT OF EDUCATION NATIONAL INSTITUTE OF EDVCATION EDUCATIONAL RESOURCES INFORMATION CENTER (ERIC, This document has been reproduced as received from the person or organization onginatino a XMinor changes have been made to improve reproduction guat

Points of view of 01)16'10ns4. stated in this docu mem do not necessarily represent official N1E position or policy,

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(7- _IL J1_ __ LLINJ __ __ EPA-430/1-80-007 1. 4 June-198Q

Microicopic Anasis of Activated Sludge

This course is for anyone who needs the skills to use a compound' microscope toanalyze microscopic communities, present in wastewater treatment processes, for operational control.Prior experience in microscopy 'is not necessary:. `After successfully.completing the course; the student will be able to relate microscopic communities present in the wastewater treatment process to operational . controls. The student will also be capable of instructing treatril4nt personnel in the more proficient use of .the compound mitroscope and re1ating communities present to operational control. 4 The training, includes classroom instruction and laboratory practice.

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% U. S. ENVIRONMENTAL PROTECTION' GENCY Office of, Water Program Operations National Training and Operational Technology Center 4. r

DISCLAIMER

., Reference to commercial products, trade names, or manufacturers` is for_purposes of example and illustration. Such reference's do not constitute ehdorgement by the Officeof Water Program Operations, U. S. Environmental 4201).. Protection Agency.

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CONTENTS

r f Title or Description -Outline Number

The Aquatic Environrdent 1

Classification of Communities, Ecosystems, .and Trophic Levels 2 Limnology and Ecology'of - 3 Biology', of Zooplankton 4

, Optics and the Microscope 5

Strubture and Function. of Cells 6

Bacteria and Protozoa as Tokicological Indicators 7 iiamentous Fungi and the "Sewage " Community

Protozoa, Neniatodes, and Rotifers' 0

Activated sludge Protozoa 11 A

Free-Living Amoebae and Nematodes 12

Animal Plankton 13

Preparation and Enumeration of Plankton in the Laboratory 14 sg Attached Growths 15

Effect of WaVewater Treatment Plant Effluent on Small 16 iol Ecology WaLste Stabilization Processes 17

Ecology Primer 18 The Laivs of Ecology 1 Application Orlifological Data 20 Signilicance of 9Limiting Factors" to POPulation Variation 21 Algae and Cultural 22 Calibration and Use of Plankton Counting Equipment 23

. . t Laboratory: Proportional Counting. of Mixed Liquor . 24 Key to Selected' Groups of Freshwater Ant . 25 ... ,A Key for the Initial Separation of Sortie Co on Plankton Organisms 26. i . .- Field Key to Some Genera of Algae Ciliated, Protozoa

r 5 / Z 'AQUATICAQUATIC ENVIRONMENT Part 1: e Nature and Behavior of Water

IINTRODUCTION .forms, but also with living Organisms' and infinite interactions that occur The earth is physicy divisible into the bet hem and their environment. lithospherekor land ma ses, and the hydrosphereiwhich includes the , C management, including , stre(ms, and subterranean waters; pollution control, thus looks to all and the atmosphere. branches of aquatic science in efforts to coordinate and improve man's toUpon the hydrospere are based a number relationship with his aquatic environment. of sciences which represent different approaches. Hydrology is the general science of water itself with its various special fields suclkas , - IISOME FACTS ABOUT WATER hydri.tros, etc.These in turn merge into physical chemistry and chemistry. AN.Water is the only abundant liquid on ota planet(It has many properties most B Limnology and combine unustial for liquids, upon which depend aspects of all of thegei and deal not oaly, most df the familiar aspebts of the world with the physical liquid water and its about us as we know it.ZSee Table 1) various naturally occurring solutions and

TABLE 1 UNIQUE PROPERTIES OF WATER . Pro2erty_ Significance Highest heat capacity (specific heat) of any Stabilizes of oilanismstand sulici or liquid (except NH3) ',geographical regions Highest latent heat of fusion (except NH / Thermostatic effect at freezing point 3 Highest heat.of evaporation of any substance Important in heat and water transfer of atmosphere The only substance that has its maximum Fresh and brackish waters have maximum density as a liquid (4°C) density abode freezing point.This is - impo ;tant in- vertical circulation pattern in lager. Highest surface tension of any liquid Controls surface and drop phenomena. iniportant in cellular physiology . Dissolves more substances in greater Makes complex biological system possible. quantity than any othei. liquid Important for transportation of materials 'in solution. Pure water has the bfgheat di- electric Leads to high dissociation of inorganic ,natant Of any liquid . substances in solution .

Very little electrolytic dissaciat Neutral, yetcontains,both ft+ and OH ions 1, Relatively transparent AbsOrbs much,energy In infra red and ultra violet ranges, bta little in visible range. Bence ."color less" . `,1

21g23. 80 4 a t, The Aquatic Envitomnent

i 4 a B ysical-Factors of Significance TABLE 2

1 Water substance EFFECTS OF ON DENSITY - OF PURE WATER AND ICE* Water is not, simply "H20" but in reality is a mixture ofsome 33 Temperature (°C) Density different substances involving three isotAeS each of hydrogen andoxygen Water (ordinary hydrogen 111,,deuterium and tritium H3; ordinary al°, -10 .99815 .9397 oxygen 17; and oxygen 18) plus 15 - 8 99869 .9360 known ty }es of ions.The molecules ,-6 9991'2 .9020 of a water mass tend to associate .99945 .9277 themselves as polymers 'rather than 2 .99970 .92,29 to remain as discrete units. '0 .99987 ---)9168 (See Figure 1) . '2 .99997 6 4 1.00000 6 .99997 .99988 SUBSTANCE OF PURE WATER 16 .99973 206: .99823 40 .99225 60 .98324 80_ .97183 I 100 '".195838

Tabular values for density, etc.; represent estimates brvarious workers rather than absolute values, due to the variability of water. * *Regular Ice is knoivn as "ice I. Fouror more other "forms" of ice are known.tq _exist (ice II, ice III, etc. ), having densities °

at 1 atm pressure ranging'from 1.1595 - I -,to 1.67.These are of.extremely restricted occurrence and. may be ignored in most Figure I routine operations. -

2Density This epsures°That ice usually forms on top of a a TeMperature and density: Ice. ( and tends'to insulate the renfain- Water is the only known substance ing water mass from fuither_loss in which the solid.state will float of heat. Did ice sink, there on the liquid state.(See Table 2) could be little or no carryover of aquatic life from.season to season in-the higher latitudes.Frazil or needle ice forms colloid fly at a few Thousandths of a- degree -below CI° C.It is adhesiye arid may build up on submerged objects as "anchor ice's', but it is still .typical-ice (ice I): The Aquatic Enviionmentt

Seasonal increase in solar Mineral-rich water from the radiation annually warms , for example,,,, surface waters in summer is mi.Red with oxygenated while other factors result in . water from the .. winter cooling. The density This usually triggers a war differences resulting establish sudden growth or.ubloom" two classic layer's: the epilimnion of. plankton organisms. .or ,' and the hypolimnion or lower layer, and 6)When stratification is present, in between.is the however, each layer belgiaves, or shear-plane. relativelytindependently, and significant quality 'differences. 2)While for certain theoretical may develop. purposes d'"thermocline" is defined as a zone in which the '7) Thermal stratification as temperature changes one described above has no degree centigrade for each reference to the size of the meter of depth, in practice, water mass; it is found In any transitional layer between oceans and puddles. two relatively stable masses of water of different temper- b The relative denSities of the atures may be regarded as a various isotopes of water thermOcline. influence its molecular com- position.For example, the 3) Obviously the greater the lighter 016 tends to go off temperature differences first in the process of evaporation, between epilimnion and leading to the relative enrichment hypolimnion and the sharper of air by, 016 acid the enrichmett the gradient in the thermOcline, of water by 017 and 018. This the more stable will the can lead to a measurably' higher situation be. 018 content in warmer climates. Also, the temperature of water 4) From information given above, in past geologic ages can be it should be evident that While closely estimated from the ratio.," the temperature of the of 018 in the. carbonate of mollusc hypolimnion rarely drops shells. much below 4° C, the epilimnion may range from cDissolved and /or suspended solids 0° C upw' ard. may,,also affect the density of natural water masses (see Table ,3) r 5) When epilimnion and hypolimnion achieve the same temperature, TAI3LE 3 stratification no longer exists. EFFECTS OF DISSOLVED SOLIDS The entire body of water)3ehayes ON D ENSITY hydrologically asaunit, and tends to assume uniform chemical Dissolved Solids Density and physical characteristics. (Grams,per liter) (at 4°C) Even a light breeze may then, 0 1.00000' cause the entire body of water 1 1.00085 to circulate,Such events. are called overturns, and usually result in . 2 1.00169 water quality changes Of consider- 3 1.00251 able physical, chernicar, and biological significance. 10. 1.00818 t. 35 (mean for water) . 1.02'822

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1 The Aquatic Environment

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d Types-of density stratification ';-____ This is important not only in situations involving the control of flowing water 1) Dersity differences produce as in a filter, but also since, - stratification which may be overcoining resistance to flow gen- ,_,permanent, transient, or erates heat, it is significant in the seasonal. heating of water by internal friction from wave and.current action. , 2) 'Permanent stratification Living organisms more easily support exists'for example where themselves in the more viscous `there is a heavy mass Of (and also denser) cold waters of the 'brine in the deeper areas of arctic than in the less vis cous warm a basin which does not respond waters of the tropic.(See Table 4). to seasonal'or other changing conditions. t' TABLE 4 3) Transient stratification may VISCOSITY OF WAT R (In millipoises at 1 attn) occur with the recurrent influx of tidal Water in an pissted solids in g/L ,for example, or the - occasional influx of cold Temp. 0 C 0 1 5 10 30 muddy water into a deep -10 26.01 .. or reservoir. I . - 5. 21.4 7,------t , 4) Seasonal stratification is 0 '17, 94 18,1 18.24 18.7 typically trynal in nature, 5 15/19 15.3 15.5 16.0 f and thyolvthe-annual establisAmt of the epilimnion, 10 13 ,10 13.2 13.4 1.3.8 hypolimnion, and thertnocline : 30 8,00 8,1 8,2 8.6 as described above. . 100 5) Density stratification is not' limited to two-layered systems; three, four, or ven more 4Surface-tension has biological as,well layers may be encountered in as physical-significance.Organisms larger bodies of water. 'I whose body gurfaces cannot be wet by water can either ride on the surface e A "pltinge line' (sometimescalled film or in some instances may be "thezmal line) niay develop at "' "trapped" on the surface film andbe the mouth of a . Heavier unable to re-enter the water., water flowing into>,a lake or reservoir plunges below the ' 4. 5 :Heat or energy lighter Water mass ofthe epiliminium to flow along at a lower level.Such Incident solar radiation is;the'prime a line is usually'marked by an Source of energy for virtually all accumulation of floating debris._ organic and most inorganic processes on earth. For the earth as a whole fStratification may be modified th'e total amount (of energy) received or ,entirely -suppressed in some _annually must exactly balance that cases *hen deemed expedient, by lost by reflection and radiation into mean- s of a simple. air lift.' space if climatic and related con- , ditions are to remain relatively 3 The viscosity of Water is greater at constant over geologic time. lower temperOures (see Table 4). 9 S The Aquatic Environment %

a' For a given body of water, significant change in surface. immediate sources of energy level is detected.Shifts in inclyle in addition to solar submerged water masses of 0 . irradiationterrestrial, heat, this type can have severe effects transformation of kinetic energy on the biota and also on human (wave and current action) to heat, water uses where withdrawals 'chemical and biochemical are.confined toa given depth, reactions, convection from the Descriptions and analyses of atmosphePe, and condensation of many other types arid sub-types ater vapor. of waves and wane -like movements may be found in the literature. bthe progortion'of light reflected depends on the angle of incidence, b the temperature, color, and other qualities of the water; and the 1) Tides are the longest waves presence or absence of films,. (' known, and are responsible for of lighter liquids such as oil. the orrce or twice a day rythmic In general, "as the ddpth increases rise'and fall of the', level arithmetically, the light fends to on most shores, around the world. decrease geometrically.Blues, greens, and yellows tend to 2) Wylie part, and parcel of the penetrate most deeply while ultra same phenomenon, it is'often violet, violets, and orange-reds Convenient to refer to the rise are most quickly absorbed. On and fall of the water level as . .0 ,the order of 90% of the total ", " and to the resulting

illUmination which penetrates the A currents as "tidal currents. ".1 surface film is absorbed in the first 10 meters of even the clearest 3) Tides are basically caused by the water, thus tending to warm the attraction of the sun and moon on upper layer,s. water masses, large and small; however, it is abnly in the oceans 6Water movements and peksibly certain of the larger lakes that time. tidal action has a Waves or rhythmic movement been,demonstrated. The patterns ortidal action are enormously 4) The best known are traveling complicated by local, topography, waves caused 'by wind. Theseare .interaction with , ancrother effective only against objects near fa,ctors.The literature on tides the surface. They have little- is very large. effect on the movement of large 'a $ masses of water. - cCurrents (except tidal currents) - are steady arythrni-c water movelneAs 2) Seiches t which have had maRir study only in oceanography although they are, Standingwaves seiches occur most often observed, in rivers and in lairs, , and other, stream's. 'They are "primarily e- ed bodies of water, but are concerned with the tranilocation of ge enough to be water masses. They May generated, observed. An " or internally by virtue of density changes, seicli" is an oscillation in a, or externally by wind or terrestrial subrhersed mass of water such topography,' Turbulence phenomena asaa hypblimmon, accompanied or currents are largely respbn- by compenSating oscillation in the Sible. for, late rals.Mixing in a curtrent. overlying Water so, thatdo, These are of far more importance in the econry of a body of water than

V . 0. mere laminar flow. 1-5 , 1 410. S. ti

The Aquatic Environment

d ° force it a result of inter- depend on the depth of the water, action between the rotation of the the velocity and duration of the earth, and the movement of masses wind, and other factors. The net or bodies on the earth. The net result is that adjacent cylinders result is a slight tendency for moving tend to rotate in oppOsite directions objects to veer to the right.in the like meshing cog wheels. Thus, northern hemisphere, and to the\ the water between two given spinals left in the southern hemisphere. maybe meeting and sinking, while While the result in fresh waters is that between spirjls on either side usually negligible, it may be con- will be meeting and rising. Water siderable in marine waters.. For over the sinking, while that between example, other factors permitting, spirals on either side will be meet- ifiebre is a tendenby in estuaries for ing and rising:, Water over.the fresh waters-to move toward the sinking areas tends to accumulate ocean faster along the right bank, flotsam and jetsam on the surface while salt tidal waterstend to 'in long conspicuous lines.' intrude farther inland,along the. left bank.Effects are even' more °aThis phenomenon is of consider- dramaticin the open oceans. able importance to those sampling forplankton (or even chemicals) e Langmuire spirals (or Langinuire near the surface when the. wind circulation) are a relatively is blowing. Grab samples from massive cylindrical motion imparte d either dance might obviously to surface waters under_ the influence differ considerably, artkeif of wind.' Thq axes of the cylinders ' a plankton tow is contemplated ..\\1 are parallel to the .direction of the. t it should be made,,across the wind, and their depth and velocity ere wind in order that thesnet may pass Through a succession of both dances.

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- b y b WATER SURFACE

WATER WATER RiSitIG SINK1N,G Figure 2. 'Langmuitilpirals h..Blue dna, water r. dance., water sinking, floating o I -swimming.,ohiects concentrated.

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The Aquatic Environment,

b Langmuire spirals. are not REFERENCES sually established until the wind has either beenk blowing Buswell, A. M. and Rodebush, W: H. for an ,extended period, or Water. Sci, Am. April 1956. , else,isblowing rather hard. Their presence can be detected 2 Dorsey, N. ,Ernest.Ribperties of by the lines of foam and Ordinary WaterSubstance. other floating material which Reinhold Publ. Corpew. York. coincide with the direction pp. 1-673.1940. of the wind. 3Fowle, Frederick E.Smithsonian PhyMcal XabIes. -Smithsonian 6 The pH of pure water hasheen deter-, Miscellaneous Collection, 71(1), mined between 5.7 and 7.01-by various. 7th revised ed.,1929. workers. The latter value is most ,,widely accepted at the present time. 4Hutcheson, George:E. A Treatise on ( Natural waters of coarse vary widely Limnology. John Wiley Company. according to circumstances. 1957. C The elements of hydrology mentioned above represent a selecton of some of the more conspicuous physical factors involved in working with water quality: Other }hems no spebificallif Mentioned include:. molecular structure of waters, ° /7. / interaction of water and radiation, 1 . internal pressur,e, acoustical charac- teristics, presVure-volume-temperature'

.relationships, refractivity, luminescence, .4 color, dielectrical characteristics and :phenomena, solubility, action and inter.- actions .of gases; liquids and 'solids, . water vapor, phenomena of hydrostatics and liy.drodynamics in general.

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Part 2: The Aquatic Environment as an Ecosystem' ) I

IINTRODUCTION IIIECOLOGY IS THE STUDY OF THE INTERRELATIONSHIPS BETWEEN Part-1 introduced the lithosphere and the ORGANISMS, AND BETWEEN ORGA- hydrosphere. Part 2 will deal with certain NISMSAND THEIR ENVIRONMENT. general aspects of th.e biosphere, or the sphere of life on this earth, which photp_-- A The ecosystem is the basic functional' graphs from space have showri is a finite unit of ecology. Any area of nature that globe in infinite spaces includ s ing organisms and nonliving substances nteracting to produce an This is the of man and the other *exchange o material8 between the livihg organisms.His relationships with tie and nonliv g pd.rtse,constitutes an aquatic biosphere are our common concern. ecosystem. (Odurp, 1959) -

1 From a structural standpoint, it is ,II THE BIOLOGICA I., NATURE OF THE- convenient to recognize four WQRLD WE LIVE IN cpnstituents as composing an ecosystem (Figure.1). 4 A We can only imagine what'this world musthave been like before there was life: a Abiotic NUTRIENT MINERALS which ape the physical stuff of B The" ircSrld as we know it is largely shaped ,which living protoilasdi will be by the forces of life. synthe.sized.

1 Primitive forms of life created organic b utotrophic (self-nourishing) or matte?.-and estab/i'shed soil. PRODUCER organisms. These are largely the -green . D Plants cover the lands and "enormously -(holophytes), .but other minor influehte the forces of erosion. groups must also be included ..(See Figure 2).They assimilate 3 The nature and rate of 'erosion affect' the nutrient, rpinerals,..by the use the redistribution of materialE . of conSiderable energy, and combine (and mass) on the Surface of -the their into living organic, substance. earth (topographic changes). ' C Heterotrophic (Ole r-nourishing) 4Organismstie up vast quantities of coNsulAgas (holozoic), are chiefly certain chemicals, such as carbon' the animals. They ingest (or eat) and oXygen. Ss, and digest organic matter, releasing . considerablk energy in the process. 5 Respiration of plants and animals releathes carbon dioxide to the . d Heterotrophic REDUCERS are chiefly atmosphere in influential quantities... bUcteria and fungi that return complex organic compounds back to 6 COaffects the heat transmisthori of 2 the originaribiotic mineral condition, the atmosphere. thereby reletising the remaining chemical energy. C ,Organisms respond to and in turn affect - their environment.. Man is one a the 2 From a functional standpoint, an, -'rhosi. influential. I ecosystem 'ha.8 two parts (Figure 2)

1 -9 The Aquatic Environment

PRODUCERS REDUCERS 1

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NUTRIENT 1 . MINERALS

FIGURE 1

a The -autotrophic or producter, 2These two groups can be `defined on organisms, which utilize light the basis of relative complexity of 1 energy or the oxidation of in- structure. organic bompounds, as their sole energy source. a The bacteria andblue-gieen

lacking a nuclear membrane are . b The heterotropic or consumer the Monerit. and reducer organisms which eutiliies organic compounds for b The single-celled algae and its energy and carbon requirements. protozoa are Protista.

3 Unless the autotrophic and hetero- 1 trophic phases of the cycle approximate C Distrilpited throughout thesegroups will a dynamic equilibrium, the ecosystem be found most of the traditional 'and the environment will change. . of classic biology. B Each dethesegtoups includes simple, single-celled representativei, persisting IVFUNCTIONING OF THE ECOSYSTEM 4arr at lower levels on the evolutionary stems of the higher organisms. (Figure 2) AA is the transfer offood energy from plants through a aeries of,organisms 1These groups span the gaps between the With repeated bating andbeing eaten. higher kingdoms with a multitude of Food chains are rrot isolatedsequences but transitional forms: They are collectively axe interconnected. called.the PROTISTA and MONERA. 14 r "

The Aquatic Environment

RELATIONSHIPS BETWEEN FREE LIVING AQUATIC ORGANISMS Energy'Flows from, eft to Right, Gal EvolutionarySequence is Upward

PRODUC S I , CONSUMERS. REDUCERS Arganic Material Ingested or Organic Material Reduced .OrganicNiaerial PrOdtced, Consumed. by Extracellular Digestion UsuallyN PhotosTifFirt Digested Ipternally and Intracellular Metabolibth, to Mineral Condition

ENERGY STORED ENERGY RELEASED ENERGY RELEASED

Flowering Plants and Arachnids Mammals Gymnosperps Basidiomycetes Insects Birds Club Mosses, Ferns Crustaceans Reptiles 4 Segmented Worms Amphibians Liverworts, Mosses Fungi Imperfecti. Molluscs Fishes Bryoz oa Primitive fylulticellular Green C'bordates Algae Rotifers Ascomycetes Roundworms Red Algae Flatworms Coelenterates p Higher Phycomycetes Sponges DEVELOPMENT OF NIULT1C'ELLULAR OR COENOCYTIC STRUCTURE.

'1* PROTISTA P r o 10 v 0 a Unicellular Green Algae Lower A nioet;oic.1 Phycoznycetes Flagellated, Suctoria - Pigmented Flagellates (nnii-pigmented)' (Chytridiales, et. al. )'

DEVELOPMENT OF A NUCLEAR MEMBRANE

MONERA Blue Green Algae Actinomycetes 1- "IpimenaMes Phototropic Bacteria 4 Saprophyt lc I Bacterial Chenwtropie Bacteria e s -

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FIGURE 2 . 131. ECO. pl. 2a. 1.69 - .

/The Aquatic EmAroliment .

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B A is the interlOcking pattern of D_Total Assimilation food chains in an ecosystein. (Figures 3, 4) In complex natural communities, organisms The amount of energy which flows through whose food is obtained by the same number a trophic level is distributed between the of steps are said to belong to the same production of biothass (living substance), trophic (feeding) level. and the demands of respiration (internal energy use by living organisms) in aratio C Trophic Levels of approximately 1:10. E Trophic Structure of the Ecosystem 1 First - Green plants (producers) (Figure 5) fix biochemical energy and The interaction of the food chain wlthesine basic organic subotances. phenomena (with energy loss at each This is . transfer) results in various communities 2 Second - Plant eating animals (herbivores) having definite trophic structure or energy depend on the producer organisms Mt levels.Trophic structure- may be food. measured and described either in terms of the standing crop per unit area or in 3 Third - Primary cazpivores, aninials terms of energy fixed per unit area per which feed on herbivores. unit time at successive trophic leWis., Trophic structure and function can be 4 Fourth --Secondary carnivores feed on shown graphically by means of ecological primary carnivores. pyraMids (Figure 5).

5 LastUltimate carnivores are the last or. ultimate level of consumers. 1

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Figure 3.Diagram cf the pond ecosystem. Basic units are asifollows: 1, abfoticsubstancesbasic inorganic and vegetation; IIB, producersphytoplankton; III-1A. primary consumers organic compounds; IIA, producers--rooted (car-.` .1 (berbivores)bottom forms; III.1B, primary con (herbivores)zooplankton; 111.2, secondary consumers decomposersbacteria and fungi of decay. &Imes); 1114, tartluy consumers (secondary carnivores); IV,

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The Aquatic Environment

k. ;04

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Nutrient E sup,* Phytoplankton

1 3 \ / Bacterial Death and decay action ti1/4 \ / ....e,A3''',;! x.....-..,_. -.

Mollusks

topr ts,1

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17410/111114. 1;11101 LI,t1

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Figure 4. A MARINE ECOSYSTEM (After Clark, 1954 and Patten, 1966)

.3 13 17 The Aquatic EfiVironment

(a) Includes bacteria, algae, .protozoa and Decomposers Carnivores (Secondary) other micnoscopic animals, and often the Carmvores (Primary" young or embryonic stages of algae' and other organisms that normally grow. up fierbworek to become a part of the (see below). Producers any planktonic types will also adhere (b) to surfaces as , and some ty.pical periphyton may break off and be collected as plankters: C' Benthos are the plants and animals living on, in, or closely associated with the bottom. They include plants and ,, -. D Nekton are the community of strong aggressive swimmers of the open waters, often called pellagic.Certain fishes, whales, and invertebrates such as 'Figtire 5.HYPOTHETICAL PYRAMIDS of shrimps and squids are included here. (a)'Numbers of individuals, (b) Bioniass, and lc) Energy (Shading Indidatet Energy:Loss). E The marsh community is based on larger "higher" plants,` floating and ernergent. Both marine and freshwater marshes are areas of enormous biological production. .Collectively known at "", they gap between the waters and the V BIOTIC COMMUNITIES dry tan s.

.pA Plankton are the macroscopic and VI PRODUCTIVITY microscopic animals, Plants, ba teria, etc., floating free in the open wat r. A The biological resultant of all physical Many clog filters, cause tastes, odors,. and chemical factors in the quantity of and other troubles in watel-supplies. Hire that may actually be present. The Eggs and larvae of larger forms are abilityto produce this "" is often presetit. often referred to as the "productivity" of a body of water.This is neither good 1 Phytoplankton are ,T4..ese nor bad per se. &water cflow'pro- are the dominarit producers of the duCtivity is a "poor" water biologically, waters, fresh and salt, "the grass, and also a relatively "pure" or "clean" of the ".' water; hence desirable as a water supply or a bathing beach. ,A productive water 2; Zooplankton are animal-like. on the other hand may be a nuisance to Includes many different animal types, man or highly desirable.it is a nuisance range in size from minute protozoa if foul odors and/or weed-chocked to gigantic marine .; waterways result, it is 'desirable if bumper crops of bass, catfish, or tst Periphyton (orAufwuchs) - The communities oysters are prodliced. Open oceans have of microscopic organisnis associated.with a low level of productivity in general. submerged surfaces of any type or depth. .

,1.+4,14 - S The Aquatic Environment

VII PERSISTENT CHEMICALS' IN THE a Oysters,Vor instance, will'con- ENVIRONMENt centrafe DDT 70,000 times higher *, \ in their tissues than it's concentration Increasingly complex manufacturing processes, in surrounding water.. They can coupled with rising industrialization, create also partially cleanse themselves health hazards for humans and aquatic life. in water free-of DDT. Compounds besides being toxic (acutely or b Fish feeding on lower organisms chrOnic) may produce 'mutagenic effects build up concentrations in their 'including Cancer, tumors, and teratogenicity visceral fat which may reach several (embryo defects).Fortunately there are,tests, thousand parts per million and levels such as the Aniis test, to screen chemical in their edible flesh of hundreds of compounds for these effects. parts per million. o Larger animals, such-as fish-eating A Metals - current levers of cadmium, lead gulls and other birds, can-further and other substances constitute a mount- concentrate the chemicals. .A survey ingconcern.Mercury pollution, as at on organochlorine residtes in aquatic Minimata., Japan has been fully documented. birds in the Canadian prairie provinces showed that California and ring-billed. Pesticides gulls were among the most contaminated. Since gulls breed in colonies, .breeding 1 A pesticide and its metabolites may , population changescan be detected and move through an ecosystem in many related to levels of chemical con- ways. Hard (pestidides which are tamination.Ecological research on persistent, having a long half-life in colonial birds to monitor the effects the environment includes the organo- of chemical pollution on the environ- -chlotines,' ex., DDT) pesticides . inent is useful. ingested or otherwise borne by the target will stay in the C "Polychlorinated biphenyls" (PCB's). 'nvironment, possibly to be recycled PCB's were.used in plasticizers, asphalt, or concentrated further through the ink, paper, and a host of other products. hattiral action of food chains if the Action was Taken to curtail their release species is eaten., Most of the volume to the environment. sincedtheir effects Of pesticides do not reach their target are similarto hard pesticides. However at all. , this doesn't solve thofroblems of con- taininated sedinients and ecosystems and 2Biological magnification final fate of the PCB's still circulating. Initially, low levels of persistent '\: D There are numerous,other compounds pesticides in air, soil, and water' may which are toxic and-accumulated in the .. be concentrated at everystep up the ecosystem. food chain.Minute aquatic organisms and , which screen water and ottom mud having pegticide levels of a few parts peribillion, can accumulate levels measured in parts per milliona. thousandfold increase. Thesediments including fecal deposits are continuously recycled by the bottom animals. A,' I

'1-15 CIA r le-

The AqUa-tic Environment

FERprcEs 5 Odum, E.P.Fundamentals of Ecology. W. B. Saunders Company, 1 Clarke;' G. L. ents of Ecology. Philadelphia and Londont1959. ',john Wiley & Sons, New York. 1954. 6Patten, B. C.Systems Ecology. 2Cooke, W. B.''rickling Filter Ecology. j_kige,Stance. 16(9). 1966. Ecology 40(2):273-291. 1959. 7Whittaker, R.H. New Concepts of 3 Hanson, E.D.Animal Diversity. Kingdoms. Science 163:150-160. 1969. Prentice-Hall, Inc New Jersey.1964. 4 Hedgpeth, J.W.Aspects of the Estuarine Ecosystem.Amer. Fish. Soc., Spec. Publ. No. 3. 1966.

a 4. e .part 3. The Freshwater Environment

IINTRODUCTION During periods of run-off after a rain or snow -melt, -buch a gulley The freshwater environment as considered -would have a flow of Water which herein refers to those inland waters not might range from torrential 'to a detectably diluted by ocean waters, although mere trickle.Erosion mayproceed the lower portions of rivers are subject to rapidly as there is no permanent certain 'tidal flow effects.. aquatic Llora or ' to stabilize streambed materials. On the other Certain atypical inland waters such as saline hand, terrestrial grass or forest or alkaline lakes,, springs, etc., are not- growth may retard 'erosion. When treated, as the main objective here is tyPical.... "Ate run-off has passed, however, inland water. - the "streambed" is dry.

All waters have certain basic biological cycles 2Youthful streams. When the and types of interactions most of Which have streambed is- eroded be/ow the already beed presented, hence this outlinT ground water level, spring or will concentrate on aspects essentially, seepage water enters, and the peculiar to fresh inland waters. stream becomes permanent. An aquatic cflora and fauna develops and water flows the year round,. IIPRESENT WATER QUALITY AS A , 14, Yout hful streams typically have a FUNCTION OF THE EVOLUTION OF relatively steep gradient, rocky.beds, FRESH.WATERS with rapids, falls°, and small pools. A The history of ,body of water determines 3 Mature streams. Maiure stred.ms its present condition.Nattficarwaterd have have wide valleys, a developed evolved in the course of geologic time flood plain, are 'deeper, more into what :we know today. turbid, and usually have warmer' water, sand, rilud, silt, O clay B Streams, bottoin materfalb Ng& shift with . 4 increase' in flow,- In Jheir more In the course of their evolution, setealiffs favorable reaches,- streams in this in general pass through four stages of oondition are at a peak of biological .development which may be called: birth,' productivity.Gradients' are moderate, youth, maturity, and old age. _riffles, or rapids are often separated

by long pools. - ' These terms or conditions may be employed or considered in two contexts: I/ 4 age, ',streams have approached temporal, or spatial.In terms of geologic 4 geologic Vase level, usually the time, a given point in a stream may pass ocean. During -flood siOgge they scour through each of the stages described below their beds and deposit. materials on or: at any given time, these various stages the flood Plain which may be very of development can be loosely identified bread and flat.During normal flow in successive reaches of a stream traveling the channel is refilled and:Many from its he dwaters tb base level in ocean shifting bars are developed. *eanders or major la e. - and ox-bow lakes are often formed. , 1 Establishment eir birth. *is might be a :'dry runt' or headwater'. stream-bed,. before it had eroded down to the level of ground watery

1-17 The A uatic Environment

4=4 f,(Under the influce of man this became a lake. Or; heglacier may,- Pattern may be b oken up, or actually scoop out a."'hole.. Landslides temporarily inteupted.Thus an may dam valleys, extinct volcanoes;may essentially "youthful" stream might... collapse, etc., etc.- take on some of the *characteristics of_ a "mature" stream following soil 2Maturing or natural eutrophication of erosion, organic enrichment, and lakets. increased surface runoff.Correction IP of these-conditions might likewise be aIf not Airoor present shoal areas followed by at least a partial reversion are developed through erosion to the "original" Condition). and deposition of the shore material by wave action and . C Lakes and Resertroirs b.currents produce bars across bays GeologiCal factors.iirhioh significantly and thud cut off irregular areas. affedt the flatting of either a stream or -lake include toe. following: cSilt brought in by tributary streams settles out in. the quiet lake water 1 The geographical locatiOn of the . ' drainage basin or watershe. ed to-eurfacet, apd floating free plankton. Dead 2 The size and shape of the drainage organic matter begins to accumulate basin. .on,the bottom.

45 The general topography, i.e.,' e Rooted aquatic plants grow on mountainous or plains. shoals and bars, and in doing so mit off bays and.coqribute to the 4The character of the bedrocks and filling of the lake. soils. fDissolyed carbonates and other 5 The character, amount, annual material's are precipitateld in the distribution, and rate' of precipitation,. deeper popions of the lake in part thfough the action o plants.' 6, the natval vegetative cover of the .1 land_ is, of course, responsive to and . gWhen filling is well.aanced,, responsible for many of the above ' mats of sphagnum mods may extend factors and is also severely subject outward from the shore.These . to.the whims of civilization.This . mats are followed by sedies and is one of the major factors determining grasses which finally con ter_ the run-off versud soil absorption, etc. lake marsh. D Lakes have a developmental history which 3 _Extinction of lakes. After lakes reach somewhat parallels that'of streams. This matukty4, their progress tolyerd process is often referred to as natural fillirig up is accelerated. They become eutrophication:' extinct through:

`41 1 The methods of formation varygreatlyf a- the. downcutting of thec outlet. a bid all influence the character and ''7 subsequent.chibitory of the lake. bFkllfng with erode from the shores or brought in b In glaciated areas, :for example, a tributary streams. huge block of ice may have been covered with till.The glacier !retreated, the cFilling by thea ccumu,lation'Ok the. ice melted, 'and the resulting hole remains of vegetable materials !!, grering.in the lake itself. . (Often two or three proceSdes may 4 act concurrently) 1-18 The Aquatic Environment

C 4, Y HP PRODUCTIVITY IN FRESH WATERS 2 As the stream flows toward a more "mature condition, nutrients tend to A Fresh waters'in general and under . accumulate, and. gradient diminishes natural conditions by definition have a ,+ and So time of flow increases, fern; lesser supply of dissolved substances perature tends to increase, and than marine waters, and thus a lesser plankton floUrish. . basic potential forthe growth.of aqUatic organiSims By tile same token, theST. _Should a heavy load of-inert silt maybe said to be more sensitive to tile develop on the other hand, the addition of extraneous materials turbidity would reduce the light (pollutants, nutrients, etc. ), The penetration and consequently the following notes are directed toward general plankton prctduction would natural geological and other environ- mental faqtorS as they ffect the . productivity`of fres% $ters. 3 As the streaniapproachesbase/lev el (old age) and the, time 'available for B Factors Affecting Stream Productivity 'plankton growth increases, the c (See Table 1) balance between to nutiie t levels; and temper ture and ther TABLE' I seasonal conditions, determines the ° overall productivity. EFFECT OF SUBSTRATE ON STRAM. PRODUCTIVITY* C -Factors Affecting the Productivity of lakes (See Tape 2) (The productivity of sand bottoms is taken as 1) 1 The size, shape, and 4epth of the lake basin.Shallow water is more productive than- deeper Water" since 1 Relative more light will.reach the bottom to Bottom Material #Productivity stimulate rooted plant grOwth, As a corollary, lakes,.with ThOre shore- Sand 1 line, having more shallow water,' Marl 6 are in general more productive. Fine Gravel 9 -c, / Broad shallow lakes and reservoirs Gravel and silt , '14 have the greateit production potential Coarse gravel ' . . 32 (and hence should be avoided for Moss on fine gravel 8g 'Nwater supplies). s Fiasidens (moss) on coarse ._.----vLit gravpl - TABLE 2. Ranunculus (water buttercup) '19'4', a Watercress 301 EFFECT OF .SUBSERA TE Elodea,(waterweed) 452 -; ON LAKE PRODUCTIVITY .. - *Selected from Tarzwell 1937 productivity of sand-bottcims is taken- s 1) 1 .0. To be productive of aquatic life, a- BottomMateHal Relative Productivity stream -must provide adequate nutrients, , . light, a 1 suitable' temperatu,re:. and time Sand. 1 , forgrowth to take place. 'Ism, -Pebbles '1. 4 ° ... ° --..\ 1 -YOUthfurstreamS; especiallyon rock Flay .ore sandSUbstrates are Zoo in-essential Flat rubble .` 's-mutrients.Temperatures in moun- Block rubble ' 11 77 tainous regions are usually low,' and. Shelving,r9ck. A ... due to thp steep,gradient, time for 4 .g.xlowth is rt.Although ample * Selected frolinTarzwell 1937 light isa,ilable; growth of true plankt is thus greatly llmited. 9

s \ t9

Aquatic Environment ,\

2 °Hard,waters are generally more . IV CULT RA EUTROPHICATION 'prOductite than soft waters as there are more plant nutrient A. The genleral1 processes of natural available.- This is often greatly in- eutroph cation, or natural enrichment. fluenced byl.the character of the soil 4 and pro uctivity have been briefly out- and rocks in the watershed'and the lined aboye. . quality and quantity of ground water .. entering Ze lake. In general; pH B Whenthe°\activitiesof 'man speed up ranges'oP6.8 to =$. 2 appear to be these enrchfnent Processes by intro- ^ most 'productive. ducing atural quantities of nutrients tc. ) the result is often called , 3 Turbidity reduces productivity as cultural e tro hication.This term is is reduced. o often exte ded beyond its original usage 0 , to include the enrichment (pollution) of 4The presence or absence of thermal streams, /estuaries, and even oceans, as stratification with its .semi-annual well as likes. turnovers affects productivity by C distributing nutrients throughout the water mass. V CLASSIFICATION OF LAKES AND RESE VOIRS . 5Glimate, temperature, prevalence of ice and snow, Of course A The productivity of lakes and impoand- important. ments is such a conspicuous feature that it is often used as a convenient of D Facto/ale ctini the uctivity of :44 :classification. Reservoirs . 1Oligotrozhic lakes are the younger, 1 The productivity of reservoirs is less productive lakes, which are deep, governed by much the same principles have clear water, and usually support as that of lakes, withoike difference Saimonoid fishes in their deeper waters. that the water level'is much more under the control of man. Fluctuations 2 Eutroyhic lakes' are more mature, in water level can be used tb de- more turbid, and richer. They are liberately increase o'rdecrease usually shallower. They are richer productivity.This can ,be aemOnstated in dissolved solids; N, P, and Ca are by a comparison.of the TVA reservoirs abundant.. Plankton is abundant and 'CN- which practice asummaidrawdown ther'e is often a rich bottom fauna. with some of those in, the 'west where ,a winter drawdown is the rule. 4 Dy strophic lakes, such as bog lakes, are low in Ph, water yellow to brown, 2The level at which watif is removed dispolved solids, N, 1', and Ca scanty from a reservoir is important to the. ;; but hun4c materials abundant, bottom productivity of the stream below. , fauna and plankton poor, and fish 'The hypolimnion may. be anaerobic species 13.re limited. while the epilimnion is aerobic, .for ...example, or...the epilimnion is poor in Reservoirs lay also beclass ifiedas nutrients. While the hypolitnnion is orage, an run of the river. relatively rich,_ 4.. orage reserNirs have a large 3Reservoir discharges also Profoundly volume in relation to their inflow. affect the DO, temperature, and turbidity in the.stream below a'dam. 2Run'of the river reservoirs have a Too much fluctuationin flow may'° iarge.flow-thrOZh in relation to their permit sections of he stream, to dry, ° storage value: or proVide inadequate dilution for ..toxic waste. ,The Aquatic Environment

I C According to 'location, lakes and reservoirs may be classified as polar, temperate, or tropical.Differences in. climatic .and geographic conditions xesult in,sdifferences in theiebiology.

a VI SUMMARY A A body of water such as a lake, stream, l, or estuary represents an intricately balanced-system in a state of dynamic eqnilibrium. Modification imposed at one point in the system automatically results in compensatory adjustments at associated' B The mca4 th rough our knowledge of the entire system the better we can judge 40. where to ivnpo e control, measures to achieve a desir d result.

REFERENCES

1 Chamberlin, Thomas C. and Salisburg, Rollin1).Geological Processes and Their ResUlts. Geology 1: pp and 1-654. Henry Holt and Company. New York.1904.

2Hutcheson, George E. A-Treatise, on Limnology Vol. I \Geography, Physics and Chemistry. 197. Vol. II. Introduction to Lake Biology and the Limnoplankton.11'l5 pp.1967. John Wiley CO. e 3Hynes, H. B. N.The Ecology of Running Waters. Univ. Toronto Press. 555 pp.1970,

4De Santo, Robert S.Concepts of Applied EpolOgy. Spinger-

Verlag.1978. A

1 to,

0 .1 -2

MO 0 .4. 4-

Part 4.The Marine Environmentand its Mile in the Total Aquatic EhAronment .I TABLE 1 l e .. '1 k. PERCENTAGE.COMPOSITION OF THE MAJOg IONS INTRODUCTION, OF TWO STREAMS AND SEA WATER . -' (Data from Cthrk, F.W., 1924, '"The CoinpOsition offfiverx A The marine emlir onmerit is arbitrarily and Lake Waters of the United Stales", U.S. . Geal. Surv, .., , Prof. Paper'No. 135; Harvey, H. W.1957. "The Chemistry 4 defined as the water massextending and Fertility of Sea Waters", Cambridge ljniversitrPretx, ?. , Cambridge) beyond the continental land masse1/4., ..., V'''' " 4 -,. including the plants and animals harbored'. 1 '1 r DeIaware River ..4-)AloGrande" , - i.e.! , .",. therein. This water`mass is large and -Ioik:,1,-.-.rat _ ,-, at '' - 0e4-41Vajec 'i'deep, coveleing 'about 70 percent of the l'ilr.as#***-.' -4 - earth's surface and being as deep as .1,ando Na 6.70 'ire- 1:143,0: ,.:,;30.°4 7 miles. The salt content averages. .. "-81.46 1.ti: :85i-4-!\.1-,,, about 35 parts per.thousand.Life extends 1r Ca - ,.1 '17.49 --. 13.I2 41- 1:16 .to all depths'. . Mg 4.81 503/ 4 3.7 .. o Cl., h44.23 2t. 65 1. 55.2 :B The general nature of the water cycle on e . earth is well knOwn. Because the largest' SO4 17.49 30.10 7.7 . . -... CO 32.99 ., 11.55 +HCO3 0.35 portion of the; surface area of the earth 3,5" is covered-with water, roughly 70 percent. of the earth's rainfall is on the seas. C 'or this presentation, the marine (Figure 1) .environment will be (1) described using an ecological, approach, (2) characterized ecolOgically by comparing it with fresh-, water and estuarine environments, and (3) considered as a functional: eqological "systerh (ecosystem). .

IFRESHWAT&,ESTUARINE)._ AND RINE ENVIRONMENTS, 7'

TIgmr, 1. TNE WATER CYCLE Distinct differences are found inphgsicare .--.--7\chemtical, dr kci biotic factors in going from a fr,enhwater to an oceanic environment. Since roughly one third of the . In general, environmental factors are more . rain which falls on the land is again constant in freswater iriVers) and oceanic' recycled through the atmospiiere. 1414,' - ',.-environments than in the highly variable (see Figure 1 again), the total amount and harsh environments of estuarine an of water washing.Over-Ithei.p.rth's surface, coastal waters.(Figure 2) , is significantly greaterethan one third c the total world rainfall.It is thusnot physical and Ch7nical Factors surprising to note that the rivers which finally empty into the sell carry'a RiVers, estfaaries, and oceans are disproportionate burden of dissolved and ..14dompared.in Figure 2 with referenee to suspended`golids picked up from the --, the relative instability (or variation) of The chemical.composition of this burden several important parameters: ',In the depends on the 'cOmpoditibn of the rocks oceans, J will be noted, very little change and soils through vi,./hIchihe river flMvs,' occurs in any,parametei.. In fivers, while' the'Pr'oximity of an ocean; the direction "Salinity" (usually, referred to as udissalbed- of prevailing winds, and otherfacars. -:salids") and teMperature,(accepting normal This latho substance of geological eroSiOn. seasokelvariation,$) changtlittle'3,the other (Table_ 1) 4f four pars:ters'vary c nsiderphly.In estuattes, they.all chan e. 1 23 AK -The Aquatic Environment -

Degree of instability . Avail- Type of environment - Water .Vertical ageneral direction Salinity Temperature Strati- - -ability Turbidity arwater movement elevation fication oS.,00- s nutrients (degree) , . -

Riverine - I

. .. .

.

_

scene .

. ,

Oceanic ,I I . c--

Figure 2. RELATIVE 'VALUES OF VARIOUS PHYSICAL AND CHEMICAL FACTORS ' FOR RIVER, ESTUARINE, AND OCEANIC ENVIRONMENTS B Biotid Factors C Zones of the Sea

.A4r- A complex of physical and ch(mical The nearshore environment is often factors determine the biotic composi- classified -in relation to tide level and tion of an environment.In general, water depth.The nearshore and offshore the number of species in a rigorous, oceanic regions isagether, are often highly variable environment tends to Ike classified with reference to light penetra- less than the number in a More stable tion and water depth. (Figure 3) environment (Hedgpeth, 1966). 1 Neritic - Relatively shallow-water The dominant animal species (in zone which extends froin the high- \ terms.of total biomass) which occur 4* tide mark to the edge of the -7 in estuaries ke often transient, . .pending only N. part of their lives in estuaries.This results in better utilization ofarich environment.

I

27

1-24 The Aquatic Environment'

*MARINE ECOLOGY PEL A 6/C

Swm Suomi 1 T/C 0 C EA N I C opf P opic

0. 011 L it !pot '70 (Intertidal) Sittilittorol $0. tee

PELAGIC (WM') 14 Ntelhe, wecop logic. 1), GIctone EPNI0Pe °we 'Ineptly& .54.1ltyptlave .r.z_r_r_rx://xxxe7-7 Atforvilopr gra 8ENTHIC (Bottom) SuproItttoral Littoral (IntHIJol) ACV Mocaor CA) Kee 14a NV SOldtatel tl scret civotle of P.* BolOrpologic Inns. hVIC. Oyler Bathyal 6. Xt. AbliSta

e ' 4/././C'2 Atisse0,109,C Jhe, txe Xt. N:N:\ 13-ENTHIC ON* FIGURE 3Classikition of marine environments

..sow . aStability of physical factors is .1) Physical factors fluctuate intermediate between estuarine less than in the . 00 and oceanic environments. 2) Producers are the phyto- b Phytoplankters are the dominant plankton and consumers are producers but in some locations the zooplankton and nekton. attached algae are also important As producers. b - From the bottom of the euptiotic zone to about c The animal consumers are 2000 meters. zooplankton, nektoil; and benthic forms. 1) _Physical factors relatively constant but light is absent. 2Oceanic - The region of the ocean . beyond the continental shelf.Divided 2.) Producers are abSent and into three parts, all relatively consumers are scarce. poorly populated compared to the neritic zone., c - All the sea below the bathyal zone, aEuEihotic zone - Waters into which sunlight penetrates (often to the 1) Physical factors'more con- bottom, in the neritic zone). The stant than in bathyal zone:. zone of primary productivity often extend's to 600 feet below the surface. 2) Producers absent and consumers ven less abundant than in the bathyal 'zone. 1-25 . The A uatic Environment

IIISEA WATER AND THE BODY FLUIDS N FACTORS AFFECTING THE DISTRI- . BUTION OF MARINE AND ESTUARINE A Sea water is a remarkably suitable ORGANISMS environment for living cells,, as it contains11 of the chemical elements A Salinity.Salinity is the single most essential to thesgrowthand maintenance.. constant and controlling factoAtn the ' of plant and animalS. The ratio and ' marine environment, probablbllowed often the concentration of the major by temperature.It ranges around salts of sea water are strikingly similar 35, 000 mg. per liter, or "35 parts per . . in the cytoplasm and body fluids of thousand" (symbol: 35 %) in the language marine organisms. This similarity is of the oceanographer. While variations also evident; although modified somewhat in the open ocean= are relatively small, in the body fluids of fresh water and salinity decreases rapidly as one terrestrial animals. For example, approaches shore'and prpceeds through sterile sea water may be usedin the estuary and up into fresh water with emergencies as a substitute for blood a salinity of "0 Too (see -Figure 2) plasma in man. B Salinity and temperature as limiting B Since marine organisms have an internal . factors in ecological distribution. salt content similar to that of their `*,r) surrounding medium (isotonic condition) 1 Organisms differ in the salinities osmoregulation poses no problem. On the and temperatures in which they other hand, fresh water organisms are prefer to live, and in the variabilities hypertonic (osmotic pressure of body ofthese parameters which they can ands is higher than that of the surround- t olerate. These preferences and ing water).Hence, fresh water animals tolerances often change with successive must constantly expend more energy to life history stages, and in turn often keep water out (i. e., high osmotic dictate where the organisms live:. 1 pressure fluids contain more salts, the their "distribution.' , action being then to dilute this concen- tration with more water). 2These requirements or preferences often lead to extensive migrations 1 Generally, marine invertebrates are of various Species for breeding, narrowly 2oildlosmotic, i.e., the salt feeding, mild growing stages. One concentration of the body fluids chatnges very important-resnkof this is that with that of the external medium. This an estuarine environment is an has 'special significance in estuarine absolute necessity for over half of situations where salt concentrations all coastal commercial and sport of the water often vary considerably related species- of fishes and invertebrates,' e. in short periods of time. for eitherall or certain portions of their life histories. (Part V; figure 8) 2 Marine bony fish (teldosts4ave loWer salt content internally than aite external 3' The Greek word roots "eury" environment (hypotonic).' In order to (meaning wide) and "steno '-(ineaning prevent dehydration, water is ingested narrow) are customarily combined, and salts are excreted through special with such Words as "haline" for salt, cells in the gills. and "thermal" for temperature, to give us "euryhaline" as an adjective to characterize an organism able to tolerate a wide range of salinity, for example; or "stenothermal" meaning one which cahnot stand -much change ti in temperature. "Meso-" is a prefix indicating an intermediate capacity.

29 ' The Aquatic Environment

4 Some will knoWn and interesting examples of migratory species which C Marine, estuarine, and fresh water change their environmental preferemes organisms. (See-Figure 4) with the-life history stage include the shrimp (mentioned above), striped bass,, many herrings and relatives, the salmons, and many others. -None are more dramatics than the salmon hordes which hatch in headwater streams; migrate far out to feed and grow, then return to the mountain stream. where they hatched 'to lay their own eggs before dying. 5 Among euryhaline animals landlocked (trapped), populations,living in lowered Nlinitieskoften have a smaller maximum 0 Salinity ",; ca. 35 size than individuals of the same species living in more saline waters. For Figure 4.Salinity Tolefance of Organisms example, the lamprey (Petromyzon marinus)attains a length of 30 - 56" 1 Offshore marine organisms are, in in the sea, while in the Great Lakes general, both stenohaline and the length is 18 -.24". stenothermal unless, as noted above, they have certain life history require- Usually the larvae of aquatic organisms ments for estuarine conditions. are more sensitive to changes in salinity than are thesodults.This Fresh water organisms are also characteristic both limits and dictates stenohaline, and (except for seasonal the distribution and size of populations. adaptation) meso- or steno ,ermal. (Figure 2) D The effects of tides on organisms.

3 Indi us or native estuarine species 1 Tidal fluctuations probably subject at normally spend their entire lives the benthic or intertidal populations in the estuary are relatively few in to the most extreme and rapid variations number. (See Figure 5).Tpey are of environmental stress encountered generally meso- or euryhaline and in any aquatic habitat. Highly specialized meso- or eurythermal, communities have developed in this zone, some adapted to the rocky surf zones of the open coast, others to the muddy inlets of protected estuaries. Tidal reaches of fresh water rivers,, sandy beaches, reefs and swamps in the tropics; all have their own floras and .All must emerge and flourish when whatever water there is rises and povers.or tears at them, all must collapse or 10 15 20 25 30 35 retract to endure drying, blazing bnityau tropical sun, or freezing arctic ice Figure 5.DISTRIBUTION OF during the low tide interval.Such a ORGANISMS IN AN ESTUARY community is depicted in Figure 6. tEuryhalirie, freshwater bIndigenous, estuarine, (mesohaline) cEuryhaline, marine

1-27 , t. the Aquatic Environment

SNAILS Littorina neritoides L. rudis I.. obtusata O.L. littorea P,A dNA CLES

0!) Chthamalus stellatus 0Balanus balanoides 13. pe Hoist uS

Figure 6 Zonation of plants, snails, and barnacles on a , While this diagram is based on the situation on the southwest coast of England, the general idea of zonation may be applied to any temper-- att. rocky ocean shore, though the species will differ,The gray zone consists largely of lichens. At the left is the zonation of rocks with exposure too extreme topport algae; at the right, on a less exposed situation, the animals re mostly obscured by the algae. Figures at the right hand margin refer to the percent of time that the zone is exposed.to the air, i.e., the time that the tide is out. 'Three major zones can be recognized: the L4ttorina zone (above the gray zone); the Balan.oid zone (between the gray zone and the laminarias); and the Laininaria zone,a. Pelvetia cankliculata; b. Fucus spiralis; c. nodosum; d. Fucus serratus; e.Laminaria digitata. (Based on Stephenson) .

0

..-/

31 The Auatic Environment

V FACTORS AFFECTINGTHE REFERENCES PRODUCTIVITY OF THE MARINE ENVIRONMENT 1 Harvey, H. W. The Chemistry and Fertility of Sea Water (2nd Ed. )., A The sea is in continuous circulation. ,With- Cambridge Univ. Press, New York. 'on circulation, nutrients' of the ocea.n would 234 pp.1957. eventually become a part of the bottom and biological Production would cease.Generally, 2Wickstead,, John.Marine Zooplankton in all oceans there.exists a warm surface Studies in Bio gy,no. 62.The Institute layer which overlies the dolder'water and of Biology. 78. forms a two-layer system of persistent stability.Nutrient concentration is-usually greatest in the lower zone. Wherever a mixing or disturbance of these two layers occurs biological production is greatest. B The estuaries are also a mixing zone of enormous importance. Here the fertility washed off the lanclis mingled with the nutrient capacity of , and many of the would's most productive waters result. C When man adds his cultural contributions of sewage, fertilizer, silt or toxic waste, it is no wonder that the dynamic. equilibriu of the ages is rudely upset, and the environmentalist. cries; "See what man hath wrought "!

ACKNOWLEDGEMENT':' This outline contains selected material from other outlines prepared by C. M. Tarzwell, Charles L. Brown, Jr.$ C. G. Gunnerson, W. Lee Trent, W. 13. Cooke, 13. H. Ketekum, J. McNulty,' J. L. Taylor, R. M. Sinclair,. and others. ON

Part 5:Wetlands

IINTRODUCTION B Estuarine pollution studies are usually .., . devoted to the dynamicsul.the'circulattng . A Broadly defined, wetlands are areas water, its chemical, physical, and which are "to wetto plough but too biological parameters, bottom, deposits, etc. thick to flow." The soil tends to be saturated with water, salt of fresh, C It is easy to overlook the intimate relation- and numerous channels or ponds of ships which exist between the bordering shaliow or open water are common. marshland, the moving waters, the tidal Due to ecological features too numerous flats, subtidal deposition, and seston ,.lid variable to list here, they comprise -, whether of locale oceanic, or riverine general ta rigorous (highly stressed) origin. habitat, occupied by a small relatively specialized indigenous (native) flora D The tidal marsh (some inlandareas also and fauna. ,i have salt marshes) is generally considered tb be the marginal areas or estuaries and B They are prodigiously productive coasts in the , 4hich are however, and many constitute an dominated by emergent vegetation. They absolutely essential habitat for some generally extend inland to the farthest portion of the life history of animal point reached by the spring tides, where forms generally recognized as residents they merge into freshwater swamps and of other (Figure ti).This is marshes 4-Figure 1).They may range in particularly true of tidal marshes as width from nonexistent on rocky coasts to mentioned below. many kilometers. C Wetlands( intoto comprise a remarkably large proportion of the earth's surface, and the total organic carbon bound in . their mass constitutes an enormous 1 sink of energy.

Freshwater D Since, our main concern here is with Channel LMud Flat Tidal Marsh Marsh the "aquatic" eironment, primary tuinMara emphasis will be directed towarda descr t ption of*vetlands as the transitional zone' etween the waters and the land, and how their desecration by human culture ?it - - ::: spreads degradation in both direction,. 47pat 0

- Blue Clay Substrate IITIDAL MARSHES AND THE. ESTUARY. 0*P...ert rtgurcl.Zonation in e positive New England estuary.1. Sprtng tide level. 2. Mean higlyide. S. Mean low tide, 4. Dog hole. S.leo eleavap pool, 4. Chunk of Spartina turf deposited by lee. T. Organic oose with associated community. O. pipes* (Zostei ra). 2;Ribbed mussels froodiolusl- A "There is no other case in nature, save clamQM);mud snailMealcommunity. 10. lettur vai in the coral reefs, where the adjustment of or anie'relations to physicardttlition a seein such a. beautiful way as the balan e between the growing marshep and the tidal streams by which thePare at once nourished and worn awe*: (Shale , ).886): The Aquatic, Envir6timent

IIIMARSH ORIGINS AND 'STRUCTURES Such banks are likely to be cliff-like, and are often undercut.Chunks of A In general, marsh substrates are high in ,peat are oen found lying about on organic content, relatively low in minerals harder sViattrate below high tide line. and trace elements. The upper layers If face of cliff is well above high water, bound together with living roots called overlying vegetation is likely to be turf, underlaid by more compacted peat typically terrestrial Of the area. type material. Marsh type vegetation is probably'. absent. 1 Rising or eroding coastlines may expose peat from ancient marsh 2 Low lying deltaic, or sinking coast= growth to wave action which cuts lines, or those with low energy wave into the soft peat rapidly (Figure 2). action are likely to have active marsh formation in progress. Sand dunes .. are also common in such areas A1)(11,1( h (Figure 34.General coastal Terrestrial turf configuration is a factor.

Salt marsh eat

Substrate . 7-:;: . Figure 2. gran.--13ta maticseion of.erodlng prat cliff

0

O Figure 3 . Development pi a Massachusetts Marsh since .1300 BC.involving an 18 foot rise in water level. Shaded area indicates sand dunes.Note imeandering marsh tidal drainage.A: 1300 BC, Br 1950 AD. 34 1132 ... The Acpiatic EnVironment

aRugged or precipitous coasts or slowly rising coasts, typically eghibit narrow shelves; sea/cliffs, fjords, massive beaches, and . relatiyely less marsh area (Figure 4). An Alaskan fjord subject to recent

I catastrophic subsidoce and rapid - deposition of glaciflour Shows eVidencelif the recent encToachment of saline waters in the presence of - `recently buried trees and other terrestrial vegetation, 'exposure . of layers of salt marsh peat 4ong the edges of channels, .and.a poorly compacted younglmarsh turf developing at the. new high water level (Figure 5).

b

Figure 4 A River Mouth on a Slowly Rising Coast. Note abseime of deltaic development' and relatively marshland although mud flats stippled are extensive,

Shifting flats Tidal marsh I'Terre stria jilt° 03 0 Et',

2 8 . 4. . it\ /..:,4 , : a ,e ';. g .. '

. .. /Vv\A

.11,

Figure 5 Some genial relationships in 4 northern fjord witla rising water level.1. ,mean low 'water,' 2, maximum high tide, 3,Bedrock, 4.Glacial flour to depths in excess or- b() meters, 5.Shifting flats and channels, 6.Channel against bedrock, 7.Buried terrestrial vegetation, 8.Outcroppings of salt marsh peat.-

F (V bLowslying coastal plains tend to be cs Deep tidal' channels fan but through fringed by barrier islands, broadc. ° irmumerablebranching and often estuaries and deltas, and broad Infer-connecting rivulets: The assqciated marshlands (Figure 3). izitervening. grassy plains are _ essentially, at mean high tide level.

A

1 -33 C. F_ The Aquatic Environment

cTropical and subtropical regions tidal mar sh is the mar.sh grass, but ver34 such as Florida, the Gulf Coast, little of it is use& flreman as grass. and Central America, are _frequented (11ab1, 1) "'by mangrove swamps. This unique _ type of growth is able to establish The nutritionaLanalysis of several. itself in shallow water and move out marsh grasses as compared to airy land" into progressively' deeper areas hay is shown in Table 2. '(Figure 6). The strong deeply .. embedded roots enable the-mangrove to resist considerable wave action at times, and the tangle of roots TABLE1,, General Order's of Magnitude &Gross Primary Productivity in Terms quickly accumulates a deep layer of of ,Dry Weight of Organic Matter Pitad Annually ., organic ... gms/M/year in the south may-be considered to Ec9system (grarns/awkre meters/year) lbs/acre/year be roughly the equivalen,t of the Landdesekts, deep sceane v Tens Hundreds Spartina marsh grassin the north Grass4ds, forests, cutrophic Hundreds Ttiousends as a land builder. Whenfully lakes, ordthary agriculture Estuaries, deltas, coral reefs, Thousands Tenthousands developed, a mangrove swamp is an intensive agriculture (sugar impenetrable thicket of roots over cane% rice) the tidal flat affording shelter to an assortment of semi-aquatic organisms such as various molluscs arid crustaceans,' and providing access from the nearby land to predaceous bir'dsa.reptiles, add mammals. , Mprigrov.es are not restricted to # estuaries, buernay develop out into . TABLE2. Analyses of Some Tidal Marsh Grasses shallow oceanic lagoons, orupstream into relatively fresh waters.-

T/A li'ercentage Composition Protein r Eat .Fiber Water Ash Nfree Extract attiosvora Dry Wt; WACO. Tit=taruS,4ASSCOCS SALtiiIrrerASSOCAO COMOOLS I Divichln spicata (Ore stand, dry) :resit 2.8 ' 5.3 1.7. 32.4 8.1 6.7 4S.5 Short Spartina alternillora and Sabccenia curopaea In standing water) IN0001 1.2 7.7 2.5 31.1 - 6.6 12.0 37.7 Is= Spartina alterndlora (tall, pure stand in standing water) 3.S 7.6' 2.0 . 29.0 8.3 15.5 37.3 an1.40.4111 Sparrinavalftns sport stand, dry) .:. .. . 3.2 , 6,0 ,.. 2.1 30.0 8.1 9.0 44.5 Sparrina a/remit/4ra and Sp.iffifla patens (mixed stand, wet) 3.4 - 6.6 1.9.4 .29.5 8.1 10.4 42.8 o Spariniakernillura (short, wet) Z2 13.0 . 2.4 30.4 81 13.3 36.3 warauravasor Comparable Analyses for Hal 0 El 0 Figure 6 1st rut 6.0 2.5 ° 36.2 ",,0 6.7 4.2 44.9 Diagrammatic transect of a mangrove swamp 2,,,roil 11.0 3.7 25.5 10.4 5.9 30.5 showing transition from marine to terrestrial a habitat.° , AnalrieeperfOrMedby Roland W.. Gilbert, Deptrtment 0 b. ofAgricultural Chemistry, U. R. I. 4O . N PRODUCTIVITY OF WETLANDS

o A Measuring the productivity of not_easy,tbecause.today,grass is seldpm Ow used directly 4isisuch by man. Itis thus . - usually 'expiessed-as production of meat, milk, or bythe case of salt marshes, 'the . total crop'of animals that obtain food per :. unit of area.'4he.prfirnary producer in a:. 3C . .

61-14 'I The Aquatic Environment

11 B The actual utjlizaiion'of marsh grass is accomplighed primarily by its decom- 11;"1 position-and ingestion by micro organ&ns. . (Figure 7) A small quantity of seeds and solids is consumed directly by hirds. VrTZtle -1 YOUNG

ISOUNJ23

N OCEAN

.eli4106A ek ADULT EGGS

Figure 8Diagram of the life cycle- . of white shrimp (after Anderson.and .Lunz .1965).

3. An effort to make an indirect , estimate,ofprogiuctivity in a ithOde Figure 7The nutritive composition of ^ Island marsh was made on a single successive stages of decomposition of - dad by recording the numbers Spartina marsh grass, showing increase in protein anddecreasept carbohydrate 1= and kinds oebirdirthat fed on a with increasing age and decreasing size, relatively -small area (Figure of detritus particles. Betwepn 700 and 1000 Wild oirdsof 12 species, ranging from 100 least 1 The quantity of micro invertebrates sandpipers to uncountable numbers which thrive on this wealth of decaying of seagulls were counted. One food marsh has not been 'estimated, nor has requirement estimate for three- the actual production'of small indigenous pound poultry in the confined inactivity fishes and invertebrates such as the of a poultry yard is approximately one ft top:minn.ows (Fundulus),or the mud ounce per pound of bird per day. -snaili (Nesse), and others. 2 Many forms of oceanic life migrate into the estuaries, especially the' marsh areas, for important portiohs ; Ps. of their life histories as is mentioned a elsewhere (Figure 8)." It has been. 0 estimated that in exgess of. 60% of the marine commercia1:4nd sport fisherIe4e are estuarine or marsh dependent in some way. . Greater Yellow legs (left)- and black duck. Nr\

Great blue heron 0 . Figure 9SomeCommon Marsh Birds

444 4

04/ 0, V. 1-35-

',- -40;: The Aquatic Environment

One-hundred black bellied plovers and geographic distribution, etc. at approximately ten ounces each Included would be the familiar cattails, would weigh on the order of sixty spike rushes, cotton grasses, sedges, pounds, At4the same rate of food trefoils, alders, and many, many'. consumption, this would indicate Others. nearly fotfr pbunds of food required for this species Lone. The much C Types of inland wetlands. greater activity of the wild birds would obviously greatly increase their 1 As noted above (Cf: Figure 1) food requirements, as would their tidal marshes often merge into relatiSely smaller size. freshwater marshes and bayous. - Deltaic tidal swamps and marshes -Considering the range of foods con- are often saline in the seaward sumed, the sizes of the birds, and the portion, and fresh in the landward fact that at certain seasons, thousands . . areas. of migrating ducks and others pause to feed here, the enormous productivity, 2River bottom wetlands differ from of such a rbarsh can be better under- -those formed from lakes, since wide stood. flood plains subject to periodic inundation are the final stages of - the erosion of river valleys, whereas lakes in general tend to be eliminated VINLAND BOAS AND MARSHES byhe geologic proceSses of natural eutrophication often involving AMuch of what has been said of tidal Sphagnum and peat formation. marshes also applies to inland wetlands. Riverbottom marshes in the southern As was mentioned earlier, not all inland United States, with favorable climates, swamps are salt-free, any more than all have luxurient growths such as the marshes affected by tidal rythms are canebrake of the lower Mississippi, saline. ° or a characteristic timber growth such as cypress. - .. 13 The specificity of specialtz'ed floras to particula types'Of wetlands is perhaps 3Aithoug\bird lift is the more spectacular in freshwater wetlands conspicuoUs animal elenerin the than in the marine, where Juncus, fauna (Cf:- izigure 9), many mammals, Spartina, and Mangroves tend to dominate. such as muskrats, beavers, otters, and others arealmarsh-oriented. t 1 Sphagnum, or peat mass,--is' Figure 12) - , probably one of the most widespe d o. .2 ,, and abundant plants on ea Deevey Q.958) quotes an estimate tai there is pt:obably upwards of 223 billions (dry weight) of tons of peat in ti( world today, derived during recent geologic time, from Sphagnum bogs. Particularly in the northern regions,eat moss t nds to overgrow to ponds An shallow defissions, eventually forming the vast turd lains-and- moores of the nort

2 Long lists of other bog and marsh plants Might be cited; each with its own special requirements, topographical, 33 r ft. S 4 The AcLuatiC Environment

VI POLLUTION 2 Marsh grasRes.may alsO be eliminated - by smothering as, for example, by A No "Single statement can summarize the deposition of dredge spoils, or the of pollution on Marshlands'as spill or discharge of sewage sludge. distinct from effects ,noted elsewhere on other habitats. 3 Considerable marsh.area has been 4 eliminated by industrial construction B .Reduction of Primary Productivity activity, such as wharf and dock con- struction, oil well construction and The primary producers in most wetlands operation, and the discharge of toxic are the grasses and peat mosses. brines and other chemicals. Production may be reduced or eliminated by: C Consumer production (animal life) has been dr a st Joan:), 're'auised by th'..dr. liberated' 1' Changes iri the- water level brought distribution of-pesticides. .lesome cases, about by flooding or drainage. this has been aimed at nearby agricultural,. , lands for economic crop pest control, in a Marshland areas are s5metimes other cases the marshes have been sprayed diked and flooded t9 produce fre or dusted directly to control noxious water ponds. This may bq for' insects. 4 aesthetic reasons, to ,suppress the ingrowth of noxious marsh inhabitating The results have been universally sects -such as mosquitoes or biting disadtrous for the marshes, and the midges, to constructan industrial bene \to the human community often waste }folding pond; a thermal or a questio able. sewagg stabilization pond, a .` "convenient" result of highway .2Pesticides designed to kill nuisance' 'causeway construction, or other insects, are atso toxic to other reason., The result is theelim- arthropods sb that in addition to the' ination of an area of marsh. A a target species, such forage staples as sitallcompensating border of the various scuds (amphipods), fiddler marsh may or may nor develop. crabs, and other macroinvertebrate have either been drastically reduced b r-Highi'dal marshes were often or entirely eliminated in many places ditcheand drained in former days For example, one familiar with fiddle to stabze the sod for salt hay or crabs can traverse miles of marsh "thatch' barvesting which was highly margins, still riddled with their burrows,) sought a sr in colonial days.This without seeing a single live crab. ,inevitably changed the character of the marsh, but it remained as a, DDT and related compouna have been' essentially marshland. COnversion "eaten up the food chain" (biological to outright agricultural land has Magnification effect) until fidh eating been less widespread because of the and other predatory birds such as herons necessity of diking to exclude the and egrets (Figure 9), have been virtually periodic floods or tidal incursions,.. eliminated from vast areas, and the / and carefully timed drainage to -accumulation of DDT in .nian himself eliminate excess precipitation. is only too well known. Mechanical tidal marshes has_not been econo'cal in this country, althoUgh the succesb of the Dutch andOthers in this regard is well known. ii

The Aquatic Environmen

1' - . .1? D Most serious of marsh enemies is E Swimming birds uch as duckd, loons, man himself.In his quest for "lebensraum" cormorants, pelicans, and many othervs near the water, he has all but killed the are severelyjeopardizedby floating water he strives fo approach.. Thus up to - pollutants such as oil.. twenty percent of the marshestuarine area in various pails of the country has already beentutterly destroyed by cut and . fill real estatedevelopment's (Figures, `10, 11).

Yt t

4 rigure 10.Diagrammatic representation of cut-and-Sill for real estatexclevelopmentmlw = m ean 'low. water

S

. . i Figure 11.Tracings of portion of map of a southern 1, : .. city. showing, extent of cut-and-fill real j.,, -f estate development. t . ) . 0 z et 6. The Aquatic Environmentr

4. Oft 0

VII SUMMARY 5Morgan, .J. P. Ephemeral Estuaries of the Deltaic Environnient in :'Estuaries; A "Wetlands comprise the marshes, swamps, pp. 115-120. Publ. No. 83, Am. bogs, and areas of the world. Assoc. =Adv. Sol,Washington, DC. 1967. They are, essential to the well-being of our surface waters and ground waters. 6 Odum, E.P. and Dela Crug, A.A. They are essential to aquatic life of Particulate Organic Detritus in'a all types living in the open waters. They Georgia Salt Marsh - Estuarine are essential as habitat for all forms of, Ecosystem. 'in:Estuaries, pp. 383- wildlife. _ 388, Publ. No. 83, Am. Assoc. Adv. Sci. Washington, DC.1957. B The tidal marsh is the area of emergent 0 = vegetation bordea.igthe,..ocean er ash 7Redfield, A. C.The .Ontogeny of a Salt. , estuary. Marsh Estuary. in: Estuaries, pp. 108-114.Pulil. No. 83, Arn, Assoc. C Marshes are highly productive areas, Adv. Sci. Washington, QC.1967. essential to the maintenance of a well rounded community of aquatic life. 8 Stuckey, 0. H. Measuring the Productivity of Salt Marshes: Maritimes (Grad D Wetlands may be destroyed by: School of Ocean. , U. R. I. )Vol. 14(1): 9-11, February 1970. 1 Degradation of the life forms of

Aft, which it is composed in the name of Williams, -11.B. _Compartmental nuisance control. Analysis of Production and Decay of Juncus reomerianus.Prog. 2Physical destruction by cut-and-fill Report, Radiobiol. Lab., Beaufort, NC, to create more land area. Fiscal Year 1968, USDI, BCF, pp, 10- 12. ,

0 This outline was prepared by H. W. Jackson, former Chief Biologist, National Training. REFERENCES Center, and revised by R. M._ Sinclair, Aquatic Anderson, VP. W. The Shrimp anti the Biologist, National Training Center, MOTD,I Shrimp Fishery of the Southern " OWPO, EPA, Cincinnati, OH 45268. United States.TJSDI, FWS, BCF. Fiihery Leaflet 5,89.1966. ° 2 ,,DeeVey,- E. S. ,. Jr. ,BogS. Sci.' Am. Vol. 199(4):115-122. October 1958. Descriptors: Aquatic Environment, Biological Estuarine Environment, Lento Enrironment, 3 "Emery, K. 0. and Stevenson. Estuaries Lotic Environment, Currents, Marshes, and Lagoons. Part II, Biological Limnology, Magnification, Water Properties

0 Aspects by J.W. Hedgepeth, pp: -693- "M. in:. Treatise on Marine Ecology and Pleoecology. Geol. Soc. Am. Mem. 6 ?.Washington, DC.' 1957. 4 Hesse, R.;; W. C. Allee, and K. P. Schmidt. Ecological Animal

6,4 GeOgraphyw John Wiley; & Sons. 1937.

6

(1-39

A** CLASSIFICATIONOF COMMUNITIES, ECOSYSTEMS, AND TROPHIC LEVELS -

IA COMMUNITY is an ashmblige of 1 Collectors strain, filter, or otherwise populations of plants, animals, bacteria, collect fine particulate organic matter and fungi that live in an environmental and from the passing current. interact with one another, forming together a destinctive living system with its own 2 Shredders feed on leaves, detritust composition, structure, environmental and coarse particulate organic matter. relations, development, and function. , 3Grazers feed on attached growths. II An ECOSYSTEM is a community and its environment treated together as a functional 4Predators feed on other organisms.' system of complementary relationships, and transfer and circulation of energy and IV Taxonomic Groupings matter.(A delightful litte essajr.on the odyssey of atoms X and Y through an A TAXOCENES, a specific group of organisms. .ecosystem is in Leopold's, A Sand County Ex. midges. For obvious reasons most Almanac. ) systematists (..xonomists) can specialize- in only one group of organisms.This fact IIITROPHIC levels are a convenient means is difficult for the non-biologist to grasp: of classify ing organisms according to nutrition, or food and feeding.(See B Size, which is often dictated by the inves- -Figure 1. )' tigator's `sampling equipment and specific interests. A PRODUCER, the photosynthetic plant or first organism on the food chain sequence. V Arbitrary due to organismhabitat prefer- Fossil fuels were produced photosynthe- ences, available 'samplingdevices, personal preference of the investigator, - and mesh sizes of nets and sieves. BHerbivore or primary CONSUMER, the first animal which feeds on plant food.. A PLANKTON, organisms suspended in a body of water and at the mercy of currents. C First carnivore or secondary CONSUMER, This group has been subject to numerous an animal feeding on a plant-eating animal. divisional schemes. Plants are PHYTO- PLANKTON, and animals, ZOOPLANKTON. D.Second carnivore or tertiary CONSUMER Those retained by nets are obviously, MET feeding on the preceding. PLANKTON. Those passing thru even the- finest meshed nets are NANNOPLANKTON: ETertiary carnivore. B PERIPHYTON, the community of micro- FQuaternary carnivore. organisms which grow on submerged objects (substrates). -Literal meaning DECOMPOSERS OR REDUCERS, bacteria "to grow around plants", however which break down the above. organisms. standard glass microslides are sub- Often called the middlemen or stokers of mersed in the aquatic habitat to the furnace of photosynthesis., standardize results.

HSaprovores-ox! which feed C BENTHat. is often used to mean on bacteria and/or fungi. MACROINVERTEBRATES; although there ....-- are tenthic organisms inother Plant, Benthic I Macroinvertebrates have been subdiVided ariiinal, and protist grolips. into trophic levels according to feeding refers strictly to the bottom substratesof habits ,(See Figure 1 from Cummints); lakes, - streams, and other water bodies. ( 42 BI. ECO, 25a. 3.80 2-1 Classification of Communities, Ecosystems and Trophic Levels

0.5' METERS) MICROSES. CPOM 46. (404 ,. 2(1-2 METERS) Az4!re mootNin P/R°

MICROIE CPOM 401 00. W 4 (10 METERS) 0 PiR 5

PRODUCERS EDATORS 6 (50-75 METE (PERIPHYTON)

MicROSES COLLECTORS

4.P/R < 1 PROODCERS (PHYTOPeasicToN)

ORS j

OLLEcTORS (COMPLANKTON) I I I -12 METERS)

.FIGURE 1

43 2-2 Classification of Communities, Ecosystems andTrophic Levels

D ./MACROINVEATEBRATES, are animals JMANIPULATED SUBSTRATE COMMUNI- retained on a No. 30 mesh screen (approx- TIES. Like the preceding community, imately 0.5 mm) and thus visible to the; theses are manipulated by man. Placing naked eye. artificial or natural substrates in a body of water will cause these communities E MACROPHYTES,. the larger aquatic plants to appear thereon. which are divided into emersed, floating, and submersed communities. Usually K We will again emphasize ARBITRARY, vascular-plants but may inclUde the larger because organisms confound our neat algae and "primitive" plants.These have little schemes to classify them. Many posed tremendous economic problems in move from one community to another the large man-made lakes, especially for variousreasims. However, all in tropical areas. these basic scheme do have intrinsic value, provided they are used with F NEKTON, in freshwater, essentially fish, - reason: salamanders, and the larger crustacea. In contrast to PLANKTON,4\ese organisms are not at the mercy of the-current. REFERENCES

G NEUSTON, or PLEUSTON, -are inhabitants 1 Cummins, Kenneth W. The Ecology ofthe surfaCe film (meniscus organisms),- of Running Waters, Theory and either supported by It hanging from, or " Practice in Proc. Sandusky River breaking through. it.Other organisms Symposium. International Joint are trapped by this neat little barrier of Commission.1975. nature.The micro members of this are easily sampled by placing a clean cover. 2Leopold, Aldo. A Sand County Almanac. slip on top of the surface film then either Oxford University Press.1966. leaving it a specified time or examining: it immediately,under_the microscope.' 3 Peters, Robert Henry. The Unpredictable Problems of Tropho-dynamics. H DRIFT, macroinvertebrates which drift Env. Biol. Fish 2:97-101.1977. with the streams current either periodically (diel or 24 hour), behaviorally, catastro- phically or incidentally. This outline was prepared by R. M. Sinclair, BIOLOCIAL FLOCS, are suspended Aquatic Biologist, National Training and

' that are formed by ,. Operational Technology Center, MOTD, various means. In wastewater treatment OWPO, USEPA, Cincinnati, Ohio 45268. plants they are encouraged in concrete aer aeration basins 'using diffused air or Descriptors: Biological Communities oxygen (the,heart of the, activated sludge . , process). .

A

44 ,2 -3

1 LIMNOLOGY AND ECOLOGY OF PLANKTON

I INTRODUCTION br outside of a lake.

A Most Interference _Organisms are a That which comes in from 4ap Small. outside (allochthonous) is predominately inert solids B Small Organisms generally have (ttripton): Short Life Histories. b That of internal origin (auto- .0 POpulations of Organisms with chthonous) tends totbe bio- Short Life Histories may Fluctuate 1ogieal in nature. Rapidly in Response to Key Environ- 4' , mental Changes. B Heat and Temperature Phenomena are Important in Aquatic Ecology. D Small Organisms are Relatively at the Mercy of the Elements 1 The total 4uantityof heat avail- .able to a body of water per year E The Following Discussion will can be calculated and is known Analyze the Nature of These'Ele- as the heat budget. .ments with Reference to the Res- . ponseof Important Organisms; 2. Heat is derived directly from in- solation; alsO by transfer from air, internal friction, and other TI PHYSICAL FACTORS OF THE ENVIRONr sources. MENT Density Phenomena A Light is a Fundamlental SoUrce.of Energy for Life and Heat," '1 Density And viscosity affect the floatation and locomotion of 1 Insolation-is affected by geo- microorganisms. graphical location and mete- orological factors. a Pure fresh water achieves its maximum density. at 4 °C 2 Light penetration in water is and its maximum viscosity affected by'angle of .incidence at 0 °C. (geographicalY, turbidity, and color.'he. propOrtion of light b The "rate of change of density reflected depends on the angle) increases with the temperature. of incidence, the temperature, color, and other qualities of 2 Density stratification affect the water. In general, as the aquatic life and water uses. depth increases arithmetically, the light tends to decrease geo-: a In summer, a mass of warm metrically, Blues greens, and surface water, the epilimnion, yellows tend to penetrate most is usually present and separated deeply while ultraviolet, vio- from a cool deeper mass, the lets, and orange-reds are most hypolimnion, bYa relatively quickly absorbed. On the order thin layer known as the of 90% of the total illumination thermocline. which penetrates the surface '41 film es absorbed in the first b Ice cover and annualspring 10 meters of even the clearest and fall overturns are due to witdr. successive seasonal changes in the relative densities of 3 TuXiditymay originate within the epilimnion and the hypo- 3,1/

45.. 3-1 Limno loley and EcologyAokPlankton

limnion, profoundly influ- D Shore development, depth, inflow - enced by prevailing meteoro7 outflow pattern, and topographic logical conditions. features affect the behavior of the water.

c The sudden exchange of E Water movements that may affect organ- water masses having differ- isms include such phenomena as waves, ent chemical characteris- currents, tides, seicheS, floods, and tics may have catastrophic others. effects on bertain biota, may cause others to bloom.

d Silt laden waters may seek 1 Waves or rhythmic movement certain levels, depending on their own specific gravity The best luiolAn are traveling in relation to existing layers waves. These are effective already present. only against objects.near the surface.. They have little Saline waters will also 4. effect on the movement of stratify according to the large masses of water. relatiye densities of the ts Various layers. bStanding waves or seiches ocdur, in all lakes but are 3 Theoriseosity of water is greater seldom-large enough to be at lowertemperatures. observed. An "internal seich" is an oscillation in a density aThis is important not Only mass within a lake with no in situations involving the surface manifestation may control of flowing water as in cause considerable water a.sand filter, but also since moVement. overcoming resistance to . flow generates eat, it is significant in the heating - of waterby internal friction from waive and current ac- tion and many delay the establishment of anchor ice under critical conditions. , b It is easier for plankton to remain suspended in cold viscous (and also dense) water than in less Viscous warm water.This is re- flected in differences in the appearance of winter vs summer forms of life (also arctic vs tropical).

4G \ ti 4

Limnology and Ecology of Plankton

2 Currents There are typically two tidal ly cycles per lunar day (approx- a Currents are arhythmic imately 25 hours), but there is water movements which have continuous gradation from this had major study only in ocean- to only one cycle per (lunar) day ography. They primarily in some places, are concerned with the trans- location of water masses. Estuarine plankton populations They may be generated inter- are extremely influenced bylocal nally by virtue of density . tidal patterns. changes, or externally by wing or runoff.. d Flood waters ranmfrom torren- . tial velocities which tear away Turbulence phenomena or and transport vast masses of eddy currents are largely re- substrate to quiet backwaters sponsible for lateral mixing which may inundate normally dry in a current.These are of and areas for extended periods far more importance in the o time. In the former case, ..._ economy of a body of water, plktonic life is flushed away than mere laminar flow. co pletely; in the latter, alocal plaon bloom may develop which 4cTides, or'rathetidal may of immediate significance, currents, are reversible or which may serve as aninoculum (or oscillatory) on a relative- for receding waters. ly long and iiredictable period. They are closely allied to F Surface Tension and the SurfaceFilm seiches. For all practical purposes, they are restricted 1 The surface film is the habitat of to oceanic (especially-coastal) the "neuston", a group of special " waters. -significance.

If there is no freshwater 2* Surface tension lowered bysurfactants" inflow involved, tidal currents may eliminate theneuston.' This can are basically "in and out;" be, a significant biological observation. if a significant amount of freshwater is added to the IIIDISSOLVED SUBSTANCES system at a constant rate, the outflowing current will in generalA Carbon dioxide is reeased by plants and exceed the inflow by the amount animals in respiron but taken in by of freshwater inpvt. plants in photosynthesis. Oxygen is the biblogical complement of carbon dioxide, 'a.nd'necessary for all animal life. C ,Nitrogen and phosphorus are fundamental nutrients for plant life. 4 ee. Occurin great dilution, Concentrated by plants.

47' 3-3 Limnology and Ecology of PlanktOn 1111.01

0 The distribution of nitrogen V BIOTIC COMMUNITIES (OR ECOSYSTEMS) compounds is generally corr , ed with the oxygen curve, esp A A biotic community will be defined.here cially in oceans. j as an assemblage of organisms living in a given ecological niche (as defined D Iron, manganese, sulphur, and silicon below). Producer (plant - like); consumer are other minerals important to aquatic (animal-like) and reducer (bacteria and life which exhibit biological stratification. fungi) organisms are usually .included. . A source of energy (nutrient, food) must E Many other minerals are present but their also be present. The essential concept biological distribution in waters is less in that each so-called 'community is a well known? fluorine, .tin, and vanadium relatively independent entity.ArCtually shave recently been added to the "essential" this position is only tenable at any given list, and more may well follow. , instant, as individuals are constantly shifting from one community to another in F Dissolved organic matter is present in response to stages in their life cycles, even the purest of lakes. physical conditions, etc. The only one tosbe considered in detail here is the BIOLOGICAL FACTORS plankton. A Isttritional Classification of Organisms B Plankton are the macroscopic and microscopic animals, *ants, bacteria, 1 Holophyt ic or independent or- etc. floating free in the open water. ganisms, like green plants, pro- Many clog filters,. cause tastes, odors, duce their own basic food elements and other troubles in water supplies. from the physical environment. , 1 Those that pass !through a plankton 2 Holozoic or dependent organisms, net (No. 25 silk bolting cloth or like animals, ingest and digest equivalent) or sand filter are often solid food particles of organic known as nannopankton (they usually greatly exceed the "net" plankton in actual quantity). 3. Saprophytic or carrion eating organisms, like many fungi and 2 Those less than four microns in bacteria, digest and assimilate length are sometimes called the dead bodies of other organ-- ultraplankton. isms or their products. There are many ways in which B The Prey-Predator Relationship is plankton may be classified: taxo- Simply one Organismating Another. nomic,- ecological, industrial:

Toxic and Horm c Relationships 4 TheMit concentration of plankton varies markedly in space and time. 1 Some organisms such as certain blue green algae apd some ar- Depth, light,. currents, and mored flagellages produce s 4 water quality profoundly affect stances poisonous to others. plankton distribution. 2 Antibiotic action in nature is The relative abundance of 'not well understood but has been plankton in the; various sea- shown,to play a very influential sons is generally: role in the economy of nature. 0 1 sprjng, 2 fall, 3 summer, 4 winter 4P

"3-4

ti . 1. Limnology and EColbgy of Plankton

ot4

5 Marine plankton include many .deeper, more turbid, and usually _larger animal forms than are warmer water, sand, mud, silt, found in freshwaters. or clay,bottorn -materials which shift with increase in flow. C Thbenthic community is generally cgixsideredto be the macroscopic life 4 In old age, streams have appi-oa- living in or on the bottom. ched base level. During flood es, stage they scour their bed and de- D The peon community might be positmaterials on the flood plain defined as the microscopic benthos, which may be very broad an/ flat. except alai they are by no Means confined During normal flow the etteneris.._ 'refilled and many shifting bars are to the bottom. Any surface, floating, or I. not, is usually covered by 'film of living developed. organisms. ,There is frequent exchange betWeen the periphytdn and plankton (Under the influence of man this communities. patternmaygle broken up, or tem- porarily interrupted. Thus as essen- E The nekton is the comMunityof larger, tially "youthful" stream might take free-swimming animals (fishes, shrimps, On some of the characteristics of a etc. ), and so is dependent on the other .'.'mature" stream following soil - communities for basic plant foods. erosion, organic enrichment, and increased surface runoff.Correction F "Neuston or Pleuston of these conditions might likewise be followed by at least a partial rever- This community inhabits the air/water sion to the "original" condition.) interface, and may be suspended above or below it or break it. 'Naturally this C Lakes have a developmental history interface is a very critical One, it being which somewhat parallels that of streams. micro molecular and allowing interchange I°1 between atmospheric contaminants and 1 The method of formation greatly the water medium./ Rich in bacteria, influences the character and sub- metals, protozoi, pesticides etc. sequent history of lakes.;

2 Maturing or natural eutrophication VITIIE EVOLUTION OF WATERS _- of lakes A, The history of a body of waterdetermines e, its present condition. Naturalswaters haye aIf not already present, shoal evolvedin the course of geologic time areas are developed through to what we know today.. ro.?iop. of the shore by wave ., -action and undertow. B In the. course of their evolution, streams in general pass through four general bCurrents produce bars across' stages of development which may be called: bays and thus cut off irregulars birth, ypvth, Maturity, and old age. areas.

. , Estallishment. of birth.In an ° c Silt brought in by tributtry .40 .extant' stream, .this might be streams settles eut in the quiet a "dry run's or headwater lake water. streambed, before it had eroded d Rooted aquatics grow on shoals down to the level of ground Water. and bars, and in doing so cut off bays and contribute to the 2 Youthfultreams; when the is eroded below the filling of thelake. 1 grouhd water level, spring water e Dissolved., carbonates and other esters and the stream becomes materials 'are precipitated in the ,,.permanent. deeper portions of the lake in part through the action of plants. 3 Mature streams; have wide valleys, a developed flood plain, .

Limno logy and of

.>

o

f When filling is well' ad.anced 1 .z Youthful streams, especially on . sphagnum mats extend Nt- rock or sand substrates are low ward from the shore.Ttibse- in essential nutrients. Tempera- mats are followed by sedges tures in mountainous regions are and grasses chfinalky usually low, and due to the steep convert thelak Into a gradient; time for growth is short. marsh. Although ample light is available, growth of true plankton is thus 3 Extinction of lakes. After lakesA' greatly limited. reach maturity their progress toward -hp is accelerated. 2 As the stream flows toward a They become extinct through: more 4'mature" condition nutrients 14 .4.4,1%tend tp accumulate, and gradient 'a The downcuitink Of the out diminishes 91 so time of flow, , let. increases, temperature tends to increase, and plankton flourish. b° Filling with detritus eroded, ' from the shores or brought _Should a heavy load of inert silt in by tributary streams. develop on the other hand, the Q turbidity would reduce the light c Fillingby theacca tion of ' penetration and, consequently the .s.the remains orvegetable- general plankton production would geowinen the 'e itself.'s

a As the stream approaches base t(Oftentiro or'threepro- ." level (old age)' and the time avail- cesses may act concurrently) able for plankton growth increases, . o . L. the balance between turbidity, When mth hastens tie abode : .," nutrient levels, and temperature process, leis Often called and other_seasonal conditions, "cultural eutrophication. " detdmines-the overall peoduc- CI tivit37.-'` VIIPRODUCTIVITY Q. C.' F-actors Affecting the Productivity A Thebiological resultant of all physical of ,Lakes i sand ctlemical factors is the quantity of . life that may actually be present. The '''10" The size, shapeand depth ability to produce this "biomass" is ,of the lake basin:Shallow water often referred to as the "produCtivity" is more productive than deeper 1' of a body of water.This-is neither good water .since more light will reach nor bad per se. Aater of low producti- stile bottom to stimulate rooted vity is a "poor" water. biologically, and plant growth. As a corollary, also a relatively "pure" or "clean" water; lakeslvith more shoreline, having hence desirable as a water supply. A more shallow water, are in general productive water on the other nand may more productive. Broad shallow be a 'nuisance to man or highly desirable. Vekes and reservoirs have the Some of the factors which influence the sreatesttproduction potential (and productivity of-waters are as ..follows: hence should_ be- avoided for water supplies). B Factors affecting stream productivity. -A*To be productive of plankton, a, stream 2 Hard waters are generally more must provide adequate nutrients, light, prodhdtive than soft waters as a suitable temperature, and time for there are more plant nutrient growth to take place. mtperaleavailable. This is often 5d O a.

FACTORS AFFECTING PRODUCTIVITY a ,) Geographic Location

t.

Human Geological Latitude Influence Pormatign Longitude Altitude

0. . Sewage Competition She of Basin Climate Agriculture of Substrate Mining

Wind

Primary Drainage Dept"... Area Bottbm Precipitation Insolation Nutritiv - Area Conforination Material I I a II h 04W.

O Nature of Inflow of Trans- --31....Light edt Penetration 4,2 Penetra Develop o Seasonal Cycle Bottom A llochthon oils pa. r en cy Penetrationand Stratification-01.- and LittoralCirculit. Stagnation Deposits Materials ptilizatiop Region Growing Season:.

tz1

16' Trophic Nature of a Lake

ro

51 'Limnology and Ecology of Plankton

. greatly influenced by the 3 Reservoir discharges also pro- character' of the soil and rocks formdly affect the DO, temperature, iiithe watershed, and the quality and turbidity lr the stream below and quantity of ground water a dam. Too much°fluctuation in 1 entering thd lake.lir general, flow may permit sections of the WOO pliTanges of 6.8 to 8.2 appear stream to dry periodically. to be most productive. VIII CLASSIFICATION OF LAKES AND 3 Turbidity'reduces produCtivity RESERVOIRS as light penetration is reduced. A The productivity. of lakes and impound- 4 The presence or absence of ments is such a conspicuous feature , thermal stratification with its that it is often used as a means of semi-;annual turnovers affect classification. Productivity by distributing nutrients throughout the water 1 Oligotroxhic lakes are the mass. geologically younger, less produc- ° a tive lakes, which are deep, have 5 Climate, temperature, pre - clear water, and usually support 6 valance of ice and snow., are Salmonoid fishes. also important., 2 Mesotroac lakes are generally Di Factors AffeCting the Productivity of intermediate between oligotrophic Reservoirs andeutrophic lakes.They are moderately productive, yet 1 The productivity of reservoirs pleasant.to be around. is governed by much the same principle's as that of lakes, with 3 Eutrophic lakes gre more mature; the difference that the water more turbid, and richer. They level is much more under the are -usually shallower. They are control of man. Fluctuations richer in dissolved solids; N; p,4; r in water levelican be used to ap d Ca are abundant. Plankton is- deliberately increase of decrease abundant and there is often a- productivity.This can be dem- * riclibottom'fauna. Nuisance onstrated by a comparison of conditions often appear. the TVA reservoirs which practice a summer drawdown with some of 4 Dystrophic lakes - bog lake?: those in the west where a winter .11 low in pH, wate yellow to brown, drawdown is the rule. Idissolved solids; N, P, and Ca scanty but humic materials abun, 2 The level at whiir water is re- . dant; ,bottom fauna and plankton moved from the /reservoir is also poor,and fish species are limited. important( The upper epilimnion dr. may have a high plankton turbi- B Reservoirs may be classified as storage, dity while lower down the plankton or run of the rider. count may be less, but a taste and odor causer {suchas M_all_o- 1 Storage reservoirs have a large I be present. There 4 volume in relation to .their inflow. - maybetwo , with 4 ). a mass of muddy water flr3wing 2 'Run of thel river reservoirs have between-a clear upper epilimnion a large flow throUgh in relation, and a clear hypolimnion. Other to their storage value. . combinations ad infinitum may A .,, occur.

53, 0 N.11A

Irmamman...... Limnology and Ecology of Plankton

a

'According to location, lakes and to maintain minimum limiting nu- reservoirs may be classified as polar, tritional factors. temperate, or tropical.Differences inclimatic and geographic conditions 2 Shading out the energy ofinsola- result in aiffvences in their biology. tion by roofing or inert turbidity; r suppresses photosynthesis. DC THE MANAGEMENT OR CONTROL-OF... ENVIRONMENTAL FACTORS 3 Introduction of substances toxic to some fundamental patt of the A Liebig's Law of the Minimum states food chain (such as copper sul- that productivity is limited by the phate) tends to temporarily inhibit nutrient present iri the least amoung productivity.. at any given time relative to the .assimilative capacity of-the organism. X .SUIVIMARY B ShelfOrd's Law of T oler.ation: A A body of water such as a lakerep- resents an Intricately balanced system ` Minimum Unlit --Wange of Optimum' Maximum limit of in a state of dyhainic equilibrium. of toleration of factor toleration Modification imposed at one point in Absent -4- Greatest abundance -t-ia- the system automatically results in Decreasing Decreasing compensatory adjustments at associated Abundance ' Abundance points. B The more thorough our knowledge*or of CThe artificial introductio,of nutrients the entire systeM, the better we can (sewage pollution or Pertzer) thus judge where to imp`ofe control measures tendsto eliminate existing iting to abhieve a desired result. minimums for some specieand create intolerable maximums for oher species. REFERENCES .

1 Known limiting minumsimay 1 Chamberlin, Thomas C., and SalisburgiO4. S. sometimes be delib rately Rollin P., Geology Vol. 1, "Geolo maintained. gical Processes:and Their Results", pp irzlix, 1.nd 1-654, Henry Holt and_ 2 As the total available engy Company, ,904. supply is increased, ductivity ; tends to increase. meat. ..r.aperties of Ordinary Water - SUbstario. 3 As productivity, increases,, the Reinhold Publ. Corp., 17ew York. whole character o; the water pp: 1-673. 1940. may be changed from a Meagerly .productive clear water lake 13Hutchesoni-George E. A Treatise on (oligotrophic).to a highly pro- LirnnOlogy.John Wiley Company. ductive and Usually turbid lake .,(eutrophia. .11 4Wuttner, Franz. Fundamentals of .4 Eutrophecation leads to tesatment Limnology. University of Tprontq troutRes. "Press. pp. 1-242.1953.

D . Control of eutrophication may be accom- 5Tarzwell, Clarence M. ExPerimezital .plisbed by various means Evidence on the Value of Trout 1937 Stmettm Improvement in Michigan.

- Watershed management, ade- . American Fisheries Society Trans, quate preparation of .reservoir. 661177-187. 1936. sites, and Pollution control tend.'- . . .1 .:317.. Limnology and EcOlogy of Plankton

,.6US DHEW. PHS. Algae and Metropolitan Wastes, transactions of a seminar held April 2I-29, 1960 at the Robert A,;' Taft Sanitary Engineer- ing Center, Cincinnati, Ohio. No. SEC TR IN61-3. 7 Ward and Whipple. Freshwater Biology (Introduction). JAn Wiley Company.1918., 8 Whittaker, 'R. Ir Communities and Ecosystems. Macmillan NeW York.162 pp.' 1970.

9 Zhadin, V. I. and perd, Sr.Fauna and Flora of the lakes and - Reservoirs of the USSR., Avail- able from the Office of Technical Ser4ices, U. S. Dept. Commerce, Washington, DC.

'41 o 10 Josephs, lifelvin and Sanders, Howard J. Chemistry and the Environment ACS Publications, WashAgton, DC. 1967. p 11 Sournia, A. (editor), Phytoplankton Manual. UNESCO. 1978. .

, This outlike"was prepared by IL W. Jackson, formai Chief Biologist,- National Training Center, and z;evisect by 'R. M. Sinclair, Aquatic .Biologist,' NationarTrairiing and Operational Technology Center, ()WYO.' ' USEPA, Cincinnati, Ohio 45268.

-40P Descriptors: Plankton, Ecology,. Limnology

11,

.t

t BIOLOGY OF ZOOPLANKTON COMMUNITIES

ICLASSIFICATION E In this presentation, a minimum of clas- sification and is used, but it A The planktonic cornmanity is composed of should be, realized that each group is , organisms that are relatively independent typified by adaptations of structureon of the bottom to complete their life history. physiology that are related to the plank- They inhabit the open water of lakes tonic mode of existence.These adapta- (). Some Species have inactive tions are reflected in the classification. or resting stages that lie ()Ole bottom and carry the species through periods of stress; e. g. , winter,A few burrow iri IIFRESHWATERZOOPLANKTON the mud and enter the pelagic zone at night, but most live in the open water all thb A The, freshwater zooplankton is dominated time that the species is present in an active by representatives of threegroups of form. animals, two of them crustaceans: Copepoda, Cladocera, Rotifera.All have B Compared to the bottom fauna and flora, feeding mechanisins that permit a high the plankton, consists of relatively few degree of selectivity of food, and twocan kinds of organisms that are consistently produce resting eggs that can withstand and abundantly present. Two major 'cat- severe environmental conditions.In egories are often called phytoplankton general the food of usual zooplanktonpop- (plants) and zooplanktonAanimals), but \ ulationsranges from bacteria and, small this is based on an outmoded classification algae to small animals. of living things.The modern tendency is to identify groupings according to their B The C.opepoda reproduce by a normal function in the ecosystem: Primary pro-. biparental process, and the females lay ducers (photosynthetic organisms), consum fertilized eggs in groups which are carried (zooplankton), and decomposers (hetero- around in sacs until they hatch. The trophiC.bacteria and fungi). immature animals go through an elaborate development with many stages.The later C, The primary difference ken is nutritional; 'stages have mouthparts that permit them phytoplankton use inorganic nutrient to collect particles.In many cases, these elements and solar radiation.Zooplankton are in the form of combs which remove feed on particles, much of which can be smallparticlps by a sort of filtration phytoplankton cells, but'can be bacteria or process.In others, they are modified to particles of dead organisms (detritus) form grasping organs by which small originating in the plankton, the shore animals or large algae are captured region; or the land surrounding the lake. 'individually. 'D The swimming powers of .planktonic organisms is so limited that their hort- C The Cladocera (represented by Daphnia) zontal distributioniis determined mostly reproduce much of the time by partheno- by movements of water. Some of the iesis, so that only females are present. viek animals are able to swim fast enough that Eggs are held by the mother in a brood they can migrate vertically tens of meters chamber until the young are developed far each day, but they are capable of little enough prrfepd for themselves. The newborn horizontal navigation.At most some animals look like and do species of crustaceans show a general not go through an elaborateseriesof avoidance of%the shore areas during,calm ,developmental stages in the water as do weather when the water is movinernore the copepods,oaphni.a,has slowly than the animals can swim.47By filtering structures on somesome of its legs, definition, animals that are able to control that act as filters. their horizontal location are nekton, not plankton.

ti

BI..Aci.29. 6. 46 ,56 -r Biology of Zooplankton Communities

D Under some environmental conditions-the' 2 Inorganic materials development of eggs is affected and males are produced.Fertilized eggs are produced Freshwater lakes vary in the content that can resist freezing and drying, and of dissolved solids according to the these carry the population thrbugh geological situation.Tile total salinity unsatisfactory conditions. and proportion of different dissolved materials in water cartIffect the pop- E The Rotifera are small animals.with'a ulation. Some species are limited to ciliated area on the head which creates soft water, others to saline iaterl, as currents used both for locomotion, and for the brine shrimp.. The maxim.= pop- bringing food 'particles to the mouth. They ulation size developed may be related too reproduce by parthenogenesis during to salinity, but thisris probably'an much of the year, but production of males .indirect effect working through `the results in fertilized, resistant resting eggs. abundance of nutrients and production Most rotifers lay eggs one at a time and of food. carry them until they hatch. 3 Food supply IIIZOOPLANKTON POPULATION DYNAMICS, Very strong corrylations have been found between reproduction and food A In general, zooplankton populations are at supply as measured by abundance of a minimum in the cold seasons, although phytoplankton. The rate of food supply some species flourish in cold water. Species can affect almost.,all aspects of pop - with similar food requirements seern'to ulation .biology including rate of indi- reproduce at different times of.the year or vidual growth-time of maturity, rate are segregated in-di#e-nent layers of lakes. , of reproduction and length of life. B There is no single, simple measurement 4Apparently in freshwater, dissolved of activity for the zooplankton as a whole organic materials are of little nutri- that can be used as an.index of production tional significance, although some as can the uptake of radioactive carbon for species can be kept -if the concentration. the phytoplankton. However, it is possible of dissolved material is high enough. to find the rate of reproduction of species Some species require definite vitamins 'that carry their eggs. The basis of the in the food. method is that the number of-eggs in a sample taken a #a given time represents 5Effect of predation on populations the number 9f animals that will be added to the population during an interval that The kind, quantity and relative pro- is equal to the length of time it takes the portions of species strongly affected eggs to develop. Thus the potential growth' by grazing by vertebrate and inverte- rate of.the populations can be determined. brate predators. The death rate of The-actual growth rate, determined by Daphnia is correlated with the abun- successive samplings and counting, is less dance of a predator.Planktivorous than the potential, and the difference is a fish (alewives) selectively feed on measure of the death rate.. larger species, so a lake with alewives is dominated by the smaller species of C Such measurements of birth and death rates crustaceads and large ones are scarce permits a more penetrating analysis to be or absent. made of the causes of population change than if data were available for population 6 Other aspects of zooplankton size alone. Many species migrate vertically con- D Following is an indication of the major siderable distances each day.Typically, environmental factors in the control of migrating species spend the daylight zooplankton. hours deep in the lake and rite 'toward t-o the surface in late afternoon and early 1 Temperature has an obvious effect in 'evening. its general control of rates. ,In addition, the production and hatching of resting Some species go through a seasonal eggs may be affected. change of form (cyclomorphosis) which is not fully understood.It may have an effect 4n reducing predation. 57 4-2 Biolo&Srof Zooplankton Communikies

REFERENCES

1 Baker, A. L. An Inexpensive Micro- 7 Lund, J. W. G.1965. The Ecology of sampler.Limnol. and Oceanogr. the Freshwater Plankton.Biological 15(5) :. .158-160.1970. Reviews, 40:231-293.

. 2Brooks, J. L. and DOdson, S. I. 8 UNESCO. Zoolplankton Sampling. Predation, ,Body, Size, and Corn- UNESCO Monogr. Oceanogr. Methodol. pqSition of Plankton. Science 150: 2.17.4 pp.1968.(UNESCO. -Place 28 -35.1965. de Fortenoy, 75, Paris 7e France). 3Dodson, Stanley I,Complementary Feeding Niches Sustained by Size.- Selective Pl-edation, Limnology and Ocenography 15(1):131-137. 4Hutchinson, G. E.1967. A Treatise on Limnology.Vol. II.IitrodUction to Lake Biology and the Limnoplankton. xi + 1115. John Wiley & Sons, Inc. New York. 5. Jossi, Jack W. Annotated Bibliography of Zooplankton Sampling peyices. USFWS. Spec, Sci. :Rep. -Fisheries. 609.90 pp.1970.

6 Likens, Gene E. and Gilbert, John J. 'This outline was prepared by W. T. Edmondson, Notes on Quantitativ,e Sampling of Professor of Zoology, University of Natural Populations of Planktonic Washington, Seattle, Washington. Rotifers.Limnol.and Oceanogr. 15(5): 816-820. . Descriptor: Zooplankton

53 4-3 ,

Biology of Zoopl nkton Communities 1111

FIGURE 1SEASONAL CHANGES OF ZOOPLANKTON IN LAKE ERKEN,S-WEDBN-

Keratella hiemali

Kellicottia longispi

Polyarthra vulgaris

L Daphnia longispina "144.1 Ceriodaphnia quadrangula

Bosmina coregoni

a a a a a a a J P M M -J J A S 0 N D 1957

Each panel shows the abundan4 of a species of animal.-Each mark on the vertical axis represents 10 individuals/liter. Nadwerck, A. 1963.11 Die Beziehungen zwischen Zooplankton and Phytoplankton im See Erken. Symbolae Botanicae Upsaltensis, 1,7:1-163.

4 59 FIGURE- 2REPRODUCTIVE RATE OF ZOOPLANKTON AS A FUNCTION OF ABUNDANCE OF FOOD

B ...

7 .

* . 7 . . Young per :Total young 0.20, 9 .. Brood 223, 1 Temperature . o -177 a ., more than 10 0 132 0 00 0.15 76

. .. .

0.10

,Temperature . . co less thanI00 Number of young producedin each brood by Daphnia living in

ae four different concentrations of food organisms, rewewed : . daily. The total numbetaproduced during the life of a ' 0.05 mother is shown by the numbers at the right. The Daphnia at the two lowest concentrations produced theirfirst batch . of eggs on the same day as the others, but the eggs degen- erated, and the first viable eggs were released two days . . . later. Richman, S. 1958. The transformation of energy by '0 Daphnia pulex. Ecol. Monogr. 28: 273-291. 0 200 400 600

Abundanceof foodorganisms ugm /1, dry weight

4Mean rate.of'laying eggs by the planktonic rotifer Keratella cochlearis in natural populationsapa function of abundance of food'organisms and temperature. W. T. Edmonton. 1965. ReprOducEive rate of planktonic rotifers as related to food and temperature in nature. Ecol. Mmogr. 35: 61-111. 61

CSI

1 Biology of Zooplankton Commuriities

<,

PROTOZOA.

9,

-

Difflugia Amoebae

ft.

Co-donella

fi `Stentor Epistyks 7.

Ciliates Biology of Zooplankton Communities

ROT I FERA

Polygarthra Brachionus

. . ARTHROPODA

crustaCea

I

Cladocera Copepoda

.-....-.- Nauplius larva of copepod 4 I 44.

0

1414." ON

Ins'ectaChaoborus 0

. , .. ..,. . . .- a .

El! , *I.*, .":1 Biology of Zooplankton Communities

s

PLANKTONIC BIV VE RVAE

0 1,1 0

380p. 864.

2 3°

spinet (fin nttached).. sim le (gill attached)

Glochidia '(Unionidae) Fish Parasites- ' (1-3)

r

1

veliger

Veliger Larva6 (Corbiculidae) Free LiArAngPlanktonic

a . ..(4-5)

fr/S-a Pedivpliger' attaches byssus lines)

,

6. 61

4 ?*i^ 1'

OPTICS AND n-1E-MICROSCOPE 4

b.

IOPTICS Strictly, speaking, of course, all reflected light, even diffuse, obey/4 Snell's Law. An understanding of elementary optics is Diffuse reflected light is made up 8l many essential to theproper use of the microscope. specularly .reflected rays, each from a The microscopist will find th4t unusual pro- a tiny element of surface, and appears blems in illumination and photomicrography diffuse when the reflecting elements are can be handled much more effectively bnce 'very numerous and very small. The terms the,underlying ideas in physical opti7 are diffuse and .specular, referring to reflection, understood. describe not so much a difference in the nature of the reflection but rather a differ- A Reflection ence in the type of surface. A polished sur- face gives specular reflection; a rough A food place to begin is-with reflection at surface gives diffuse reflection. a 'surface or interface.Specular (on regular) reflection results when d beam It is also important to note and remember of light leaves a surface at-the same angle that specularly reflected light tends to'be at which itreached it.This type of .strongly polarized in the plane of the reflect- reflection occurs with highly, polished ing surface. This is due to the fact that smooth surfaces.It Is stated more pre- those rays whose vibration directions lie cisely as Snell's Law, is e. ,'the angle of closest to the plane of the reflection surface incidence, i, ,is equal to the angle .of are most strongly reflected.This effect is reflection, r (Figure1). Diffuse (or strongest when the angle of incidence is -scattered) reflection results when abeam such that the tangent of the angle is equal of light strikes a rough or irregular sur- to the refractive index of the reflecting sur- face and different portions of the incident face.This particular angle of incidence is light are reflected from the surface at called the Brewster angle. different 'angles.The light reflected from a piece of white paper or a ground glass is B Image Formation on Reflection.

an'example of diffuse reflection. , Considering reflection by mirrors, we find (Figure 2) that a plane mirror formsa virtual image behind the mirror, revered right to left but of the same sizeas the object: The word virtualmeans that the ,image appears to be in a Oren plane but that a ground glass screen or a photographic film placed in that plane would stiOwno image.' The converse of a virtuariniage is a real image. '

Spherical 'mirrors are either convex orcon- cave with the surface of the mirror repre- senting a portion of the surface ofa sphere. The center of Curvature is the...center of the Sphere, part orwhose surfaceforms The . Figure 1 mirror: The Tocus,lies halfway bb'tween the SPECULAR REFLECTION SINTELLIS'-center of curvature 1144:14he milk9F surface. ,., - .7, LAW ,BI. MIC. 18..2. 79 5-1 Opttr..5 and the Microscope

,< 3 A ray of light which passes through the focus is reflected parallel to the axis of the mirror. . 4. - The image from an object can tie located using the familiar lens formula:

it I I + 1 °bloat Virtual Imago where p = distanCe from the object to -. the mirror Mirror q = distance from the image to t the mirror *Figure 2 f = focal length IMAGE FORMATION BY PLANE MIRROR C Spherical Aberration No spheyical surface can be paeftct in its Construction of an image by a concave image-forming ability.The most serious mirror follows from. the ,two premises of the imperfections,' spherical aberration, given below (Figure 3): occurs in spherical mirrors of large aperture (Figure 4).The rays of light ,making up an image point from the outer zone of a sphericd.1 mirror do not pass through the same point as the more central rays.This type of aberration is reduced by blocking the outer zone rays from the image area or by-using aspheric'surfaces.

111

c_ ( Figure -3

.IMAGE FORMATION BY CONCAVE MIRROR 1 A ray of light parallel to the axis of the mirror must pass through thd' foCus after: reflection. Figure 4 2 A ray of light which passes through the O center of. curvature mast return along SPHERICAL ABERRATION BY SPHERICAL MIRROR the sarrie pat11.-

11.N A corollary of the firstpremiseis: < , 5-2

a 66. `' .3

Optics and the Micrpscope

D Refiaction Of Light into focus and the new micrometer reading is taken.Finally, the microscope is re- Turning now.to lenses rather than mirrors focused until the surface of. the liquid appears we find that the inost`ininortant t liaicte- in sharp focus.The micrometer reading., istic is refraction.Reratilni releys to is taken again and, with this information,. the change of dirlction and/or velocity Of the 'refractive index may be calculated from light as it passed from one 111..diutil to the simplified equation: another. The ratio of the veloc it) in air ,(or more corrcu.tly in a vacuum) to, the active index actual depth velocity ip-th«.-; 111 rdit1111 i, called the apparent depth C Lte frac ti've index. Some.. typical values of refractive index measured with mono- , chromatic' light (sodium I) line) are listed 'Table I REFRACTIVE INDICES OE COMMON in Table 1. MATERIALS MEASURED WITH SODIUM LIGHT .Refraction causes. an object inimer,s(el in a medium of higher refractive index than V a't uu m 1.0,000000Crown glass.1.48 to 1.61 air to appear Olosert.to the surface thah it Air 1.00.029 i 8Rock salt)oirs:.1.5443 actually is (Figure 5).This effect CO2, L0004498Diamond 2.417 Wate 'I. 3330 Lead sulfide1E3.912

When the situation' is reversed, add a ray of light from a medium of high refractive index p'a4es through the interfaCe of a medium of lower index, the ray is refracted , until a critical angle.is reached beyond which Air all pf the ,light is reflected frOm the interface (Figure 6).This critical angle, C, has the folldwing relationship to the refracti've irdices Apparent Medhim of the two Media: Actual-. depth depth , 'TOO sin C=n9, where\n2

114 s. F 1.

be used to dete'rimine th,e,gpfractive index or a.liquid:with the nlivipscope.A flat '.;,t .vial`with a. scHtcl).eiti Otte bottomcipside) cr ,. is Placed,ern the' stage of themic"rbscope.e. .. ale stratch f The' microscope iS fo'cubed on and the fine adjustment micrometerreading unknotArn. . 1. ..is noted. A small amount of the liquid is added: the scratch is again brought . , ..,. , Figure6 .., . ... 4 , . -4 . REFLECTIONAT CRITICAL ANGLE . a, -... A I 67 t .3

Kst , a,

Optics and the Microscope

iss 0

.16 E F Lenses Dispersiodis another important property .There are two cPasses of lenses, con- of transparent materials. This is the verg ing and diverging, called also convex variation of refraetive index with color, and concave., respectively. The focal (or wavelength)of light.When white'light point of a converging lens is defined as passes` thro6gh a glass prism, the light the pqint at which a bundle of light rays rays are-reffraCted'hy different amounts ...parallel to the axis of the lens appearA to and separated into the colors of the .-,- 'converge aftter,passing through the lens. spectrum.' This spreading of light into The focal lerigth of the lens is the distance itg component, colors is due to disperses from the lens to the focal point (Figure 7):, which,. in turn' is due to the fact that the .refractive index of transparent substances, .liquids and solids, is lower for long wave- lengths than for short wavelength. BeCause of dispersion, determination of the refractive index of a substance re- quires designation of the particula, ye- length'used.Light from a sodium lamp has a strong, closely spaced doublet with an average wavelength of 5893A, called the D line, which is commonly used as a reference wavelength. Table 2 illustrates the change, of refractive index with wave -' length for a few common substrCes. f . '

Table 2.DISPERSION (iF REFRACTIV ., . IALS :, . , Figure 7 INDICES OF SEVERAL COMMONMAT . CONVERGENCE OFLIGI-IT AT FOCAL POINT Fefractive index 0 F line D line C line. blue (yellow) '(red) , 4861A 5893A 6563A G Image FoAnation by Refraction Carbondisulfide 1.65211,62761.6182 Image formation by lenses (Figure 8) Crown glass 1.5240'1%5172-.1.5145 .follows rules analogous to those already given above for mirrors: Flint glass 1.:63911.6210\..1.t221/ Water 1. 33721.'33301. 3312 1 Light traveling parallel to the axis of . the lens Will he refracted so as to pass through the foeus of the lens. mr - . The dispersion Qt a material can be defined t Light traveling through the geometrical quanittativel§ as: center of the lens will be unrefracted. n (yellow) - 1 v dispersion z-b1n (blue)-- n (red) he,pos itionof the, image c e determined' . by remembering that a light r y passing n (593mii) - 1' through itcus,',F, will be parallel to n (486mv) - n(656mv) the axis of the lens.ori the opposite "sine' of the lens and that a 'ray passing through the where, n is the refractive index, of The geometrical. center of the lenswill be material at the par+icular wavelength unrefracted. w noted( in the parentheses. Optics and.the° Microscope . a. $ object points are riot located on the optical axis of the lens and results in the formation- of an indistinct image. The simplest remedy for astigmatism is to place ?tie ' objey.t close to the dxis of the lens system.

IIntCrft.rence Phenomena Interference and diffraction are two phe=- nomena which are due *to thq wave character- isties of light.The superposition of two . light rays arriving simultaneously At a given point will give rise td interference effects, whereby the intensity at that point will vary Figure 8 from. dark to bright depending on the, phase different es between the two light rays.. IMAGE FORMATION BY A CONVEX LENS The first requirement for interferenceis The magnification,.M, of an image° of an that the light must come from a single object,prothiced bya lens is given by the source. The light may be split into any relationship: number of-paths but must originate from the same poipt (or .coherent source). Two image size image distance q .M = light waves from a coherent source arriv- object size object digtance p ing at .a.'point in phase agreement will reinforce each other (Figure 9a). Two where q = distance.;rom image to lens light waves,from a coherent source arriv- and p =. distancefrom object to lens. ing at a point in opposite phase will cancel each other (Figure 9b). H Aberrations of Lendm Lenses have aberrations of several types which, unless corrected, cause loss of detail in the image. Spherical aberration appears in lenses with-sphericalsurfaces. Reduction bf spheriCal aberration cane accomplished by.diaphragming the outer zones of the lens ,or by designing special asplferical surfaces in the lens system.

Chromatic aberration is -a lifienomkwrion caused by the vaiiation of refractiVe. index with wavelength (dispersion).Thus' a lens receiving white light from an object will farm a violet image closer to the lens' and a red one father away.AchroznA'CiC' . lenses arb,employed to minimize this . Figure9a. Twolight rays, I and .2, of effect...The lenses ar4"cornliiratizins of the same:frequency'but dif- two or more lenselements made tip.of, ferent amplitudes, are in phaie .1 materials having different dispersive in the upper diagram.in the lower diagram, rays l 'and 2 titers: The use of monochromatic light- .44.t fs. obvious way of eliminating interfere constructively to give chromatic aberration.. , , . a' single wave of the same fre- quency aricP.with an, amplitude Aistigmatism-is a third aberration of equal to the summation of the spherical lens. syStems.It occurs' when two former waves.,

9 ak 'Optics and the Microscope

a

otb

Figure 9b.Rays 1 and 2 are no.v 180° Figur,e 19 -"out of phase and interfere destructively.The resultant, INTERFERENCE IN A THIN FILM in the bottom diagram, is of the same frequency but is of A simple interferometer can be made,by reduced amplitude (a is partially silvering a microscope slide and negative and is subtracted cover slip.A preparation between the two from b). partially silvered surfaces will show inter- ference fringes, when viewed with mono- chromatic light, either transmitted or by The reflection of a monochromatic light vertical illuminator. The fringes will be beam by a thin film results in two beanis, close together with a wedge-shaped prep- one reflected from the top surface and one aration and will reflect-refractive index from the bottom surface. The distance. differences due to Temperature variations, traveled by the latter beam in excess of concentration differences, different solid the first is twice the ,thickness of the film phases, etc.The method has been used to . and equivalent air path is: measure quantitatively the concsrgration of solute around a growing crystalui(Figure 11). 2 nt

, where n is.the refractive index and t is'the thickness-of the film. 50% Morrom/.. The second beam, however, upon reflection N . at the bottom surface, undergoes a half wavelength shift and now the total retaril- --Cover s-p - tapon of the second beam with respect to Specimen °the-first is given as: retardation = 2 nt +4 0 b, 000 Where k is the wavelength of the light Figure 11 e 4 beam. MICROSCOPICAL METHOD QF VIEWING INTERFERENCE IMAGES When .retardation is_ exactly an *odd -Timber a Examination is by transmitted light. of half wavelengths, destructive interfer- Light ray undergoes multiple . ence takes place resulting in darkaess. ' reflections and producesdark and When it is zero di an even number-of half light frifiges in the-field.A speci- wavelengths,, constructive interference men introduces a phase 'shift and results in brightness (Figure 10). Changes the fringepattern. b Illumination is fronithe top.The principle is the same but fringes show greater contrast. Optics and the Microscope

2 44 f X Each dark band represents an equivalent d air thickness of an odd number of half D wavelengths.Conversely, each bright band is the result of an` even number' of where f is the focal length of thelens, X the wavelength, and D the., diamete half wavelengths.' of the lens. Witlyinterference illumination, the effect of a transparent object of different re- It is seen that in order to maintain fractive index than the medium in the small diffraction disc at a given wave- length, the diameter of the lens should microscope fittia is: '4 be as large as possible with respect to the focal length.It should be noted, 1 a change of light intensity of the object if the background is uniformly illumi- also, that a shorter wavelength produces nated (parallel cover slip), or a smaller disc. If two pin points of light are to be distin- 2 a shift of the ipterference bands within guished in an image, their diffraction discs the object if the background consists must not overlap more than one 'half their of bands ( tilted cover slip). diameters.The ability to distingvish such image points is called resolving power and The relationship of refractive indices of is expressed, as one half of thepreceding the surrounding medium and the object is expression: as folloWs: resolving power =1.22 f ex D =nm( 360t es where ns = refractive index of the IITHE.°C.OMPOUND MICI:PSCOk'E specimen nITI= refractive index of the The compound mCcoscope isan extension in surrounding_medium 'principle ofheimple magnifying glass; = phase shift of thostwo hence it is e ntial-toutidefstand "fully the beams, degrees properties of this simple lens system. X = wavelengthof the light t= thickness of the specimen. A Image Formation by the Simple Magnifier J Diffraction' The apparent size of an object is determined' In geometrical optics; it is assumed that, by the angle that is, formed at the eye by the light travels in straight lines.This is not extreme rays of the object.By bringing the always true.We note that a beam.passing 4 object closer to the eye, that angle (called through a slit toward a screen creates a the visual angle) is increased.This_also bright band wider than the slit with alter- increases the apparent size. HoWever a nate bright and dark bands appearing on limit of accommodation of the eye is reached, either side of the central bright Band, at which distance the eye can no longer focus. :a This limiting distance is about 10-inches or 25 decreasing in intensity as a function of . the distance from the center.Diffraction centimeters.It is at this distance that the describes this phenomenon and, as one of magnification of an object observed, by the its-practical'consequences, limits the unaided eye is said:to be unity.Theeye can, lens in its ability to reproduce an image. of course, be focused at shorter distances but For example, the image of a pin point of not usually in a relaxed condition, light produced by a-lens is not a pin point but ,is revealed to be a somewhat larger A positive, or converging, lens can be'used patch of light surrounded by dark and to'permit placing an object closer than 10 bright rings.The diameter, d, of this inches to the eye (Figure 12). By this means diffraction disc ( to the first dark ring) the visual angle of the object is increased is given as: (as is its apparent size) while the image of g4k, ... 5 -7° 71 Optics and the Microscope

Eye

1. 111060 t'---1 r Eyepiece 4 IMO.. ObjeCI

Ilast414, EV, Image 4 t% Figure 12 VIRTUAL IMAGE FORMATION BY CONVEX LENS the object appears to be 10 inches from the eye, where it is best accommodated. 4 ob,.... 111 BMagnification by a Single Lens System t The magnification, M, of a simple magni- Object fyinass is given Py: 25 , M = +1

v'tug Image where f = focal length of the lensin centimeters. Figure 13 Theoretically the magnification can be IMAGE FORMA ION IN increage-d-with-shorterlocal-lengtirienses7 COMPOUND MIC OSCOPE However such lenses require placing the eye very close to the lens surface and lengths to give differentagnifications have much 'mage distortion and other (Table 3).The magncation is calculated optical aber ations. The practical limit from the focal 1 by dividing the latter for a simple magnifying glass is about into the tube length, usually 160 mm. 20X. The numerical aperture (N. A.) is a measuz4 In order to go to magnifications higher of the ability of an objective to resolve detail'' than 20X, the compound microscope is This is more fully discussed in the next required. Two lens systems are used section. The working distance is in the free to form an enlarged image of an object space between the objective and the cover ..,.(Figure 13).This is accamplished in slip and varies slightly for objectives of the two steps, the first by a lehs called the same focal length depending upgn the degree objective and the second by a lens known of correction and the manufacturer. as the eyepiece (or ocplar). There are three basic classifitations of C The Objective objectives: achromats, fluorites and apochromats, listed in the order of their The objective is the lens or lens system) complexity. The achromats are good fOr closestito the object.Its function' is -to. routine work' while the fluorites andapo- reproduce an enlarged image of tht object chromats offer additional optical corrections in the body tube of the Microscope. to compensate for spherical, chromatic and' .Objectives are available in various focal other aberrations. . 5 -8 72 Optics and the Microscope,

a Table 3.NOMINAL CHARACTERISTICS OF USUAI. MICROSCOPE OBJECTIVES Working Depth Diam. of, Resolving MaximumEyepiece Nominal 'Nominal focal length N. A. distance focus field power, white useful for niax, magnif. mm mm ii mm. light,p. magnif.,useful magnif.

56 2. 5X 0.08 .40 50 8.5' 4..4 80X 30X 16 5 3.9 el )X 20X 32 4 1 4... 'O. 10 ,25*

16 \ 10 0.25 7 8- 2. 1.4 250X 25X

8 20 0.50 1.3 2 1 0.7 500X 25X

4 43. 0.66 0.7 1 0.5 0.4 660X 15X 4 \45, / 0.85 0,5 1 0.4 0.35 850X 20X 1.8 90 1.30 0.2, 0.4 0.2 0.21 1250X 12X

Another tem of objectives employs at one time (there are some nosepieces refytti surfaces in the shape of concave that accept five and even six objectives). and vexx mirrors.ReflectionRe optics, In this system, the objectives are ...1.--eSuse they have no refracting elements, usually nccncenterable and the stage is do not suffer from chromatic aberrations centerable.Several manufacturers pro- as ordinary refraction objectives do.Based vide centerable objective mounts so.that . entirely on reflection,reflecting objectives each objective on the nosepiece need be are extremely useful in the infrared and centered only'once to the fixed rotating 'ultraviolet regions of the spectrums They stage. The insides of objectives are also have a much longer working distance better protected from dust by the rotating than the refracting objectives. nosepiece. This,yas well as the incon- venience of the so-called "quick-change" The body tube of the microscope supports objective holder, makes it worthwhile the objective at the bogom (over the object) to have one's microscope fitted with and the eyepiece at the top.The tube rotating nosepiece. length i's maintained at 160 mm except for Leitz instruments, which have a 170-mm .DTheOcular tube length. The eyepiece, or ocular, is necessary in The objective support may be of two kinds, the second step of the magnification process. an objective clutch changer or a rotating The eyepiece functions as a simple magni- nosepiece: fier viewing the image formed by the objective.

1 The objective clutch changer ("quick- change" holder) permits the mbunting There are three classes of eyepieces in of only one objective at a time on the common use: huyghenian,,compensating microscope.It has a tentering arrange- and flat-field.The huyghenian (or huyghens) nient, so that each objective need be eyepiece is designed4to, be used with centered only once with respect to the achromats while the compensating type is stage rotation. _.The changing of objec- used with fluorite and apochromatic tives with this system is somewhat objectives.Flat-field eyepieces, as the awkward compared with the rotating name implies, are employed in photo- nosepiece., -micrography or projection and can be used with most objectives,It is best to follow 2' The revolving nosepiece allows mounting the recoramendations ofthe manufacturer three or four objectiV,es on the microscope as to the proper combination of objective and eyepiece. -.

5-9 .1

Optics and the Microscope

The usual magnification available in eyepiece gives insufficient magnification oculars run from abouttBiC up to 25 or for the eye to see detail actually resolved 30X. The 6X is generally too lo;,v to be of by the objective., any real value while the ?5 and 30X oculars have sligh y poorer imagery than medium F Focusing the Microscope powers an have avery low eyepoint.The most useful eyepieces lie in the 10 to 20X The coarse adjustment is*used to roughly magnification range. positron the body tube (in some newer microscopes, the stage) to bring the image E Magnification of the Microscope into focus.The fine adjustment is used after the coarse adjustment to bring the The total magnification of the objective- image into perfect focus and to maintain eyepiece combination is simply the product the focus as the slide is moved across the of the two individual magnifications. A stage.Most microscope objectives are convenient working rule to assist in the parfocal so that once they are focused any proper choice of eyepiecesl states that the other objective can be swung into position maximum useful magnification (MUM) for without the necessity of refocusing except, the microscope is 1,000 times the numeri- with the fine adjustment. cal apei-ture (N. A.) of the objective. The MUM is related to resolving power The student of the microscope should first in that magnification in excess of MUM learn to focus in the following fashion, to gives little or no additional resolving prevent damage to a specimen ox objective: power and results in what is termed empty magnification. Table 4 shows the results 1 Raise the body tqbe and place the speci- of such combinations and a comparison men on the stage. with the 1000X N.A. rule. The under:. lined figure Shows the magnification near- 2Never focus the body tube down,(or the estto the MUM and the eyepiece required stage up) while observing the field with each objective to achieve the MUM. through. the eyepiece. From this table it is al_parent that only higher power eyepieces can give full use 3 Lower the body tube (or raise the stage) of the resolving power of the objectives. with the coarse adjustment while care- It is obvious that a 10X, or even a 15X, * fully observing the space between the

Table 4.MICROSCOPE MAGNIFICATION CALCULATED FOR VARIOUS OBJECTIVE-EYEPIECE COMBINATIONS Objective Focal Magni- Eyepiece MU Ma lengthfication 5X 10X 15X 28X 25X (1000 NA) 56mm 3X 1.5X 30X 46X .-60X 75X 80X 32 5 25X 50X 75X 100X 125X 100X. 16 10 50X100X 150X -200/ 250X 250X 8 20 .200X . 300X 400RI .500X 500X 4 40 . 200X400X OOX 800X 1000X 660X

1.8 90 . 450X900X 135')X ,1800X 22504 1250X 'aMUM= maximum useful magnificati9p

. 74 Optics and the Microscope

objective, and slide and permitting the while in research microscopes, the two to come close together wiothout poWrizer can be rotated. - Modern instru- touching. Inints haveA,larizing filters (such as Pblaroid)eplacing the older calcite 4 Looking through the microscope and prisms.Polarizing fgters are preferred

turning the fine adjustment in such a .,N beause they: way as to move the objective away from the specimen, bring the image into I are low-cost; sharp focus. 2 require no maintenance; The fine adjustment is VR ua11y calibrated in one- or two-micron steps to indicate, 3 permit use of the full condenser thvertical movement of the body tube. aperture. ThN feature is useful in making depth measurements but should not be relied An analyzer, of the same construction as upon for accuracy. the polarizer, is fitted in the body tube of the microscope on a slider So that it may G The Substage Condenser - be easily removed from the optical path. It is oriented with its plane of vibration The substage holds the condenser and perpendicular to the corresponding direction polarizer.It can usually be focused in a of the polarizer. vertical direction so that the condenser can be brought intethe.correct position with J The Bertrand Lens respect to the specimen for proper 'illumination.In some models, the conden- The Bertrand lens is usually found only on ser is centerable so that itay be set the polarizing microscope although some exactly in the axis of rotatiof the stage, manufacturers are beginning to include it otherwise. it will have been centered at on phase microscopes.It is located in the- the factory and should be permanent. body tube above the analyzer on a slider (or pivot) to permit quick removal from H The Microscope Stage the optical path.The Bertrand lens is used to observe the back fooal plane of the objective. The stage of the microscope supports the It is convenient for checking cgickly the type specimen between the condenser and and quality of illumination, for observing objective, and may offer a mechanical stage interference figures of crystals, for adjust- as an attachment to provide a means of ing tphase annuli in phase microscopy moving the slide methodically during obser- and foadjusting the annular and central vation.The_polarizipg mica °scope is stops inaspersion staining. fitted with a circular rotating stage to which a mechanical stage may be added. K The Compensator Slot The rotating stage, which is used fowbject orientation to observe opticakeffects, will The compensator slot receives compensators have centering screws if the objectives are (quarter:wave, first-order red and quartz- not centerable, or vice versa.It is un- wedge) for observation of the optical prop- desirable to have both objectives and stage ertiesOf crystalline materials.It is usually centerable as this does not provide a fixed placed at the lower end of the body tube just reference axis. above the objective mount, and is oriented 45° from the vibration directions of the iThe Polarizing Elements polarizer and analyzer. A polarizer is fitted to,tlie`condenser of all L The Stereoscopic Microscope polarizing microscopes.In routine instru- ments, the polarizer is fixed with its The stereoscopic microscope, also called vibration direction oriented north-south the binocular, wide-field, dissecting or (east-vr-st for most European instruments) 541 Optics and the Microscope

- Greenough binocular microscope, is in good illumination system for the microscope reality a combination of two separate .is to have uniform intensity of illumination, compound microscopes. The two micro- over the entire field of view with independent . scopes,_ usually mounted in one body, havi control of intensity and of the angular aperture their optical axes inclined from the vertical of the illuminating cone. by about 7° and from each other by twice' this angle. When an object is placed onthe A Basic Types of. Illumination stage of a stereoscopic microscope, the optical systems view it from slightly There are three types 'of ilfuminatiop different angles, presenting a stereoscopic (Table 5) used generally: pair of images to the eyes, which fuse the two into a single three-dimensional image.,, 1 Critical.This is7used when high levels 1 of illumination intensity are necessary ,The objectives are supplied in pairs, either for"oil immersion, darkfield, fluores- *.,T4 as separate units to be mounted on the cence, low birefringence or photo- microscope or, as in,the"new instrtments,, micrographic studies.Since the lamp built into a rotating drum. Bausch and filament is imadd in the plane of the' Lomb was the first manufacturer to have a specimen, a ribbon filament or arc zooni Iens system which gives a continuous lamp is required. The lamp must be Change in magnification over the full range. focusable and have an iris diaphragm; Objectives for the stereomicroscope run the position of the filament must also from about 0.4X to 12X, well below the be adjustable in all directions. magnification range of objectives available for single-objective microscopes. 2 KOhler. Also useful for intense illumi- nation, Kohler.illumination may be The eyepieces supplied with stereoscopic obtained with any lamp not fitted with'a microscopes run from 10 to 25Xand have ground glass.' The illuminator must fAt.wider fields than their counterparts in the however, be focusable, it.must have an single-objective microscopes. adjustable field diaphragm (iris) and the lamp filament position must b\adjust- Because of mechanical limitations, the able in all directions. stereomicroscope is, limited to about 200X magnification and usually does not permit 3 "Poor man's".So-called because a low- more than about 120X.It is most useful priced illuminator may be used, this at rela,tively low powers in observing method gives illumination of high quality shape and surface texture, relegating the although of lower intensity becauseqf the study of greater detail to the monocular presence of a ground glass in the system. microscope. The .stereomicroscope is No adjustments are necessary on the also helpful in manipulating small samples, illuminator or lamp filament although separating ingredients of mixtures, pre- an adjustable diaphragm on the illuminator paring specimens for detailed study at is helpful. higher magnifications and performing various mechanical operations under micro- All three types of illumination require that scopical observation, e. g. micromanipulation. the microscope substage condenser focus the image of the illuminator aperture in the plane of the specimen.In each case, then, HI ILLUMINATION AND RESOLVING POWER the lamp iris acts as a field diaphragm and 41110 - should be closed to just illuminate the field Good resolving power and optimum specimen of view. The differencein these three contrast are prerequisites for .good microscopy. types o'f illumination lie the adjustment Assuming the availability of suitable optics of the lamp condensing le .With poor (ocular, Objectives and substage condensei) man's illumination there is'no lamp conden- it is still of paramount importance to use ser,.hence no adjustment. The lamp should proper illumination. The requirement for a be placed close to the microscope so that

5-12. 76 0 Optics and the Microscope°

Table 5.COMPARISON OF CRITICAL, OHLER AND POOR MAN'S ILLUMINATION

Critical Kohler Poor man's

Lamp filamnent ribbon filamentany type any type ° .Lamp condensing leris required t.required none Lamp( iris ° required required useful. Ground `glass at lamp none none . present Image of light source in object plane. °at substage none iris Image of field iris nedr object in object neap object plane plane cplane IMage.of substage iris back focal Planeback focal' planeback focal plane of objective of objective of objective

the entire field of view is always the substage condenser iris (also coincident illuminated.If the surface structure of the with the anterior Meal plahe of the substage. ground glass becomes apparent in he field condenser).The functions of the lamp of view the substage condenser very condenser iris and the substage condenser slightly defocused: iris in controlling, respectively, thearea . of the illuminated field of view and the Critical Illumination angular apertureof the illuminating cone are precisely alike for all three types of Withcritical illumination the lamp conden-; illumination. a ser is focused to give parhllel rays; focur ing the lamp filament on a far wathis. Critical illumination is seldom used because sufficient.Aimed, then,. at the substage it requires a special-lamp filament and be- Mirror, the substage condenser will focus cause, wtren used, it,phows no advantage the lamp filament in the.object plane. 'The over well-adjusted-Kohler illumination. substage condenser iris will now be found 8 II imaged in the back focal plane of the ob- Kohler Illumination jective; it serves as a control over con- v'ergence.of, the illumination.Althdligh the To arrange the microscope and illuminato.° substage4ris also affects the light intensity for Kohler illumination it is well to proceed over the field of view it should most decid- through the following steps: edly not be used for this purpose. The intensity of illumination may be varied by a ,Removelffediffusers and filters the use of neutral density filters and, unless from the lamp: color photomicrography is anticipated, by the use of variable voltage on the lamp b Turn the lamp on and aim at-acon- filament. venient wall'or vertical screen about '19' inches away. Open thelamp Kohler illumination (Figure 14) differs diaphragiri. from critical ilhitmination in the use of the lamp condenser. \With'critical illumination c By moving the lamp condenser, focus the lamp condenser focuse§ the lamp a sharp image,of the filament.It filament at infinity; with Kohler illumination should be of such a sizeas to fill, the lamp filament-ts focused in the plane of not necessarily evenly, the microscope

5r13 77 O ,Optics and the Microscope

O

"Critical Poor man's

Eye Eyepoint

Ocular

Focal plane

'I /

Focal plane .1)

Objective Preparation O Substage . condenser /NJ 'I Substage' , IriS

Lainp iris Lamp condenser Light source,

O

5-14 1

Optic's and the Microscope

. . substage ,condenser opening.If it as the center of movement (again does note -move the lamp away ("von endeavoring to keep the lamp dia- the wall to enlarge the filament image; phragm in the cienivred position), -c refocus. If you have mastered this step, you 4 have Zieromplishd the must difficult d Turn the lamp zuurain it attlie micro- portion.(}letter tilierosopd-larits scope mirror-so as,to maintain th.: have adjustments to move the bulb, same, 18 inches (oradjuided lamp 'indepindently of the lamp housing to distance).` simplify this step,.) . - ePlace a specimen oil' the iirospe. Putthe specinulri in place, replace `stage and focus sharp1.wilh.0 I ti -mm the eyepiece and the desired olSjec- (lox) objective. Open fully the live and refocus. aperture diaphragm in the substage condenser.Irthe light is too bright, kOpen orlose. tIm.rfield diaphragm temporarily place a neutral density until it jtist disappears fi:om.the field. filter or a diffuser in the lamp. Observe -the back aperture of the fClose the lamp diiphragm, or field. objective, preferably with the Bertrand diaphragm, to about a 1-cm operringi lbns or the auxiliary telescope,, and Rack the Microscope substage con- close the apeiture diaphragm on the denser up and down to focus the substage condenser until it is about field diaphragm sharply in the same four-fifths the diameter of the back plane as the.speeimen.. aperture-.This fs the best position for the aperture diaphragm, a posi- I g. Adjust the mirror to center the field tion which minimizes glare and maxi-, diaphragm in The field Of view. mizes:the resolving power.It is instruttiVe to°.vary the aperturedia- solro h Remove the 16-misr,,objectiveand phragm'and observe the image criti-- replace with.a4-mm objective,Move cally during the manipulation: the specimen sb that a clear area is under observation, -Place the m If the illumination is too great, :' Bertrand lens in the optical path, or insert an appropriate neutradennity remove the eyepiece and insert an filter between the illuminator and . auxiliary telescope (sold with phase the condenser. Do not use the con. contrast accessories) in itefilace, -denser aperture d.iaphragm or .the orkremove the eyepiece and observe, lamp field diaphrAgm to controLthe theback aperture of the objective intensity of illumination. au ; directly. Remove any ground glass diffusers from the limp. Now Poor Man's Illumination observe the lamp filament through the microscope. . Both critical ana 'Kohler illumination re- quireexpens illuminatorg' with adjust- .iIf the filament does not appear to be able focus, lamp 'iris and adjustable lamp centered,'-swing the lamp housing in © mounts. Poor man's illumination requires a horizontil,arc whoire center is at- a cheap illuminator although an expensive Effie field-diaphragm. The putpose illuminator may be used if its expensive is.to maintain the field diaphragm on features are negated by inserting a ground "the lamp in_jti3.centered position,If glash diffuser or by rising arosted bulb. a -.vertical movement of the filament Admittedly an iris diaphragm on the lamp is required, loblieri the bulb base and would be a help though it is not necessary. slide it up or down.If the base is'. ;, - fixed,7-2tilt-theTlamP housing a The illuminator must have a kOsted bulb 'vertical arc with`thefieI8diaphragm ,or a ground glass diffuser. ' 7' 5-15 ta 79

APF Orlties.and the Miernope . , r

( should be possible to.direet it in liItcsoWing Power the general direction of the substage mirror, very close thereto or in The resolving pow r of the microscope is place thereof. its ability to distinguish ,separate details of closoly tpaced microscopic structures.. b Focus on ahy preparation after The theoretical limit of resolving two' ' tilting the mirror to illumin9te the dim:rely points, a. Ilistance X apart,',is: 'field. X1. 22 k c RemOve the top lens of the condenser 2 N. A. ;, and, by racking the condenser pp*, maze often, down, bring into focus wIn.re = waveleniftlmtf light, used to (in the same plane as the specimen) illuminate °the specimen a finger, iclneil or other object placed N, A. = nktnericalaperture of the in the same general region as the objective ? groundtlass diffuser on the lamp. The glass surface itself can ern be Substituting a wavelength of 4,500 focused in the plane of the specimr.n. Angstroms ana numerical aperture of 0 1. 3, about est that can be done with . visible light, we find that two points about d Ideally the grotknd glass surface will, just fill `the field of view when centered' 2, 000A (or 0.2 fnicron) apart can be=seen by the substage mirror; adjustment as two separate points.Further increase may be made by moving the lamp in resolving power can be achieved far the closer to or farther from the micro- light microscope by using light or shorter scope (the position 'Might be marked wavelength.Ultraviolet light near 2; 000, for each objective used) or by tutting Anqtroms lowers the- limit to about 0.1 paper diaphragms of fixed aperture micron, the lower limit for the light (one for each objective used).In this microscope. instance a lamp iris would be usejul. The.numerical aperture of an objectiveis et e .Low 9i condenser just sufficiently usually engraved on the objective and is to defocus the ground glass surface related to the angular aperture,AA and render the field of illumhtation (Figure 15), by the formula: even. N.A. r n sinAA 2 f,' Observe the back aperture of the objective and open the substage con- where n = the lowest index in the spacp denser iris about 75-percent of the between the object and the way... The final adjustment of the objective. substage iris is made 'while observing the preparation; the iris should be open as far as possible, still giving good contrast. g The intensity of illumination should -be adjusted °WI, with neutral density filters or by changing the lamp voltage. Proper illumination is one of the most im- portant operations in microscopy.It is

easy to.41:dgemicroscopist's ability by 4 a glance at his field of view and the objec- 4 tive back lens. Figure 15 0 ANGULARAPERTURE OF MICROSCOPE OBJECTIVE

so

s 2" I Optide and the Microscope e'

1 Maximum useful magnification. A helpful rule of thumb is that the use- ful magdificatio'n will not exceed 1,000 times the numerical aperture of the objective (see Tables 3 and 4).Although somewhat higher magnification may be used in specific cases, no additional 10111 1111114,- detail will be resolved. It is curious, considering,the figures a b . in the table,that most, if ilot all, manu- Figure 16 facturers of microscopes furnish a 10X eyepiece as the highest power. 4-10X ABBE THEORY OF RESOLUTION eyepiece is useftiltlit anyone interested in critical work should use a 1-L2SX eye- e piece; the 5-10X eyepieces are best for b The conde.nser shotild be well- scanning purposes. . ra) corrected and have a numerical aperture as high as the objective to 2 Abbe's theory of resolulion be used. '4, One of the ,mostcdgent theories,bf (c An apochromatic oil-immersion resolution is due to Ernst Abbe, who objective should be used with a come- suggested that mtroscopic objects act` pensating eyepiece of at least 15X like diffraction gratings (figure 16) and magnification. The immersion oil that the angle of diffraction, therefore, should have an index close to 1.515 increases' withthe fineness of the detail. and have proper dispersion for the He proposed,tiltit a given microscope objective being used. objective Would resolve a particular detail if at least two or three' transmitted (cf--"Immersion oil should be placed rays (one direct and two diffracted rays) between the' condenser and slide and "°.entered the objective; In Figure )6 the between cover slip and objective._ detail shown would be resolved in A and The preparation itself should be C but not in B.This theory, which can surrounded by a liquid having a be,borne ougy simple experiment, is refractive index of 1.515 or more. 'useful in shoWing how to improve resolu- tion.Since shoker wavelengths will e The illumination should be reasonably give a smaller diffraction angle, there monochromatic and as short in wave- is more chance of resolving fine detail length as possible. An interference with short wavelengths.. Also, since filter transmittinga wavelength of only two.of the transmitted rays are about 480-500 millimicrons is a needed -Oblique lightmand a high N.A. suitable answer to this problem. condenser will aid in resolving fine detail. Ideally, of course, ultraviolet light 0 4 3 should be used to decrease the wave- o 3Improv,ing resolving power length still further. .. The folToeing list summarize the The practical effect of many of these practical approaches to higher resolu- factors is critically discussed by tion with the light microscope: Loveland(2) in a paper on the optics of object a The specimen should be illuminated by either critical or Kohler -illumination.

d. 5-17

81 Optics and the Microscope

IV PHOTOMICROGRAPHY curvature of field and depth of field.The photographic plate, however, lies in one A Introduction plane; hence the greatest care must be used to focus sharply on the subject plane -Photo (gra h , as distinct from micro- of interest and to select optics to give photograpy," is tie art of ta iiii-TAC--tures------minimtim_a.mounts of field curvature and thrtugh microscope. A microphoto- chromatic aberrations. graph is a small photograph; a photomicro- graph is a photograph of a small object. With black and white film, color filters Photomicrography is a valuable tool-ill may be used to enhance the contrast of recording the results of microscopical \some portions of the specimen -while mini- stud. It 6nables the microscopist to: mizing chrdmatic aberrations of the lenses. In color work, however, filters cannot 1 describe a 'microscopic field objectively usually be used for this purpose and better )bithout resorting to written descriptions, optics may be,required. 2 record a particular field for future Photomicrographic cameras which fit reference, directly onto the microscope are available. in 35-min or up to 3-1/4 X 4-1/4 inch sizes. 3 make particlesl,ze counts and counting Others are made which accommodate larger analyses easily and without tyingp a film sizes and which have their own support microscope, independent of microscope: The former, however, are preferred for ease of handling 4 enhance or exaggerate the visual micro- and lower cost. The latter system is-pre- scopic field to bring out or emphasize ferred for greater flexibility and versatility certain details not readily apparent and lack of vibration.The Polaroid camera visually,. has-many applications in Microscopy and can be ilised on the microscope directly but, 5 record images in ultraviolet andinfra- becauSe of its weight, only when the micro- red microscopy, which are otherwise scope his a vertically moving stage for invisible to the unaidecl-eye. focusing rader than a focusing body tube. There are two general approaches to photo- B Determination of COrrect Exposure micrography; one requires only a plate or film holder supported above the eyepiece ;Correct exposure .gitterntinatisincan be of the microscope with a light -tight be,illows; accomplished by tfial and error, by. relating the other utilizes any ordinary camera with new conditions to previously used successful its own lens system, supported with a light- ,conditions and by photometry. tight adaor above the eyepiece.It is best, in the latter case, to use a reflex With the trial and error method a series of 'camera so that the image can be carefully trial exposures is made, noting the type of foCused on the ground glass.photomi- subject, illumination, filters, objective, crography of this type can be regarded eyepiece, magnification, 'film and. shutter simply as replacing the eye with the camera /speed. The best 'exposure is Selected. The lens system. The camera should be focused .fillowing parameters can be changed'and at infinity, just as the eye is for visual The exposure time-adjusted accordingly: observation, and it shoal be positioned close to and over the eyepiece. 1 Magnification. Exposure time.vvies . as the square of the magnification. . the requirements for photomicrography; hOivever, are more rigorous than those Example:Good exposure was obtained for visual work. The eye can norinally . .; With a 1/19-second exposure compensate for varying light intensities; and a Inagnification of 100)1. If the'magnification is now:

5.48 'OP 82 4 ' i Optics and the Microscope '

° o ... 1 2004, the' correct exposure, Kodachrome II-Type A __ ..._ I__is calculated as follows.,, Professional is 40. .,;,.... 4 . -. ifew exposure time= 'old exposure time new exposure time = old exposuie time . new ma gnification0 (20012 A. S. A. of old film 0 - X 11100(400/40) old magnification ' 100' A. S. A. of new film _ 4/10 or say,' 1/2''sRcond. ° .` `° 10/100 or 1/10 second.

1 should-hem- .hOWever, that the 4Other parameters may be varied but the above calculation carl:be made only when prediction of exposure time cannot be there has been no change IQ the illumi- made readily.Experience and photo- nation system including the condenser electric devices at'e the best guides to orAthe objective.Only changes in magni- 'the proper exposur'e. ' . fication due to changing eyepieces or ;!' hand- bellows extension distance. can a Photoelectric devices are excellent for led in the abovg manner. determining correct,exposure.Since brdinary photographic.exposure meters *4.- 'Numerical aperture:- Exposure time, \ 'Vre not sensitive enough for photomi- varies inversely as the square ofm:the-% crography, more sensitive instruments, smallest for king _hume B id ai!:"ap having a galvanometer'or electronic . of the condenser and objective. amplifyined'ircuit,4 are required. Some .., photosensitive cells are inserted in the Example;Good exposure wad obtained body tithe in place of the eyepiece for . at 1/10 'geeorld wit)fi tide, light intensity roadings. This has the lt° objective, N.A. 0.25, '°4t-0 advantage of delecting the light level at a 4 o full aperture., With a 204,r point of high intensity but &les not take tobjective, N,A. 0.25, at iqo account the eyepiece, the distance to .. full aperture and the same ; thalm or the filar speed. final tnagnificatioV what is -the correct expcialire time? The cell May be placed just above4 the eye- iece so that it. 'Co P 5egisters 'the total amount new exposure time = olde. pure time alight leav,i0Vthe eyepliCk. 'Again, .the effec,ts of film stgaetoatiinte44eidti*rojection . ;old N.A. 1 ..°1/19 11'40;or, distake are no "'r!..., The-04n new N. A.' O.0 s ip.11dial,vbet.Fk with the total _light -.1:=,% / , I -,, shy,.1450 second. measurihk-photomefer'is ipe difficulty' of ,. , taking into account the arkaof field covered.: -!`,,' .

It it -Oeen that moreeliglit reaches the - ake, for example, a.bright fieldin Mich , 1 ,,,t .° photographiF filttkitytth highat humeri- P lyfew crystalsappear,t perhaps 1 per- CP cal apertures at tlic pagnemagnifiCation. derhf of the light entering the field, of view is .1 scatter0,03/ the crystals and the photomete..y :,. '" 3Film. Etpbsure time varies inversely. shows closAto a ma9imum reading!' Now - . .1. with the Aifierican Standard's Association asstime that evetrythihg r_e_2mains_constant-, speed index of the film. t-the hunib-Ei. of crYitals and, conse- . .quently-,,the amount of light.-scattered:' ' Exainple : A'gdeid.picture was obtained The'pt6torpe'ter rdadini couId easily drop I with Edstman'Tri-XfilrrYat by 501:lerdent., yet ttiii° proper,,xposure is second. What is the' unchanged.. The.,:;ituation for C° Correct ext)surefor photoTrdb.rograp y wi *thcrb s,ed polarp since, , Eastman Kodachrome .. . ' the photometer reading depends. oh thei r . intensity -of lildrninationvOn the bire- Type A. .The A. S.A. speed . - for Tri-X it 400 and for fringence andstlicckness of the,,crystals and . . ;". ./ ,. 14;'t t t 44, 8 ° Optics and the Microscope e

on the number and size of the crystals in same film and projection field or, alternatively, on the area of distance) and that the new the field covered by birefringent crystals. meter reading is-16; therefore: One of the best solutions to this problem is to measure the photometer reading with expVsure time = k/meter no preparation on the stage: A first-order reading = 8/16 = 1/2 second. red compensator or,xqirartz wedge is in- serted when crosoRfOlars are being used illuminate -the entire field.- .V MICROMETRY An alternative to place the cell on the A Particle Size Determination ground,glass ere the film will be located. ever, although all variables Linear distances and-areas can be except m speed are now taken into measured with the microscope. This account, measurements in the image plane permits determination of particle size 6 have the disadvantage of requiring a more and qUantitative analysis of physical sensiOve electronic photoelectric apparatus. mixtures. The usual unit of length for, microscopical measurements is to micron No matter what- m ?thod is used for placing' (1 X 10-3mm or about 4 X 10-5incffl. the photocell, the exposure time can ,,W$ Measuring partici% in electron microscopy determined by the general formula: requires an even smaller unit the milli- micron (1 X 10-3 micron or 10 Angstrom exposure time units).Table 6 shows 'the approximate meter reading average size of a few common airborne The constant k will'depend on the physical materials:' arranerdnt and film used.; To determine.' k for anyrticular system, fillst set up the microsccr totake a picture.- Record Table 6.. APPROXIMATEPARTICLE SIZE OF the meter' reading anci take a series of SEVERAL COMMON PART,IcULATtS N S trial exposures.Pick out the best exposure

. and calcylate k.- Then the k which was determaned holds as Iongas no change is pollen 25 micr ons made lit* right pathpeyond the photocell,- Fc.:,g droplets 20 microns e. g: chAiging to a faster film or changing PnW Pr-plant flyash 5 rriic rons 'thF668atiOridratance.,. Thus the, objective, (after prIcipitators) 'condenser position or illuminator may be changed without affectinili if the cell is Tobacco smoke 0.2 micron ;,used as deicribed above. (200.millimicrons)

Foundry fumes .. ' Example:With one particular arrange- 0.1 - 1 micron-- ment of photocell and (IUO =-1b00 rfaiiiinicrons) . found to o be 40.A.serfes of photographs arelakensat 1/2, 1/5, 1/10, .IL2,5 and 1/N secon *. The. The practical lower Unlit of acaurate ph9toinicrograpil taken at 1/5 particle size measurement withthe 4ght, second is judged to be. the best; microscope is about 0.5,micron: The ',hence k is calculAea as follows: measurementeasurement of a particle,smaller than this with the light microscope leads to W4nieter.reading' X exposure errors which, under the best cirertm- time = 40X 1/5,..= 8. stances, increase to about + 100, percent ,(usually +). 7 4t Assume,,novithat. a new picture er, . .is to be takenat.sinot)ier Qne of the.princijIal-uses res.oltring magnification (hut witty-the power is in the precise measurement of r -* t - .4 .;,t -4 5--20 Optics and 'the Microscope

Po'

*fr partide size.. There are, 'however,a in an'appropriate direction until the variety otapproximategand useful proce- second trailing edge just touches the dures -as well. cross-hair line. A second reading is taken- and the difference in the two` 1 Methocid of particle size measurement readings is the distah-Ce mov- r the size of the particle.This method is. a Knowing he Magnification of the especially useful when the particle microscope (product of the magni.- is larger than the field, or when the fication of objective and ey'epicce), optics give a distorted image near the the siie -of.particles-can be esti- - edge of the field. mated. For example, with a.10X eyepieceind a 16-mm (or 10X10X)' d The above method can be extended to . objective', the, total magnification projection or photography. The image is 100X. A particle that appears to .of the particles can be projected on a be 10-mm at 10 inches from the eye screen with a suitable light source or has an actual.size of ,l0 mm divided they may be photographed. The final by 100 or 0.10 mm or 100 microns. magnification, M, on the projection This is in no sense an accurate surface (or film plane) is given approxi- method, but it does permit quick mately' by estimation of partible size; the error in this estimation is usually 10-25 M = D X O. M. X E. /25 percent. where 0. M. = objective magnific4on b'An9ther approxitrote method i's also E. M. =eyepiece magnifica ion based on the use of known data'.If. D = projection distanC0 we know approximately the diameter from the' eyepiece'in of the microscopelield, we can Centimeters. .6- estimate the percentage of'the diameter occupied by the object to The image detail can theft be measured, be measured and calctlate from - 'in centimeters and the actual size com;) these figures the approximate size puted by dividing by M. This methods Aof the object. The size of the micro- is usually accurate to within 2-5 percent scope field depends on both the objec- depending on the size range of the detail p. tive and the ocular although the latter measured. is. a minor influence. The size of the field should be determined with e The stated magnifications and/or focal a millimeter scale for each objective lengths of the microscopeoiltics_a.re .lend ocular.If this is done, eiti- nominal and bit from objective tiori .0.fizes-by-comparigiiii with- to objective or eyepiece to eyepiece. entire field diameter carbe quite To obtain accurate measurements, .a ( accurate (5-10%). stage micrometer is used to calibrate each Combination of eyepiece ant c The movement of a graduated mechan- objective. T1* stage micrometer is V ical stage can also be used for rough a glass microscope slide that.has, Measurement of diameters of largt accurately engraved in the center, a larticles. Stages are usually grad scale., usually 2 millimeter slong, aid (with vernier) tO'read to 0.1 divided into goo parts, each part repre- mittimeter, or 100 microns.In senting 0.01 millimeter.Thus 'when . ..,practb-b,' the leading 'edge of the this scale is observed, projected.or particlelis brought to one of the lines ,..,photbgraPhed, the exact image inagni-. of the cross hair in.thp eyepiece and fication can be:determined.For a.reading iSe,t4100 bf theAtAkeposition. 'example, if 5 spaces of theg,stage micro- Then the particle if mimed across the meter measure 6 millimeters when' fieldby moving the mOf tartirCailttage' 'projected, the'actual magnification is op \\'' ot_ ` '' O .

vo'

4 '4 .4 tics and the Microsc

6 120 times. The calibration consists, then, of 5 (0. 01) calculating. the number of microns per This magnification figure can eyepiece Scale division. To make the used to improve the accuracy of comparison as accurate as possible': a method 4 above. large part of each scale must be used (see Figure 17).Let's assume that The simplest px.iXedure and the most with the-low power 16-mm objective. accurate is based. on the use of a 6 large divisions of the stage scale _ micrometereyepiece.: Since the (s.m.d.)-are equal to 38'divisions of eyepiece, magnifies a real image the eyepiece scale. This means that from the objective, it is possible 38 eyepiece micrometer divisions (e.m.. to place a transparent scale in the are equivalent to 600 microns. Hence: same plane as the image from the objective and thus have a scale 1 e. m. d. = 600/38 superimposed over the image.This = 15. 8g. is done by first piecing an eyepiece micrometer scale disc in the eyepiece. The eyepiece micrometer has an arbitrary scale and mast be cali- brated with each objective used. The simplest way to do this.is to place . the stake micrometer -on the stage a and note a convenient whole number Step litcrentieler Scale of eyepiec4 micrometer divisions., The value inmicrons for each eye- 14./4,14.A.16L4.1.1 .1.4.1411, piece micrometer division is then 0 easily computed.. When the stage Eyepiece blieremeise lids . micrometer is removed and replaced by the specimen; the ,superimposedi eyepiece scale can be used for accu- , rate measurement of any feature in the spec:igen by direct observation, photogrFWor projection. 4 Figure 17 2Calibration of eyepiece micrometry . COMPARISON OF STAGE MICROMETER Each znicgometer 'stage scale has SCALE WITH EYEPIECE MICROMETER SCALE divisions 100g (0. 1mm) apa t; qne t or two of these aregusuallsubdivided 'Thus tvhen that micrometer eyepiece into '10g (0. 01-mm) div ions.These is ased.with that 1.6 -nim objective each form the standard against which the division of the eyepiece scale is eqUivalent arbitiary divisions in the micrometer to 15.8µ, and it can be used to 'make.an eyepiece are to be calibrated. Each accurate measurement of any object on objective must be "calibrated-separately the microscope stage. A particle, for by noting the correspondence between example, 'observed with the 16-mm objec- the stage scale and the eyepiece scale. tive and measuring 8.5 divisions do the Starting with the lowest power objective eyepiece scale is 8.5 (15.8) or 135g in focus -on the stage/scale, arrange/ the diameter, two scales parallel- and in goodldQus. It should be pcishible to determine the Each objective on your microscope angst number of eyepiece Azisions exactly be calibrated in tis manner. 0. ,equal to some whole umber of divisibns of the stage scale, a distance A- convenient way to record_the. necessary: readily expressed in microns. data and tct calinlate g/emd is by. means of a table.

5-22

e Optics ancrtlie Microscope

0

Table The more pafticles counted, the more accurate will be the average particle Objective size.Platelike and needlelike particles nb. emd-no. emd1 emd * should have a correctioir factor applied 32-mm 18 = 44, 1800 = 4440.9F., to account for the third dimension since all such particles ale restricted in their 16-mm 6 = 38 600 = 3815.. 8Fi orientation on the rrTicroscope slide. 4-mm 1= 30 100 30 3.33µ Whet; particle site is report11, the . _ 'general shape of the particles as well as the metboditied-to-delefrifine- the "diameter" should be noted.

3 Determination of particle size Particle size distribution is determined .distribution routinely by moving a preparation of particles past an eyepiece micrometer The measurement of particle size can scale in such a way that their Martin's vary in complexity pending on parti- tdiameter-.can be tallied.All particles cle shape'.Ttfe ze of a sphere ma e whose centers fall within two fixed denoted by divisions on the scale are tallied.Move- cube may be'expressed by engt Ment of the preparation is usually 4n edge or diagonal.Be d these two accomplished by means of a mechanical configurations, the p-cle "size" must stage but may be carried out by rotation , include information'out the shape of of an off-center rotatingstage. A sample the particle in quest on, and the tabulationtab rn rTea .obnloe s8.sTohe ear - expression'ofthis s ape .takes a more piece complicated form. t .at least-six, but not more than tweive,, size classes are required and sufficierit Martin'S diameter ise simplest means pArticies are counted to give a smooth of measuring and expr ssing the dia- curve. The actual number tallied 200 meters of irregular paicles and is 000) depends on particle shape sufficlVtly accurate wb n averaged for relaOty and the' range of sizes.The a largeltAnber of partic es.In this ze tallied fqr each Particle is that 0 methodi the horizontal east-west number of eyepiece micrometer divisions dimension of each icle which _divides most closely approximating Martin's the projected,area into halves-is taken as diameter for that par rticle. Martin's diameter (Figure 18): 4 4 C alculation of.sizeaverages 1 1-74 I 1----4 1 The size dat'a maybe treated in a variety 1 of ways; one simple, straightforward i 1 1 t 1 1 I treatment .is shown in Table 9.For a I i more complete discussion of the treat- ment of particle size data see Chamot and Masdn;s, bybook of Chemical /Vlicroscopy(aage 26. re Figure 18 The averages with resperugiiier, 9 7:11-, surface, d3; and weight or volume, . MARTIN'SDIAMETER, d4, are calculated as follows for the data in Table 9.

5 -23 , ,;1;

87 ,- 4 Optics and the Microscope

4 Tags S. PARTICLE SIZE TALLY FORA SAMPLE OF STARCH 9RA1NS &so class Numb** of particles ° . (sods) Total

4 1 r1=4.1r-r44rt.4.4 1$ rt44 214..111-14r-144r144ri-44 9$ ! r-t-s4- rt.4.4 re-4.4 1-1-44 rt-1-4 ft4.1#1'S-1.111,1

rt-i4 1-14.4 1"-11-4.1 110 re-44rt-44 2444ri--1.4r-24.4 r-1-44 2-14.1 rt-4-4

4 -2-1-4.111-44 11-541 1S7 0 r 1-4.11-1-14rt-4-11-t-4-1 r1-4.3 rt-44 lit -4.l 11

ri-44 .7-1 rt44rt-s4 rt.44

1

45 1-144rt-4.1 S rtZ1.1 .

7 t71-1..1 I-I-1414-3-1 -1 . 11

*findeiralide microrneteL dizialeas

di = En'd/En = 1758/470 , = 3. 74 emd X 2. 82* =7. 10. 5p.

.713 = End3/ End2 = 37440 /7662 = 4. 89 emd X 2. 82 = 13. 81.1. d4 = End4/End8 =1991941 37440 . = 5: 32 emd X 2. 82 = 15. OR *2:82 micronSper emd for the. cumulative;wei4ht or volume (determined by calibration of the curve, are plotted against d.Finally, .eyepiece - objective combingtion the.specific surface, Sm, .in square used for the determination). meters per gram, m, maykbe calculated if thedensity, D, is known; the surface Cumulative percents by number, average ;53, Is used. surface and weight (or volume) may be - plotted' frOm the data in Table 9.The If D 1, Sm = 6 /d3D = 6/13. 8(1. 1) calculated percentages, -e.g. = 0, 395m2ig.

5-24

5 83 Optics and the Microscope

n. / Table 9. CALCULATIONS FOR PARTICLE SIZE AVERAGE' d (Aver. diam. n nd nd2 nd2 nd4 in emd)

1 16 16 ^ 16 16 16 98 196,392 784 . 1588

3 -.1.10 330.990 2970 . 8910 4 '107 4281712 6848 27392 5 3551775 8875 "'"44375 -..4"\ ,1' 6 45 2701620, , 58320 7. 2f 1471029'''7203. 50421 8 2 16 128 1024 8192

-470 17587662 37440. 199194, ". c B Counting Analysis 4 .e..g.seounting the percent of raxon fibers , ip a sample of "silk". Mixtures of\of patteulates. can often be quantitatively aniprzed by counting the Example 1:"A dot-count of,a mixture ,of total number of particulates from each fiberglass and nylcin shows: component in a' representative sample. ' The calculations are,' however, comPli.- nylon 262 cated by three factors: average particle fiberglass . 168 ,size, particle shape and the density of the componints:If all of tht compon- Therefore: ents were equivalent in particle size, % nylon = 262/(262 + 168) X shape and density then the weight.per- = 60.9i by number. centage would'be identical tothe number_ perceritige.llsally, however, it is -However, although both fibers are smooth necessary to determine correction factors cylinders, they do have different densities to account for the differences. and usually differegt diqieters. To correct for diameter one must measure. - When pxoperly applied, this method cal the average diameter of each type of fiber be-accurate to within + 1 pirced and, and calctilatethe volume of a unit length in special cases, even better.It is often applied toe analysis of fibefmixtures aver. diam. volume of and is thenually called a dpt-count 4 1-p, slice, p.3 because the y of fibers is kept aathe nylon 185' 268 preparation isoved past a point or dot in the epiece. fiberglass 13.2 117 A variety of methods n be used to "le percent by volume is, then: simplify recognition of the different " 2620( 26,8 % nylon X 100 Components.-----Theseinclude chemical (262 X 268)+(168 X 1 .17) staineor dyes and enhancementOrOptical- --- differences such as refracWe indices, = 7; 1%-by-volume. _ _ dispersion or color.Often, however, one .relled.cc.thedifferences,in morphology, , Still we must take into account the density of each in order to calculate the weight percent. ,

4 5-25 89 Optics and the Microscope

If the densities are 4.6 for nylon and 2.2 efor glass then the percent by weight is: 262 X 268 X.]. 6. % nylon X 100 :-.,(462N268 XL4)+068 >c,417 TEI3

, = 72% by weight. ElamP11%.2: A count of quartz and gypsumshows: _ "283 gypsum.dypsum. 467 I To calculate the percent by weight we must take into account the average particle size, the shape and the-density'of The average particle size with respect to weight, d4,. must be measured for each and the shape factor must be determined. Since gypsum is more platelike than quartz each particle of gypsuni is thinner.The shape factor can be approximated or can be roughly calculated by measuring the actual, thickness of a number of particles. We might find, for exaMple, that gypsum parti- cles average 80% of the volttme of the aver- - age-quartz particle; this is our shape factor. The final equation for the weight percent is: 283 X itc14/6 X Dq % quartz = X ipo 238 X it-a4/6 X f)q.+ 467 X it cit/6 X 0. 80 XIDg where Dq and Dg are the densities of quartz and gypsum respelcitrely;--0 : 80 is the shape,. -.factor and d4 and d4, are the average parti- cle sizes with respect to weight for quaytz and gypsum respectively..

- / ACMIOWLEZIGNIEN. T: This ogtlih'ewas 2 Loveland. R. P.. J. Roy. Micros.'O Sc. preparicl.by the U.S. Public Health Service. 79, 59. (1980). Departmenfjctf-- Health. Education and Welfare,' for use in its Training Program. 3Chamot.Emile.Monnin and Mason. Clyde Walter, Handbook of Chemical. Microscopy, Vol.1, third ed. John Wiley and Sons. New York (1959). lEhmn. C. W. Crystal Grovrth from Solution.Discussions of the Faraday DESCRIPTORS: Microscope and Optical eociety No.5. 132.Glittery and Jackson. propertieP - g _

4, . 5-26. 5.

atr STRUCTURE AND FUNCTION .0E- CELLS5-11-. s.

IINTRODUCTION membrane may be thought of as the outermost layer f protoplasm. What are cells?Cells may. be defined as the basic structural units of life. The dell has b In plant cells the most-conspicuous- many different parts which'carry on the protoplasmic structures are, the . various functions of cell-life.- These are "chloroplasts", which contain- called organelles ("little organs")..' . highly organized menibeane systems bearing the photosynthetic pigments A The branch of biology which deals With the (chlorophylls, carotenoids, and form and structure of plants and animals xanthophylls) and enzymes. is called "MorPho loci." The study of the .1 arrangement of their several parts is cThe. "nucleus" is a spherical body called "anatomy", and the study ,pf cells whicli regulates cell function by ,is called "cytology','.. controlling enzyme synthesis. B "There is no "typical" cell, for cells differ d"Granules". structures of small from ,each'other in detail, and these size and. be "living" or 1 differences are in part respdhAble for the non-living' material. variety of life that exists on the earth. . . e, "Flagella are whip-likestructures 'found in both plant and animal cells. IIFUND'AMENTAI;S OF CELL STRUCTURE Thy flagella are used for locomotion, or to circulate the surrounding A tiow do, we recognize a structure as a cell? medium. We must look for certain characteristi-ea.-- and/or -structures which have been found f"Cilia" resemble short flagella, found to occur in cells. The cell is composed almost exclusively-on animal cells. of a variety of substanc.es and structures, In the lower animals, tilts are used some of which result from cellular for,Aocomotion and food gathering. activities. These include both.living and - non-living materialS. , g The "pseudopod", or false foot, is an extension of the protoplasm of Non-living components incltide: certain protozoa, in' which the - colloidal state of the protoplasm a. A "cell Wall" composed of cellulose alternates from a "soh' to a "jel" may be found as the outermost condition from titne-to time to covering a many plant cells. facilitate cell movement.

.

b t "Vacuoles" are chambers in .the . h "Ribosomesuare,protoplaarnic bodies protoplatm which contain.fluidS of which are the site of protein ; different 'densities (i. e., *different synthesis.' They are too small from the surrounding protoplasm). (150 A in diameter)ito be seen with 7 * a light microscope.' . 2 The "living" partii of the cell areicsilled "protoplasm.", The following structures i"Mitochondria" are small. mem- are, included: . branoUs structures containing . enzymes that oxidize food to producel ienergy' transfer cdmppunds, (A TP ) a. A thin "cell Membrane" is located yG just inside the ref wall. This 7,

k

BI. CEL. 6. 76 6-1 I Structure.- and Function of Cells

B how basic strutture is expressed in some 3Thp green algae as a group include a. major types of organisms. ' great variety of .Structural types,. ranging from singleq-celled non-motile We call better visualize the variety Of cell forms to large motile colonies.Some, 4 strUCture'by considering several spedifip types are large enough to resemble cells-. higher aquatic plants. o I tt' ... a 1. li,4Cieriahave fevrorganelle's;_ and are a. The' chloroplasts.aremoditied irkbos so minute that under the light - A variety of: shapes and ire loomed a , MicroscOPe only general morpholOgical in different positions, Examples, types (ire., the three basic shapes; of dh3.oropla.st shape and position.aret rods, spheres-, and spirals) can be recognized: The following structures have been defined: : 1) Parietal - located c5xrthe r- a periphery of the cell;' usually aThe. "capsul e'',*s a thick protective pup- shaped and may extend cove0ng,of the cell ekterior., con- 1 completely around the inner sisting of pArsacci-bifi, cie or surface of the plasnia membrane. polypeptide. 2)Discoid - also located oq the b The dell watt and plasma membrane" periphery of the cell; but are. - are present.. plate-shaped; usually many per . cell: c Although no well.defined nucleus is .sokt, visible in bacterial cells, the ; .3)Axial - lying in the central axis- electron microscope has revealed of the cell; may be ribbon-like areas pfdeoxyribone nucleic acid pr star-shaped. (DNA).con8entration.' This sub- ..*- stance is piese,nt within the nucleus of 4) Radial - have-os or processes of higher cellk, and is'the genetic that extend'outwa-i'.rpd from the or hereditary material: ; scenter of the cell (radiate), er"reaching the plasma melnlYrane.. d Some types -Of pa-cttriaTkynt.iin .

special type of chlorophyll 1 5) Reticulate - a mesh -like network_ (bacteriocrophyll ) and carry on that extends throughout Volume itotosyn esis, ofthe cell. 2 ue-green algae are similar to the b Located in the chloroplasts may be bactbria. in structure,. but contain the dense, proteinaceous, starch. pho ospithgtic pigment chlorophyll) a. forming bodies call "pyrenoids". atke the bacteria, these forms also 4 The flagellated algae .pbasess dne-to- tlackan organized nucleus (the ,eight flagella per cell., The chloro- nuclear region is not bounded by a plants may contain brown and/or red merhbrane). 7 pigments in addition to chlorophyll. 41. 4,1 +he Chlorophyll,b,ea.ringmembranes a ReserVe food may be stored as aide not localized in distinct bodies . . starch (Chlamydomonas)paramylon 0 IChloroplasts), but are diaperded- (EUglena), Or as oil. 4 throughout the. cell...... ' 5 The protozoa are single-celled Gas -filled structures called arlimals whidh exhibit a variety of "pse'Udovacuoles"are found iii some cell structure. type g.of blue-greens:,

It /' .92 /. Structure and Function of Cells

44 ,;

*ift .: 4. a The amoebae move.by means of a higher concentration of asubstance , pseudopodia,-, as described "(Such as, phosphate) inside the cell than -previously. is found outside. Algae are able to r- synthesis 'organic matter from inorganic b The flagellated prbtozoa raw.materials (carbon dioxide and , (Mastigophora) possess one or more water), with the aid of energy derived from light; whereW3 animal cells must obtain their organic matterready- . c The ciliates are the Most highly made" by consuming other organisms, ". -friodifiedprotozdans. The cilia may organic debris,. or dissolved organics. . ,be more or less evenly distributed . _ ,bver:the entire surface of the cell, or mapbeelocalized.. , IV SUMMARY The cell is made up of many highly special-, IIIFUNCTIONS OF CELLS izedgilbstructures. The types ofsub- structures present, and then. appearance What are the functions of Cells and their (shape, color, etc, ) are very imnortant, in structural components? Cellular function, understandinethe role of the organisth in is called "life", and life is difficult to define. the aquatic comMunity4 and in classification.

Ltfe..is charadterized1:89moccsse ommonly %.- referred to as rejarbduction, growth, photo- . syntflegis, etc. ° \REFERENCES - ° . 'A" Microorganisms living in surface waters -1. Bold, H. C.. Cytology of algae.In: G.M: - Smith, (ed. ), Manual of Phyvlogy. are subjected to corratant fluctuations in 4 4 . r the physical and chemical characteristic , Rona ld Press. 1951. 1 . . 4 of tthe h e e n v i r ondAt, and mus t constantly . Im O d ifr t h e i r activities. " 2 B. otn.e)Geoff fry Hed° :, Cytology aid ' .3rd ed.,' AAcademic . ,Cll. Physiology. The. cell requtres'oe. source of chemical, "Press.1964:- o; . . 4 ,.. . energy to, carry On life proceies and .. .. °' successfullZoompete with other-, 3 :lara_chet, Jean.The Living Cell: ° oFgarnsms. Plant cells may, obtain ' Scientific American.r 205(3):n61. . 4 .,.. 1" this energy from light, ,which is, absorbed by chlorOphyll and converted .4Corliss, John O.Ciliated Protozoa., into ATP or food reserves, 'dr from, - '...Pegamon. 1961. - the pxidation of food s tiffs. Animal cells obtain energy on y from the i'rittch; Fre- The: structure and . o oxidation oflood.,- ° treproduction.of the algae.Cambridge

F.', ,. , 0 0 0e Unik( Press. , 1965, . ,P ells. must obtain *ravio5ixaci eriars, from 6 the *envirourpent In:ordeF to grovrand t`6Frobisher, .M.I'luilamentals of carryout otheilifeyfilnctiOris:Inorgaiitc, itliepdbiology .7th edition.W.B. s . Saunders Philaalphia; ;1962.0 and'orgailic..inatelials may, be to:keit-up ... . by passive diffusion' or by . 44,. .* ;-:. C transport".Irl,theaFter process, '7 Round, F'. E.The biology of-the algae. energy Is used to,laulc1 up and maintain. p St. Martin's .Prese. 'New York. 1999*

.. `.14'4 .. . a ; .4 4 ;'Ili .. re ,, o o . 4.. : 1 ,. ca., '-; ... Tlis°outline,originally.prepared by ttilhaer . .4- ° s 9' E. Bencier;Biologist, /orfnierlyth 9 . 4.1 0 ,,;:. , c .0 . Training ACtivities; FWPCA, SEC. and

A revised by Cornelius I. Weber, March 19_700. - $ '' .. 4.04 ; o Descriptor: °Cytological Studies - ' '44 9 .1

., BACTERIA AND PRO'TOZOA AS TOXICOLOGICAL INDICATORS

2Green photOsynthetic bacteria IINTRODUCTION - Micvochloris, Chlorobium, and The. WINOGRAD,SKy COLENIN is anexcellent Chlorochromatum; methane bacteria; simple classroom experiment aswklfas a and SO4 reducers. miniature_ecosystem which yields a variety of-photosynthetic and other prbtista -especially 13 Photosynthetic pUrple sulfur bacterfa- the bacterial forms important inphotosynthesis° Thiopedia and Thiosarcina. II research. These photosynthetic bacteria, as pointed out by Dr. Hui r, are ubiquitous in 4 Filamentous sulfur bacteria, wet soils and natural waters but ordinarily . escape notice. 5Non-sulfur photosynthetic bacteria, Rhodopseudomonas. PREPARING THE COLUMN - , II 6 Blue-green algae, Schizothrix and The materials needed are simple laboratory Oscillatoria, . items. \ . Diatoms, Nitzschia. A The'ino6ulurit la back sludge)*may be easily obtained From a lOcal'sewage 8 Coccoid green algae,Ankitrodesmus; treatment plant or the bottom of a pond and flagellate greens, Chlamy'domonas; oil lake.Because the USPHS document, , similar to a stabilization'pond flora, contairiing Dr. Hutner's paper, is out of print\itreproduced, ere. 9 Filamentous green algae, Ulothrix. 0 B Directqls for preparing thecofiren,and C Besides the phbtosyntheticbacteria and' other'useful inforination are given in that, ather.piotista there will be a variety of ° paper. peotozoa found in the aobic levels (app. 6 through,9). Many of,these 01/4.12r. Butte1s*bittliography Should be protozoans are typical fauna of activated sufficient fr these who wish" more sludge. ° .information: D The possibilities are endless forfurther experimentation. These ecosystems ax's ECOSYSTEM DEVELOPMENT also conyeniehf and inexpensiye seurces iIII for protozoa and,ottterltiodsta for class, . A yactoi' such as the substrate used, the arid laboratory instruction. ° . inoculum, the overlying supernatant water, end labOrafory, condiUsi- as temperature , 4 o., MICRIDAQUARIA light: all;ittfltred440 p&rticul Ir. 4 , type of biota forfaing successive layers or zones..The.4.cdtsmpanying figure is A Fenchel describes amicrolquariunc thier efore 'generalized, and is notintended '"° (1.5 X 54u) which may be observed under . 2y- , to beabsolute. , a .compounernicroscope. (Figure B Some. representative forms are listedfor.' Be, The development of cpmrnunities of. general inforination, The numbers - 'orkanisMs is, quits?similar-to the correspondtO those,on the figur. Winogradsky Column..,(Figure 3) . - o" Inorganic substrate on towe/ing.-f., tit - o a.9.72 0 1. ,7:1 , Bacteria -aildIProtozoa as Toxicological Indifators 4

C The basic media consists of one liter of 3 Burbanck, W. D. and Spoon, D. M. natural water 10 g CaSO4, k g glucose, The Use of Sessile Ciliates Collected 1 g of peptone utoclaved and stored at in Plastic Petri Dishes for Rapid So C; Assessment of Water Pollution. Jour, of Protozoology.14(4):739-744. \ Befo*re use agar is boiled with the media. 1967. White hot7 the media is introduced into one end of the "micr6 aquarium" with a 4 Curds, C.R. and Cockburn, A. _pip et ._After. tiv agar _congeals, natural Protozoa in Biological Sewage Treat- water samples are added.During ,Pnent Processes.I.A Survey of the incubation and When not being observed, Protozoan Fauna of BritiSh Percolating the-micro aquaria are kept_in a moist . FiltermandActivated Sludge Plants. chamber.. Water Research 4:224-236. 1970.

E Although Fenchel used a 'seawater medium 5 Curds, C.R. and Cockburn, A. and inoculum, freshwater sources would II.Protozoa as Indicators in the be equally useful. Activated Sludge Process. Water Research 4:237-249.1970. F In these microaquaria simple ecosystems develop when they are kept in complete. 6Curds, C. R.An Illustrated Key to the darkness. Complex photosynthetic British Freshwater Ciliated Protozoa communities develop when they are Commonly Found in Activated Sludge. illuminated, A natural ecosystem is Water Poll. Research Laboratory. figured by FenChel (Figure 4), ald a Stevenage, England.90 mi..

related food web is shown in Figure-5 . (both from Fenchel). 7Fenchelk Tom.The Ecology of Marine-- 4 "I . . Microbenthom'IV.Structure and G Microaquariae Using PAitic Petri Dishe's ..1i4=1._..onof the nenthic EeosAtem, . its, Chemical and Phisical Factors Sessile ciliates have been successfully and the`Micrpfauna Communities with collected, cultured, and used for bioassay\ Special Reference to the Ciliated 'using the' same petri dish (membrane filter' Protozoa,01:Melia 6:1-182.1969.* type, with tight fitting lids). 3- . , t ,. ,, Botanical Gardens and ,.H. IVIicro*Aluartia,usindisilltorfe.cementsillies rings . # ,Hol-izong in Ald3.1.Rese,.arcIA.In Challenge ,0,,*hilh Allow;diffiision of 'gases throtth the for Survival.Pierre )5anseteau*ed. -' pi/icohe: cultures win $12.erfbyflemp.in ..... ColumbiaUniversity Press. -1970. , . active ipdelinirgy. 4. % . . s. . .G . . . .., 9-. Hutner, S.11. ; Baker, H. ;,031nColi, D.. . . %tti' ,g 0. . 4 . ., , ... Nutrition and Metabolism ro ozoa. SO '. ADDr l'IONAL REFERENCES...,-I Chapter inictlogy *pp: 85-177. . - Adited rprganton.35*ress. i :A r Hutner, S.H.i?rotoxoa as.Toxicokgical 197.2.' 15' (roofs: The Jour. of Frotozoology. *". 4?* "*" .-*At : 11(1):1:6.'l9B4. :' ',, ft',; 10 Hu S. H. The Urban.tiot°431ical:Giffae*A° 4, t ,ers A cadeknis Wildlife,.B..vesekirct. ,barden 2Spoor; D. M. and.lierpanc It,, W. Dr n.19L2V7,-40:1969. 4; A New Method X9r Collecting Sessile wpw .** '4 Ciliates in Plastic Petri Dishes with Tight - Fitting bids.Jour, of . Thisilyntlinevran prepared by Ralph M. Sindlair, Protozoology J4(10:735:730.1967.' 4quati4 Biologist, National Training Center, - ,Water Pi'ograms Operations, EPA, Cincinnati, 01# 45268.- ,. a 6

,'7 -2 .95 *1.

, Bacteria and Protgzoa Toxicolngical Indicators.

O

J

du 1

. o .WINOGRADSKY :COUMN

41. GENgRALI2ED.

S. a o. 4 . ... . ' 0 a %. . tl ' ',40 A

'' ^7. %

green FILAMENTOUS GREEN 'ALGAE, ,.. . 4 ,--.. . 411. vtt t ., c , S. . IS SUPERNATE , 7 sis- °

41' a° 4 1 ' .` ' s. °

green .' CPCCOID GREEN' akbdA,E '.41/4 )V! ... ''''.'/ 6- : ,tt / " 6 .14*. ..;..i e' - , +. A ' A . . . ' ; . : C % i 4 O o k 0 '.. 6 . . a 4, I -iipt1.01041% .4)4.1.016AS; 45 , 74. I.J_EpREEN ALGAE .=P. r m'clge.n o N01.LLLFUt PHOTO, ----FIL-KM EN:TO-USSU L-FUR is rest' PURPLE_SULFUR. 0 B*CTERIA - 04 . PREEN PHOTO. , gyttja . A $ INORGAtildSUBP'RATE,

J

, 4 FIGEIRE 1 . 1 Aft' 411W Did 4 Bacteria and Protozoa aS Toxicological Indicators'

t f

FIGURE 2 4 A micro aquarium fitted with electrodes and the redox conditions through 17 days.

_i , , .., .,,,, ,,`2 I ,---, . , j:,,..4.-- /T c .ezt',_'r/-A-,-4,1- r- r o, 1 '.. . i?":-.-,:i,i_...) -4; ),) / ".is-'----,1 ,1. t % V C. --,to 221 c"' :'t - "--- " -...... , (< r---1.,V\ / t1,,,,..,-,,r,, ,, .2,,-- 4,j, -----,__./.-.....,-::-. :.--/'4.'d , -,i

>1. \ -"; - \ 4

ul

ic

c FIGURE 3. brawing made by tracing a micrograpirof the bacterial plate in a micro aquarium (same experiment as shown pn Figure t.) Most conspicuous are the filaments of Beggiatoa and thg ciliates byclidium citrpllus, Euplotes elegans and Holosticha Bp: Below the Oscillatoria filament (lower left) a Plagiogogon loricatus is seen. Eafteria (exctpt Beggiatoa) 'arse not shoWn, , .

4 Bacteria and Protozoa as Tokicolosica.1 Indicators

..

r

300 it FIGURE 4 The microflora and fauna in the surface of the Beggiatoa patches.(Oscillatoria, Beggiatoa, Thiovolum, diatoms, euglenoids, nematode, Tracheloraphis sp., Frontonia marina, Diophrys scutum, Trochiloides recta).

1

4.1ACaffLA S. ;ONO' (9STom S.

v. F...tv-7nus etczettuLA.& Kt.tAlt. /11 c34.4/24./0_2Psf_

FKKONIA ,I.ONO°6 . M'AA MU A S. etaramAti2 RN Li. T V F ht),0*.4A VADLOSTS nClEvA ' ealre.S&...61121P 5AMtatayjiczpauyi isSrfPO, sPe. =4=S Lif Mf.n220.11±.M

The food relationships ofthe most common ciliates in"estuarine" and in sulphureta.

5 fi 93

-t ,

,

ENVIRONMENTAL REQUIREMENTS OF FRESH- WATER INVERTEBRATES L. A. Chambers, *Chairman Bacteria:- Protozoa as Toxicological Indicators in Purifying Water S. H. Hutner,, 'Herman Bakers S. Aaronson and A. C.Zahalsky+

There is a cynical age that all travelers The food -chain pyramids of sewage plants become entomologi ts.But now with DDT and polluted waters haverbeen adequately and detergents, t 4Velars and stay-at-homes described (Hynes, 196e; Hawkes, 1960). _alike are beco4 g toxicologists. We have A problem treated here is hoW to scale those an immediate in Brest in pollution problems: microcosms to experimentally manipulable Our laboratory receiiies, like the Eait Riyer microcosms yielding dependable predictions and adjoining United Nations buildings, a for the behavior of sewage-plant microcosms. generous sootfall from a nearby power plant. Also, we have'seen a superb fishing ground, in New York City, become a THE WINOGRADSKY COLUMN AS 'SOURCE sewer. (Jainaica Bay is, however, being OF INOCULA FOR MINERALIgATIONS AND restored to its pristine cleanlines§but not AS TOXICOLOGICAL INDICATOR SYSTEM the U. N.'read. )" We take our'theme neverthel ss not from aesthetics but from Total toxicity °depends on intrinsic toxicity- k statemes by Berger (1961): {1)It is an persistence relationship,.Techniques for expensive, timq- consuming project". .. t testing the degradability, of organic compounds- predict with confidence -a'new waste's and so their persistenoe in soil arilwater-- probable iinpact on certain important' down- are' still haphazard. The enrichment culture ! strewn water uses." And(2) "The technique, in which one seeks microbes that toxicological.phase,octie study is p1rhaps use the compound in.question as sole substrate- - its Mod perplexing aspect'The specialized hence degrades it and even "mineralizes" Services and cost necessary for determining it - -was developed by the Dutch school of the effect of repeated upoSure -to low con- microbiologists. Enrichment cultures are/ centrations of the waste forlong periods of- used routinely by biochemits wishing to. dine would inevitably plaCe this job out of work out the microbial catabolic metabolic reach of most public agencies. Equally pathway for a compound of biochemical discouraging, ,perhaps, is the probability interest.Since the compounds dealt with by that the, toxicological study may take as long' biochemists are of biological origin, micro- as two years." bial degradability 'can be assumed. Still, finding a microbe to degrade a rare biochemical As describtd here, advances in niicrobiology, is not always easy: Dubos, in a classical offer Hopes, of lightening this burden. The hunt for a microbe able to live off the capsules first question is: What kind of microcosm of pneumococci, found the bacterium only, I wilAserve'for toxicological surveys, especially after a long search which ended in a cranberry foripredictinithe pOisOning of biological means bog. Stkh.difficulty in finding mierobes that of waste disposal? The second is: Cap the degrade razbiochernrcale implies an even protozoa of this microcosm predict toxicity greater difficulty in finding microbes that to highei aninials?,, degrade many products of the synthetic , *Iiirect6r,' Allan Hancock Foundation and Head, Bio. Dept.-, Univ. South. Calif. +Haskins Laboratories, 305 E. 43rd St., New York 17, K. y.,; a/id Seton Hall College of 1Viedidine and Dentistry, Jersey City, N.J. Pharmacological work from Haskins LabdrafoPies discussed here vitas assisted by grant R6-9103, Div. of General Medical. Sciences, of the Nationtillnstitutes of Health. Paperresented by Hutner.

7-6 '.99 O 't 1, , Environmental Requirements of Fresh-Water Invertebrates

organic chemicals indu"Stry, since such As-pointed out by our colleague, Dr. L. , compounds may embody4oiochemically rare Provasoli41961), the heterotrophic,, or biochemically nonexistent linkages,. capacities of algae are very imperfectly Intimations of the frnpTaante of inoculum known.This is underscored by recent abound in the literature, e.g., Ross and studies of the green flagellate Chlamydomonas Sheppard (19561 could not at first obtain . mundane. as a dominant in sewage lagoons in phenol oxidizerslfrom ordinary inocula the Imperial Valley of California (Eppley and (presumably soil and water); but manure MaciasR,' 1962); other than that it prefers

A-and a trickling filter from a diemicar_plant______acetate among the few substrates tried; its proved abundant sources of active-bacteria.-\ heterotrophic capacities are unknown. Onelvonders how extensive a study underlies \ More unexpectedly, some- strains of the - the statement mloted by Actixander (1961)tilat photosynthetic bacterium Rhodopseudomona Z "soils treated witI 2,4, 5-T (trichlorophenoxy- palustris use benzoic acid arferobically as acetic acid) still retain the pesticide Meng the reduclant in photosynthesce--(SchF,_Scher, after all vestiges of toxicity due to equivalent and Hiitner, 1962) narrowing the gap between quantities of 2, 4-D have disappeared.',' the photosynthetic pseudomonads and the ubiquitous pseu,domonada-so often represented , What then is a reasonable inoculum for testing among bacteria attacking resistant substrates a compound's susceptibility to microbial (e.g.', hydrocarbons) as well as highly ...attack? The size range is wide: from the _vulnerable subtra s.For the widely traditional crumb or gram of soil or mud to studied, strongly h erotrophic photosynthetic the scow-load ot activated sludge.contributed flagellates Euglena. gracilia'aind E. viridis,./ by New York City to inaugurate the Yonkers, common in sewage, no speceic enrichment ..)_ sewage-disppsal plant. We suggest that to procedure is known, .meaning that their ' strike a practical mean in getting a profile ecological niches are unknon but labotatoiy of Soil, mud, or sludge to bye used as inoculum data provide hints. , ,i the uses of the Winogradsky column should be explored.Directions for Winogradsky column The increasing use of oxidation ponds would and bacteriological enrichments have been in any case urge a greater use of Sealed- detailed (Hutner, 1962) and so only an outline' down ecological systems in which development is given hei.e.The column is prepared by of none of the photosynthesizers present in putting a paste of shredded:paper, CaCO3, the original inoculum was suppressed; and CaS0at ine bottom of a hydrometer jar, Conceivably, some of the rare microbes Ming the4jarwith mud smelling H2S, coveving attacklikg rare substrates- -and such micrObes with a..shallow layer of water, and illuminating are likely to represent a sourcei of degiaders from the side With an'incandescent lamp. In of resistant non-biochemicals--are specialists 2 or 3 weeks sharp zones, appear: a green- in attacking products of photosynthetic and-black anaerobic zone at,the bottom organisms. (a mixture of green photosynth 'tics bacteria along with SO4-reducers, meth roducers, Traditionally, the inoculum for a Winogradsky and the like); over tha0. red zonesk '....sf column is a ma.rincorgbrackish mud (as (predominantly photosinthetic bacteria); New Yorkers we Would be partial to mud from above this garnet or magenta spots or zone flats of the Harlem River).Little is known (pre dominantly non- sulfur ,photo synthetic about the effectiveness as inocula of freshwater bacteria); above this a layer rich in blue- muds or water-logged soils,It would be green algae (the transition to the aerobic valuable to know how complete a cohimn zones); above this aerobic bacteria along with '',coUld develop from material from a trickling green algae, diatoms, other algae, and an filter or an activated-sludge plant.' A practical -assortment of protozoa. This makes an iattue is: Might the poisoning of 'a sewagea 4 excellent siTple classroom experiment to oxidation system be paralleled, by the poisoning defhonstrate the different kinds of photo- of a Winogradsky column}-whgre'the poison syntheticorganisnis, especially the bacterial Was Mixed with the inoculum for the column? forms that are important in photosynthesis Might tile, variously 5t111 photosyntlietic- research and that ordinarily escape notice zones of the column and the.'aerobic population yet are ubiquitous in wet soils and natural on top procride sensitive indicators for the

waters. . 0 Environmental Requirements of Fresh -Water Invertebrates I

,performance of a sewage-oxidation system The.anticonvulsant primidone provides a subjected to chern,ical wastes? clekr indication of how protozoa can be used to pinpoint the location of a metabolic lesion. If a,particular compoundmixed with inoculation Primidpne had been known to cause folic acid-. mud Fr sludge suppressed development of the responsive-anemias.It is therefore ea+to lull Winogradsky pattern, one might assume find that with joint use of a thymine-dependent. that the compound at the test concentration Escherichia coli and the flagellate Crithidia was poisonous and persisteirt.BiologicalNv/ fasciculata reversal of growth inhibition by ,destruction of such poisons, if at all possible, folic acid and related compounds permitted might demand a long hunt for Suitable micro- the charting of interferences with the inter- organisms, tHexi buildup of the culture to a connected folic acid, biopterin, and'DNA pActical scale.This might best be done with function (Baker et al., 1962), which amply illuminateti shake or aerated' cultures, with accounted for the megaloblasic anemias. the inoculg coming from a variety of environ- Lactiohacithis casei, a bacterium much used ments.. Optimism that microbes can be found in chemotherapeutic research, was unaffected capable Of breaking almost all the linkages of b§ the drug. organic chemistry is fostered by the study of antibiotics, which include a wealth of In another instance, Where the mode pf action previously "unphysiological" linkages--azo of the drug in higher animals was Unknown, compounds, oximes, N-oxides, aliphatic and growth inhibition property of the anticancer . aromatic nitro and halogen compounds, and compound 1-aminocyclopentane-l-carboxylic strang9 heterocyclic ring systems. Some acid was. reyersed for Ochromonas danica natural heterocycles, e. g,, pulcherriminic by L-alanine and glycine, as wag-th-e'rnhibition acid and 2-n-nony1-4-hydroxyquinoline, have property of 1-amino-3-methyl-cyclohexane- a disquieting resemblancelto the potent 1-carboxylic acid by L-leucine (Aaronson carcinogen 4-nitroquinolineN-oxide. and Bensky, 1962). @ Growth irklvdbition of Euglena bythe, potent PROTOZOA AS TOXICOLOGICAL TOOLS carcinogen 4-nitroquinoline N-oxide was annulled by a combination of tryptophan,. A difficult pfoblerri is one mentioned earlier: tyrosine, nicotinic acid, phenylalanine, persistence joined with lr-grade toxicity to uracil (Zahalsky et al, ; .,1962) and, in more higher animals. Recent developments in the recent experiments, the vitamin K relative use of protozoa as pharmacological tools show phthiocol.These N-oxides are of interest that protozoa can serve as sensitive detectors because of recentworkindicating that perhaps of metabolic lesions ("side actions " ?) of a the main way in which:the body converts such wide assortment of "safe" drugs.The list compounds as the amino hydrocarbons to the includes the "antich,olesterol" triparanol actual carcinogens m4y be by an initial (MER/29). TriParanol toxicity manifested byoxylation of the nitrogen, e. g., work . itself with severalprotozoa, including b Miller et al., (19610. Whether the Ochromonas danica (Aaronson et al., 1962) pe oxides in photo - chemical smogs of the and Tetral1mm (Holz et al., 1962). Los geles type action hydrocarbons to

Tkparanolwas. not acting simply as an anti- produ e carcinogen' N-oxides is entirely cholesterol for its obvious toxicity to protozoa unkno n.Leighton (961) ,lists, an array of was annulled by fatty,acids as well as' by peroxy reactions pro uced by sunlight in sterols. The connection between the protozoan polluted ail.. results and the-ttside actions" of triparanol-- baldness, impotence, and cataracts- -are of Our aforementioned experience with primidone, course Unclear, but protozoal toxicity might a ketonic, heterocycle, leus to test the serve as an initial warning That it might not sedative thalidomide.Itas toxic for ,be as harmless as assumed from short-term Ochromonas danica O. m.emensis and experiments with higher animals. Tetrahymenapyriformis; this toxicity was

A ry 101

. 7-8 Environmental Requirements of Fresh-Water Invertebrtes 0

annulled by nicotinicacid (or nico inam de) Since the embryos appear to lack the or vitamin K (menadione) (Frank et al, 1963). detoxication mechanisms of the adult animal We do not know whether a similar protection (Brodie, 1962), toxicity for protozoa (which _- could have been conferred on human embryos presumably lack these detoxication or polynedritis in the adult. mechanisms) should be compared with that fOr the embryo, not the adult, as emphasized Many widely used herbicides of the dinitrophenol by the thalidomide disaster. type are powttrful poisons for higher animals. We do not know how sensitive protdzoa would There 'are further limitations on the use of be Rix- detecting their persigtence. However, microbes as detectors of toxicity.High- since somewhat similar thyro-,active com- molecular toxins seem inert to microbes, pounds can be Sensitively detected by their- and antihormOnes (with the exception of exaggeration of the B12 requirement of anti-thyroid compounds) are generally inert. Ochromonas malhamensis (Baker of al, 1961), 'The main usefulness of microbial indices bf this flagellate might be a useful test object toxicity would-appear, then, to be for detecting for dinitrophenols and congeners. low-molecular poisons acting oncellilar targets rather than on cell` systOms and The PararneCium (ai-id perhaps too the. , organs. These are precisely the poisons Tetrahymena) test for polynuclear benzenoid liketo'put out of: business a pollution- carcinogens has remarkable sensitivity and contr 1 installation primarily dependent on spedificite (Epstein and Btirroughs, 1962; Hull, microbial activity. 1962): This test depends on the carcinogen- sensitized phot9dynamic destruction of paramecia by ultraviolet light.This test is REFERENCES approaching practicality for air, and there is fr .noTeason to suppose it cannot be applied to Aaronson, S. and Bens*, B. 1962. ;benzene extracts of foodstuffs and water. _protozoological: studies of the cellular 'action of drugs.I.Effect of 1-aminocyclopentane-1- carboxylic acid CONCLUSIONS and 1-4amino -3 -methylcyclo-hexanel-1 carboxylic acid on the phytoflagellate We have suggested here new procedures for Ochromonas danica.Biochem.` Pharmacol. examining the intrinsic toxicity-persistence 11:983-6.

relationship, using as test organisms the, 4. protists represented conspicuously in a Aaronson; S., Bensky, B., Shifrine, M. and Winogradsky column. The new field of Baker, -H. 1962.Effect of hypotholes- micro - toxicology is virtually undeveloped. teremic agents on protozoa. Proc. Soc. Th4rgent need for detecting chronic, low- Exptl. Biol. Med. 109:130=2. grade toxicities is evident from many sides. I This is not the place for a detailed discussion..,AleXander, M.1961."Introduction to Soil of the medical implications of phis area of Microbiologir", John Wiley & Sons, N. Y..' research, but it should be eplphasiied that (see p. 240). chronic toxicities and carcinogenesis are' related.Conversely, Umezawa (1961) has Bhker, H., Frank, O., Hutrier, S. H., remarked that most antitumor subStance's Aaronson, S., Ziffer, H. and Sobotka,. 1t. have chronic toxicities and that elaborate 1962.Lesions in foljc acid ,metabolism testing procedures for toxicity are required induced by sprimidone'Ekperientia tq, fix the daily,tolerable dose; appa'rently this 18:224-6. problem is a central theme in medical as well as pollution research.Inhibition of girth of Baker, H., Frank, O., Pasher, ,Ziffer, H., anarray of protozoa is now in practiCal use Hutner, S.H. and Sobotka, H, 1961. Usi a means of detecting anticancer substances Growth inhibition of microorganisms by in antibiotic beers (Johnson et al, 1962). thyroid hormones. Proc. Soc. Exptl. . . Bio. Med. 107:965-8.

1 f. 102. 7-0 I. Enviromtental Requirements of Fresh-Water Invertebrates

5 Berger, B.B. +1961. Research needs inter Johnson, I. S. , Simpson, P..J, and Cline, 'J. C. quality conservation.In "Algae and 1962.Comparative studies Aith 'MetriSpOlitan Wastes", Trans. 1960 chemotherapeutic agents in biologically Seminar, Robt. A. 'itrftSanitazy Eng. diverse in vitro cell systems. Cancer Ceriter, Cincinnati 26, Ohio, p. 156-9. Research 22:617-26.

Brodie, B. D,1102. Drug Metabolism- Leighton, P.A.1961."Photochemistry of subcellular mechanisms. In "Enzymes Air Pollution ", Academic Press, N.Y. and Drug Action", J. L. Mongar and A. V. S. de Reuck, eds., Ciba Foundation Miller, J. A.,Wyatt, C. S. ,Miller, E. C. Symposium, J. & A. Churchill Ltd., and Hartman, -H. A,.1961.The p. 317-40. N-hydroxylaiion of 4- acetylamino- bifheyl by the rat and dog and the Eppley; .R. W. and MaciasR, F. M.19.62. s g carcinogenicity of N-hydroxy-4: Rapid growth of sewage lagoon cety ,sminobiphenyl in the rat,Cancer Chlamydomonas with acetate.Physiol. Research 21:1465-73. Pls.ntarum 15:72-9. Provasoll, L.1961.Micronutrients f4md Epstein, S. S. and Burroughs., M. ' 1962. heterotrophy as possible factors in bloom Some factors influencing the photodynamic production in natural waters.In "Algae1 response of Paramecium caudatum to an Metropolitan. Wastes", Trans. 1960 3,4-benzypyrene. Nature 193:337-8. Seminar, .Robt. A. Taft Sanitary Eng. Center, 'Cincinnati 26; Ohio, p. 48-56. Frank, 0.4 Baker, H., Ziffer, H.,- --- Aaronson, S., and Hutner, H. 1963. .Ross, W. K. and Sheppard, A . A.1956. Metabolic deficiencies in protozoa induced Biological oxidation of petroleum phenolic b-§ Thalidomide. Science 139:110-1: wastewaters. In "Biological Treatment of Sewage-4nd IndUstrial Wastes", J. Hawkes, IL A.1960.Ecology of activated McCabe and W. W. Eckenfelder, Jr., eds., sludge and bacteria beds.In "Waste . 1:376=8. Reinhold Publ.. Co., N. Y.. .) Treatment ", P. C. G. Isaac, ed., Pergamon, Nr. Y., Oxford, etc., p.'52 -97; Scher, S., Scher, B. and hutner, S. H. 1963. discussion p. 97-8. Notes on the natural history of , RhodOpseudomonas galustris.In Holz, G. . Jr., Erwin, J., Rosenblum, LT. "Symposium on Marine Microbiology", and Aarson, S.1962.Triparanol ' ed.C.H. Oppenheimer, C: C. Thomas, Inhibition of Tetrahymena and its prevention Springfield, Ill., p. '580-7. of lipids. Arch. Biochem.. Biophys. 98:312-22. Umezawa, M.1961.Test methods for -antitumor substances.Sci. Repts. 1st. Hull, R.W.1962. Using the "Paramecium Super-.- Sanita1:437-38 4. assay' to screen carcinogenic hydrocarbons. J. Protozool. (Suppl) 9:18. Zahalsky,. A. C., -Keane, K., 'Hutner', H., Kittrell, M, and Amsterdam, D.I962. '- Hutner; S. H. .1962.Nutrition'of protists. Protozoan response to anticarCinogenic In "This is Life: Essays in Modern heterocyclic N-oxides: toxicity and Biology", W. I. Johnson and W, C. Steere, temporary bleaching.J. Protozool. eds., Holt, Rinehart and Winston, N.Y. (Suppl.) 9:42. P. 109-37. - Hynes, H. B. N.1960. "Biological Problems in Water Pollution" The Biology of 13olltited (Third Seminar 1962) U.S. Public Health Waters.Liverpool Univ. Press. Service-Pub. No. 999-WP-25, Cincinnati, OH .1965, II

.5 %.

1-10 110 3 FILAMENTOUS BACTERIA

1 -\ I /INTRODUCTION Transverse separations within the sheath indicate that a row of 'cells is included in There are a number of types of filamentous one sheath.The sheath may be clearly bacteria that occur in the aquatic environment. visible or so slight that only special-staining They include the sheathed sulfur and iron will indicate that it is present. bacteria such as B_ esgiatoa, Crenothrix and Sphaerotilus, the actinomycetes-which are The organism grovis as a ii,hite slimy or. unicellular microorganisms that form chains felted cover on the, surface of various objects of cells with special branchings, and undergoing decompdsition or on the surface Gallionellae a unicellular organisrli that of stagnant areas of a stream receiving secretes a long twisted ribbon-like stalk. sewage.It has also been observed.on the These filamentous forms have at times base of a trickling filter and in contact created serious problems in rivers, aerators. reservoirs, wells, and water distribution systems. It is most commonly found in sulfur strings or polluted waters where H2S is present. 3eggiatoa is distinguished by its ability to IIBEGGIATOA deposit sulfur within its cells; the sulfur deposits appear as large retractile globules. Beggiatoa is a sheathed bacterium that grows (Figure 2) as a long filamentous form. The flexible filaments may beias-large as 25 microns wide and 100 microns long: (Figure 1) Figure 2 Filaments-of Beggiatoa contaihing granules of sulphur.

t When H2S is no longer present in the environ- ment, the sulfur deposits disappear. Dr. Pringsheim of Germany has recently proved that the organisin can groiv as a true Stitotroph obtaining all its energy from the oxidation of H 2S and using.this energy to fix , _ CO,,' into alkmaterial.It can also use . certain t Materials.if they are present Begglatoa albk along with.he H2S. ,1 151.1 X Faust and k\rolfe, and Scotten and Stokes have grownpe organism in pure culture. in up to 1.000pi, this country'... Beigiatoa exhibits a motility that is quite different,from the typical flagellated motility of most bacteria; the filaments have a flexible gliding motion.

,=,13.A.: 8a. 5.80 8-1 104 Filamentous Bacteria

The only major nuisanceeffect of Beggiatoa known has been overgrowth on.trickling filters receiving waste waters rich in H2S. The Figure 4 normalinicroflora of the filterwas suppressed and the filter failed to give good treatment. Egg albumin isolation plate. Redi Oval of the H2S from the water by blbwing 'A' an actinomycete colony. air through the water before it reached the and 'B'.a bacterial colony filters caused the slow decline of the Beggiatoa anda recovery of the normal, microflora.Beggiatoa usually indicates polluted conditions with the presence of H2S rather than being a direct nuisance. 1 IIIA CTINOMYCETES! AND EARTHY ODORS IN WATER

Actinomycetes are unicellular microorganisms, `11 lsmicron in diameter, filamentous,non- 4viof sheathed, branching monopodially,and Appearance: reproduced by fission or by meanstof special coniqia.... (Figure 3) duII and powdery/smooth. and MUcOld

1, These organisMs may be consideredas a group intermediate between the fungi and the bacteria. They require organic matter for growth but can use a wide variety of substances and are widely distributed. Actinomycetes have been implicated as the cause of earthy-odors in some drinlqg waters (Romano and Safferman, Sily and. Roach) and in earthy smelling substance has been isolated from several members of the Figure 3 Filamente. of Actlnomycetes. group by Gerber and Lechevalier.Safferman and Morris have reported on a method for the - "Isolation and Enumeration of Actinomycetes Related toyWateeSupplies. " But the actino- Their filamentous habit and method of mycetes are primarily soilinici-oorganisms sporulation is reminiscent of fungi.However, and often grow in field's or on the banks ofa -their size, chemical cbrigiosition,4and other. river or lake used for the water supply. characteristics are more similar to,bacteria. Although residual. chlorination will kill the (Figure 4) organisms in the treatment plant or distribqion

105

o

- . r

. Filamentous 'Bacteria

system, the odors often are present-before the water enters the plant. Use of perman- ganate oxidation and activated carbon filters shave been most successful of the methods tried to rJmove the odors from the water. control procedures to prevent the odorous ma rial from being washed into the water sup151 rains or to prevent possible develop- men of t e actinomycetes in water rich in deca ing organic matter is still'needed.

IV FILAMENTOUS IRON BACTERIA Figure.5 Crenothrix polyspora The filamentous iron bacteria of the Sehaerotilue- Lept othrix group, Crenothrix, cells are very variable in and Gallionella have the ability, to either sizettom small cocci or oxidize manganous or ferrous ions to innic polyspores ta cells 3x12/.4 or ferricsalts-or are able to precipitates of these compounds within the sheaths of the organisms.Extensive growths or accumulations of the empty, metallic encrusted sheaths devoid of cells, have created much trouble in well's or water dis- tribution systems. Pumps and back-surge valves have been clogged with masses of material, taste and odor problems have occurred, and rust colored masses of material have spoiled products in contact with water. Crenothrix polyspora has only been examined under the microscope as we have never been able to grow it in the laboratory. The orga- nism is 'easily recognized by its special morphology.Dr. Wolfe of-the University-of Illinois has published photomicrographs of the organism. (Figure 5) Orgapisms of the S haerotilus-Leptothrix group have been extensivel studied by many investigators (Dondero .al., Dondero, Stokes,- Weitz and 1,0.ey, Mulder and van ,Veen, and Amberg an Cormack.) Under jdiffereht, ,environmental conditions the mok- .phological se- ance of the organism varies, , -The usual farm f in polluted streams or bulke vated sludge is Sphaerotilus natans; Figure 6 (Flyre 6) Sphaerotllus natans

3-8 x 1.21.8fp

. cells

..10G. 8-3 e Filamentous Bacteria

This is a sheathed bacterium consisting of , long, unbranched filaments', .whereby individual rod- shaped bacterial cells are enclosed in a linear order within the sheath. The individual ,cells. are .31 microns long and 1.2-1.8 microns wide.Sphaerotilus grows in great masses; at times in streams or rivers that receive wastes from pulp mills,' sugar refineries; distilleries, slaughterhouses, orilk prOoessing plants.In these conditions, it apars as large masses or tufts attached to rocks, twigs, or other projections and -the masses may vary In color from light grey to ,reddish brOwn.In sortie rivers large tnasses of , Sphaerotilus break loose and clog watet intake pipes or foul fishing nets. When the Figure 7 cells 'die, taste and odor proble4 may also Galionella furrugfnea occur in the wa , OA X PI -1.1p Amberg, Cormack, and Rivers and McKean Cells have reported on methods to try to limit 'the development of Sphaerotilus in rivers by intermittant discharge of wastes. Adequate control will probably only be achieved once .the wastes are treated before discharge to REFERE ES such an extent that the growth of Sphaerotilus is no longer favored in the river. Sphaerotilus Beggiatoi. grows welt at cool temperatures-and slightly low DO levels in streams receiving these r- Faust, L. and Wolfe; R. $.Enrichment wastes and domestic sewage. Growth is slow .*; and Cultivation of Beggiatoa Alba. where the only nitrogen present is inorganic Jour. Bact., 81:99-106. 1961. nitrogen; peptones and proteins are utilized 2 Scotterrr-H. L. and Stokes, J. L. preferentially. Isdlation and. Propertifs of Beiatoa. Arch Fur. Microbiol.42:3537368. Gallionella is an iron bacterium which appears .. as a kidney-shaped cell with a 'twisted ribbon- 1962. like stalk emanating from the concavity of the cell.Gallionella obtains its energrby 3 -Kowallitk, U. and Pringsheim, .B. G. oxidizing ferrous ironlnerric iron and uses The Oxidation of Hydrogen Sulfide by only CO2 and inorg)cnic salts to forth all- of a Beggiatoa: Amer. Jour. of Botany: 53:801-805. 1966, . the. cell material; it is an antotroph.Large. masses of Gallionella may clause problems in wells or accumulate in low-flow how= Actinomycetes. and Earthy Odors pressure water mains. Super chlorination (up..to-p0, ppm of sodium hypochlorite for ,.4Silvey, J.,K. G: et al. A ctinomycetes and 48 hours) followed by flushingill often Common Tastes and Odors. JAWWA, rer hove tile masses of growth and'periodic 42:1018-1026. 1950. treatmen will preirentthe nuislince,effeCts of the extensive masses1/4& Gallionella. 5Safferman, R. 5; and Morris, M. E% A Method for the Isolation and, (Figure 7) Enunieration of ActinomyCetes Related 4 to Water Supplies.Robert A. Taft Sanitary Engineering Center Tech. Report W-62-10. 1962.

1Q7 00 Filamentous Bacteria

< .6Gerber, N. N. and Lebhevaliert, H.A. 15Donlero, N. C.Sphaeretilust Its Natiire _j Geosmin, an Earthy-Smelling Substance and Economic Signifirianee. Advances Isolated from Actinomycetee. Appl. Appl. Microbiol. 3:717-107. 1961. Microbiol.13:935-938. 1965. , . 16 ° Mulder', E. G. akid van Veen, W. L., Filamektous Iron Bacteria Investigations on the SphaerotilUs ' Leptothrix Group. Antonie van 7Wolfe, R. S.Cultivation, Morphology, and Leewenhoek29:121-153. 963. Classification of the Iron Bacteria.: r.- kip). . JAWWA. 50 :1241 -1249. 1958. 17 Amberg, H.R. and Cormac ,J. F._ Factors Affecting Slime Growth in .E1 Kucera, S. and Wolfe, R. S.A Selective the Lowers Columbia 'River and Enrichment Method foi Gallionella Evaluation of Some Possible ContrOl. ferruginea.Jour. Bacteriol.74 :344- Mea'sures. Pulp and Paper Mag. of 349. 1957. Canada.61.;T70:-T80.1960.

9Wolfe, R. S.Observations and Studies 18 Amberg, H.R.,Cormabk, J. F. and of Crenothrix polyspora. JAWWA, Rivers, M. R.Slifne. Growth Control 52:915-918. 1960. by Intermittant Discharge of Spent ^1. Sulfite Liquor..Tappi.45':770-779. 10 Wolfe, R. S.,Microbibl.Concentration 1962. of Iron and Manganese in Water with . Low Concentrations of theiefrElements. 19 McKeown, 4. J. -The Sonttol of JpWAr. 521335=1337. 1860. Sphaerotii.us natans. Watchr and Wastes, 47:137 19-22 and 11Stokes,c4.,L.Studies on the Filamentous 8:(4)30-33'. 1963. Sheathed Iron Baetiriurn Sphaferotilus natans. Jour; Bacteriol.67:278-291. -20CtiPtis,,LJ. C:, Sco,a3e Fun-,:us; 41954. : Nature.and Effects. Wat. Res. .3:289.4311. 1969, 1Z, 'Waltz, S. and Lackey; J. B.Kerphologicfti and Biochemical Siudies on the _ 21Lechevalier, Hubert A., P:ctinomycefes OrIganismSphaerotilus natans. arf. of Sewage 'treatment Plants.Envir. Joy. Fla. Acad. -Sci. 24(4)13 Prot. Tech.Series USEPA, 1958. '600/r-75-031.1975. " o I ° 13'Dondero, N.C., Philips, R.A. anr . This outline was prepared by R. F. 'Lewis Henkelkian, H.Isolatidn and Bacteriologist, Advanc aste Treatment Preservation of Cultures of Sohaerotill ResearchLaboratory, NER,C, 'USEPA, Appl. 9:2191-227% 1961. Ciricinniliz-OhiO 45268. re- 14 Eikelboom D. H. ,Identification-QS__ Descriptors: Aquatic Bacteria, Sphs.erotilusl Filamentous Organisms in Bulking Actinomycetes, Nocardia Activated Sludge.- Frog. Water

Te0.,h. 8(8):155-161.' . 4 Pergamon Press. I

FUNGI AND TUE SEWAGE FUNGUS" COMMUNITY

I INTRODUCTION \st IIIECOLOGY

Descri tion A Distribution Fungi a e heterotrophicachylorophyllous plant-like organiSms which possess-true Fungi are ubiquitous in nature and mem- nuclei with nuclear membranes andnu- bers of all classes may occur in large cleoli.Dependent upon the species and numb aquatic habitats.Sparrow in some instances the environmental (1968) hasriefly reviewed the ecology conditions, the body of the fungus, the of fungi in reshwaters with particular thallus, varies from a microscopic emphasis on the zoosporic phycomycetes. single cell` to an extensive plasmodium The occurrence a.p.d ecology of fungi in or mycelium. Numerous forms produce r 'marine and estuarine waters hasbeen j. . ,macroscopicfruiting bodies. examined recently by a number of in- vestigators (Johnson and Sparrow, 1961; B Life Cycle Johnson, 1968; Myers,1968; van Uden and Fell, 1968). Thelife cycles of fungi vary from simple to complex and may include sexual and asexual stages with varying spore types B -Relation to Pollution as the reproductive units'. Wm; Br'cllxe Cooke,in a series of in; C ClairSifica.tion- vestiions (Cooke,1965), has estab- ,lished that fungi other than phycomycetes Traditiorially, true fungi are classified _occur in high numbers 'in sewage and within the Division Etnnycotina of the pollutedAters.His reports on organic Phylum Mycota of the plant kingdom. pollution of streams (eooke, 1961; 1967) Some authorities consider the fungi an show that the variety of the Deuteromy- essentially monophyletic group distinct cete flora is decreased at the .immediate ( from the classical plant and animal sites a pollution, but dramatically in- kingdoms. creaseddownstream from these regions.

U AC' IVITY Yeasts,- in particular, have been found innarge numbers in organically enriched Igen eral, -fungi possess:btoad enzymatic waters (Cooke, et al., 1960;- Cooke and. apacities; Various species are able- to .Matsuura, 1963; 'Cooke, 1965b; Ahearn, a.ctiOely, degrade such compounds as. et al.,1968). > Centain yeasts are of complex polysaccharides (e. g., cellulose, special interest. due-to their potential° chitin, and glycogen), proteins (casein, use as "indicator" organisms and their albumin, keratin), hydrocarbons (kerosene) ability to degrade or utilize proteins, and pesticides. 'Most species, possess an various hydrocarbons,. straight and oxidative or microaerophilicmetahq4111, branch, chained alkyl-benzene sulfonates, bit anaerobic catabolism is not uncommon. Tats, .metaphosPhates, and wood sugars. A fespeCie.s.iihow anaerobic metabolism and growth.

109 BI. FU. 6a. 5. 80 9-1* Fungi

C "Sewage Fungus" Community (Plate. I) 2 Leom. itus lacteus also ,produces ex ensi, e slimes and fouling flocs , A few microorganisms have Tong been Ines water's. This speciesiorrns termed "sewage fungi. "The mbst thal ified by regular constrictions. common microorganisms includsidin this group are the iron bcterk aMorphology Sphaerotilus natans and the phycomy- cete- Leptomitus' lacteus. Cellulin plugs may be present * \ near the constrictions and there 1 Sphaerotilus natans is nota fungus;' may be nurrterous granules in rather it'is a sheath bacteritlyn of the cytoplasm. The basal cell the order -chlamydobacterialts. of the thallus may possess This polymorphic bactersium occurs rhizoids. .clommonly in organically enriched' streams where it may produce b Reproduction extensive slimes. The segments delimited by the.. aMorphology partial constrictions are con- verted basipetally to sporangia. Characteristically, S. natans The zoospo'res are diplanetic forms chains of rod shaped (i.e., dimorphic) and each o cells (1. - 2.04 x 2.5 - 174). possesses one whiplash and one within 'a clear sheath or tri- tinsel flagelluth.No sexual clome composed of a protein- stage has been demonstrated poly saccharidae-lipid complex. for this species. The rod cells are frequently motile upon release from the .cDistribution sheath; the flagella are lophO- trichous.Occasionally two For further information on the row.s-of cells may be present distribution and systematics in a single sheath. Single tri- of L. lacteus see Sparrow (1960), chomes may be several mm Yerkes I1h66) and Emerson and in lerigth and bent at various Weston (1967).Both S. natans angles. Empty sheaths, ap- and L. lacteus appear to thrive - pearirig like thin cellophane in organically enriched cold straws, may be present. waters (59-22°C) and both seem

- $ incapable of extensive growth at 13'Attached growths temperatures of about 30°C. The trichomes are cemented d Gross morphology at one end to solid substrata such as stone or metal, and Their metabolism is oxidative their cross attachment and and growth of both specie ay bending gives a .superficial appear as reddish brown flocs similarity to true fungal hYphae. or stringy.slimes of 30 cm or The ability to attach firmly to more in length. solid substrates gives S. natans a selective advantage in the eNUtritive requirements population of flowing streams. Sphaerotilus natans is able to For more thorough reviews of utilize a wide variety of organic S. natans see Prigisheim (1949) compounds, whereas L. lacteus and Stokes (1954). does not assimilate simple

4.. Fungi .

PLATE, I

"SEWAGE FUNGUS" COMMUNITY OR "SLIME GROWTHS" (Attached "filamentous" and slime growths)

14. 3 Sphaetotilus natans

9

Begeatoa alba BACTERIA

.

:

7 Fusarium aqueductum Leptomitus lacteus

1--I 44- FUNGI

ro 9 OpercUlaria CarchesiuM PROTOZOA Fungi

.° PLATE II REPRESENTATIVE FUNGI

Figure Fusariumaquaeductuum (Radlmacher and Rabenhorst) Saccardo Figure Microconidia (A) produced Leptomitus lacteus (R from phialides as in Cephalo- Agardh IPOriUM. remaining in slime balls. Mac:soma& (B), with Cells of theqy hae show. onetoseveralcrosswalls, ing_constrictionVivithcellulm produced from collared phial. plugs.In ,one/cell large zoo. ides. Drawn from culture. spores hive been deliraid. .Redrawn from Coker, 1

Figure.3 Figure 4).- Geouichum candulum Zoophagus in:idiom Link ex Persoon Sommerstorff Mycelium with short cells Mycelium with hyphal pegs and arthrospores. Youngby (A) on which rotiferswill pha (A) ; and mature arthro- become impaled; gemmae (B) spores (B). Drawn from cul produced as conidis on short ture. hyphal branches; and rotifer impaled on hyphal peg (C) fromwhich hyphae have grown into the rotifer whose thell will be discarded after thecontents areduisumed. Drawn from culture.

Figure S nichlya americana Humphrey Oeogonium with three oo- spores (A); young rmosier- r wrier/dub rolm M11111111111 121011M with delimited Zoo- spores (R)and zeosporangia (C) with released zoospores that remain encysted in dus- ters at the month of the dis- charge tube. Drawn from ma- ture-

A

FIGUR);7Ilapinsporideum ccWale. spots;

Figures 1 th;ough 5 from Cpoke;FigUres 6 and 7 from Galtsoff. Fungi

sugars and grows most lu,iniriantly:in IV CLASSIFICATION the pres1,-nce of organio nitrogenous 4,. wastes. In recent classification schemes, classes of fungi are distinguished primarily onthe 3 Ecological roles basis of the morphology of the sexual and zoosporic stages.In practical schematics, ' Although the "sewage fUgg?"_olt however, numerous fungi do not dernonstVe occasion attain visdally notic.eable these stas, -.Classification must therefore concentrations,,the less obitious be based nlhe sum total of the morphological populations. of cfeuteromycetes may arid/or physiological characteristics. The be more important in the ecology of extensive review by Cooke (1963) on methods the aquatic habitat.Investigations of of isolation and classification of fungi from the past decade indicate that numerous sewage and polluted waters precludes the fungi and of primary importance in the need herein of extensive keys and species mineralization of organic wastes; the -illustrations. A brief synopsis key of the overall significance and exact roles of fungi adapted in part from Alexopholous fungi in this process are yet to be (1962) j.s presented on the following pages. established. D Predacious Fungi 1,58 1 Zoophagus insidians (Plate H, Figure 4) has been observed . toimpair functioning of laboratory activated sludge units (see Cooke and 'Ludzack). ,

2 Arthrobotrys is usually found along with Zoophagus in laboratory activated sludge units.This fungus is pretiacioua This outline was prepared by Dr.DontldG.' , uponnematode's.Loops rather than Ahearn, Professor of Biology, Georgia State "pegs" are used in snaring nematodes. College; 'Atlanta, Georgia 30303. Aquatic Fungi Des Cr iPtbr41,

,

PLATE II (Figure 4) -1

9-5 . 113 'Fungi

.KEY TO THE MAJOR TAXA OF FUNGI .A.

Definite cell walls lacking. somatic phase a free li;)ingPlasmodium : :$41=n Sub-phylum Myxomycotina. (true slimecnolcfs)..Class Myxomycete' Cell walls usuallyiwell defined, somatic phase not a free-living Plasvmodikm. a (true fungi) Sub-phylum Eumycotina ...... 2 t.

2 Hyphal filaments usually coenoctytic, rarely septate, sex sElls when pciesent farming oospores or zygospores, aquatic species propagating asexually by zoospores, terrestrial species by zoospores, sporangiospores conidia or conidia-like sporangia .VPhycomycetes".: .3 The phycomycetes, are generally considered to include the most primitive of the true fungi.As a whole, they encompass S wide diversity of forms with some ,showing relation-

t ships to the flagellates, while others closely resemble colorless algae, and still others are true molds. The Vegetative body (thallus) maybe non-specialized and entirely con- verted into a reproductive. organ (holocarpic), or it may bear tapering rhizoids, or be mycelial and v?ry extensive. The outstanding characteristics of the thallus is a tendency to be nonseptate andin most groups, multinuNiate; cross walls are laid down in vigorously growing material only to delimit the rAfeorductive organs. The spore unit of nonsexual re- production is borne in a siporangium, and, in aquatic and semiaquatic orders, is provided with a single postdrioror anterior flagellum or two laterally attached ones.Sexual activity in the phycomycetes characteristically results in the formation of resting spores.

2.t1'1 Hyphal filaments when present sefitate. without zoospores. with or without sporangia. usually with conida; sexual reproduction absent pr culminating in the formation of asci or basiaia 8

3 (2) Flagellated cells characteristically produced 4 3' Flagellated cells lacking or rarely produced .7

4 (3) Motile cells uniflagellate 5 6 . 4'. Motile cells biflagellate . 5 (4) Zoospores posteriorly uniflagellate, formed inside the sporangium class.Chytridiomycetes The Chytridiomycetesoroduce asexual zoospores with a single posterior whiplash flagellum.The thallus is highly variable, the Most primitive forms are unicellular and holocarpic and in their early stages of development are plasmodial (lack cell walls). more advanced forms develop rhizoids and with further evollikonary progress develop mycelium The principle chemical 'component of the cell wall is chitin, but cellulose is also present. Chytrids are typically an atic organisms but may be found in other habitats. Some species are re chitinolytic and/or k ratinolytic.Chytrids may be isolated from nature by baiting (et. g. hemp seeds or-pine pollen) ytrids occur both in marine and fresh water habitats and are of some economic importance due to their parasitism of algae and animals.The genus Dermocystidium may be provisionally grouped with the chytrids.Species Of this genus cause serious epidemics of oysters and marine and freshzaterfish,

5' Zoospores anteriorly uniflage4late, formed inside or outside the spbrangium. class Hyphochyt r iciiomycetes

These fungi are. aquatic-(fresh water or marine) Chytfid-like force whbtZe motile cells posses; a single anterior flagellum of thetinseThYpe (feather-like).They are parasitic on algae and fungi or may be saprobic.Cell walls contain chitin with some species also demon- strating cellulosecontent.Little information is available on the biology of this class and at present it is limited to less than 20 species. a ', t!16. . ,re ' ' 6 (4!) Flagella nearly equal, poe whiplash the other tinsel claws...... . .a.,, . ... A number of representatives of the ; cAve been shown tp have cellulosic cell * walls.rt The mycelium is coenocytic, branched and well doieloped in most cases,The sexual process results in the formation of a restingspot4 of the.00gamous type, i:e., a type of fertilization.in which two heterogametangia cqme fri contact'and fuse their contents through..., a po4s or tube.Tke-thalli in this class r e from unicellular to profusely branched filamentous types. Most forms arc eucarpizoospores arc produced throughput the class except in the more highly advanced speciesCertain species are of economic ithportance due e, 'ec: their destruction of food crops (potatoes and grapes) while others cause serious diseases of a fish (e. g. Saprolegina parasitical. Members of the family Saprolegniaceae arethe common

6 4

Fungi

water moldsand are among the most ubiquitous fungi in nature, The order Lagenidta es includes only a few species which are parasitic on algae, small'inimal, and other aqua is life.The somatic structures of this taxon are holocarpic and endobiotic.The sewage f ngi4 are classified in'the order Leptomitales.Fungi of this order are ch:aracte rued by the formation of retractile constrictions.cellulin plugs ' occur throughout the thalli or, at 1ast, at, the bases o( hyphae or to cut off reproductive structures.Leptomitus lacteus may produce rather extensive fouling floes or slimes in organically enriched waters.

II Flagella,of unequal size, both whiplash ...... class....Pfasmodiophoromycetes Members of this cla e obligate endoparasites of vascular plants, algae. and fungi The thalls consists of a pla chum which develops within the host cells.Nuclear division at some stages of the life cycle of a type found in no other fungi but known to occur in protozoa.Zoosporangia which ari e directly from the plasmodium bear zoospores with two" unequal anterior falgella.The cell waifs of thefungi apparently lack cellulose.

7 13') Mainly .sapro,bic. sex cell when present a zygospore...... class . 7%vomycetes This class has well developed mycelium with septa developed in portions of the older hyphae, actively growing hyphae are normally non-septate.The asexual spore,. at E non-motile sporangioipores (aplanospore,$).Such spores lack flagella and are usually aerialy disseminated. Sexual reproduction is initiated by the fusion of two gametangia with resultant formation of a thick-walled, resting spore, the zmospore.In the more advanced species. the sporangia or the sporangiospores are conidia -like Many of the , Zygomycetes are of economic importance due to their ability to synthesize commercially valuable organic acids and alcofiols. to transform steroids such as cortisone, and to parasitize and destroy food crops. A few species are capable of causing disease in man avid animals (zygomycosis) Obligate commensals of arthropods, zygospores usually lacking chss ...Trichomycetes The Trichomycetes are an ill-studied group of fudgi which appeao be obligate commensals of arthropods. The trichomyceteare associated with a wide variety of insecta. 2111iplopods, and crirstacea of terrestrial and aquitic (fresh and marine) habitats.None of the members of this class have been cultured in vitro for continued periods of times with any success.Asexual reproduction is by means of sporangiosporesZygospures have been observed in species of several orders.

8.(2'1 Sexual spores borne in asci class ..fikscOmycetes In the Ascomycetes the products'of meiosis; 'the ascospores, are borree in sac .like structures termed asci.The ascus usually contains eight ascospores. but the number produced may vary,with the species or strain.Most sp6cles produce extensive sepiate mycelium.This large class is divided into two subclasses on the presence or absence of an.ascocarp. The Hemiasconcetidae lack arLascocarp and do not produce ascogenous hy'phae; this subclass includes the true yeasts. The Euascomycetidae usually are divided into'three series (Plectomycetes, Pyrenomycetes, and Discomycetes) on the basis of ascocarp structure.

8' Sexual spores borne on basidia . class...4113asidiomycetes The Basidiomycetes generally are considered the most highly evolved of the fungi. Karyogamy and meiosis occur in the basidium which bears sexual exogenous spores, basidiospores. The mushrooms. toadstools, rusts, and smuts are included in this class. _Sexual stage.14cking . .Form clarri.(Fungilmperfecti).Deuteromycetes The Deuteromycetes is a form class for those fungi (withorphological affinities to the Ascomycetes or Basidiomycetes) which have,not demon trated a sexual stage. The generally employed classification scheme for these fungis based on the morphology iY 'and color of the asexual reproductive etages.This scheme is briefly outlined be'low Nehver concepts of the classification based on conidium development after the classical atoirk_of S. J. Hughes (1953) may eventually replace the gross morphology system (see Barron 1968).

7 A F 14.

1

-J

r

, -KEY TO THE,FORM-ORDERS OF THE FUNGI IMPERFEC1i

1 . Reproduction by means of conidia, oidia, or by budding 2 1' No reproductive structures present Mycelia Sterilia

2 (1) Reproduction by means of conidia borne in pycnidia Sphae rolisidales 2' Conidia, when formed, not in cycnidia 3 , .

'3 (2') Conidia borne in acervuli t;',. ,. , . Melanconiales 1 3' Conidia borne otherwise, or rep'roduction by oidia or by budding, Moniliales .-.i KEY TO THE FORM - FAMILIES OF THE MONILIALES

1 Reproduction mainly by unicellular budding, yekst-like; mycelia' phase, if present, secondary, arthrospores occasionally produced, manifest nielanin pigmentation lacking 2 Manus mainly filamentous; dark melanin pigments sometimes produced 3

2 (1) Ballistospores produced .'Sporobolornycetceae 2' No ballistospores Cryptococeaceae ' 3 COnidiophore*, if present, not united into 8porodochia or synnernata 4 3' Sporodochia present Tuberculariaceae 3" Synnemata present Stilbellaceae

4 (3) Conidia and conidiophores or oidia hyaline or brightly colored Monies liacCae 4' Conidia and/or conitliophores, containing dark melanin pigment Dematiaceae

O 4

0

e

444

8 Fungi and the Sewage Fungus Community'

4:4

SELECTED REFERENCES, Stagnant. waters.I.Morphology and I. Occurrence in Nature. Arher. J. Ahearn, D. G., Roth,F. J. Jr . ; Meyers; S.P. Botany '54:702-719." 1967 Ecology and Charact erization Of Yeasts t. from Aquatic Regions of South Florida. Hughes, S. J.Conidiophores, Conidia and . 1:291-308.1968 Classification.Can. J. Bot. 3 r:577 - Alexopoulos J. C.' Introductory Mycology. 659.1953 2nd ed. John Wileyand Sons, New York, Johnson, T.W., Jr.Saprobic . 613 pp.1962 plzt-:-.95-104.In Ainsworth, G. C. and Sussman, A. The Fungi, III. Harron, G. L.The Genera of Hyphomycetes _Academic Press, New York. 1968 frbm Soil.Williams and Wilkins Co., - Baltimore.364 pp.1968 and Sparrow, F. K., Jr.Fungi Population Effects on the in ceans and Estuaries. Weinheim, Cooke, W. B. Germany. 668 pp.1961 Fungus Population of a Stream. /# Ecology 42:1-18.1961 Meyers, .S.P. Observations on the Physio-7 '1 logicalltcology of Marine Fungi. Bull. . A Laboratory Guide to Fungi in Misaki Mr. Biol. Inst. 12:207-.228.'1968 Polluted Waters, Sewage, and Sewage . , Treatmept Systems.U. S. Dept: of Prigshei, E.G.Iron Bacteria.Biol. Revs. Health, Education and Welfare, Cincinnati, Cambridge Phil: Soc. 24:200-245. 1949 132 pp.1963 1 Sparrow, F. K., Jr. A quatic Phypomycetes. Fungi in Sludge Digesters. 2nd'ed. lUniv. Mich. press, AnnArbor. \ Purdue Univ. Proc. 20th Industrial Waste Conference, pp 6-17.. 1965a 1187pp:1960.

. Ecology of Fr_Fshwatertungi: 4The Enumeration oreast pp. 41 -93k. Ainsworth G.C. and Populations in a Sewage Treatment' }dart._ SusSman, A.S. The Fungi., III.Acad Mycologia 57:6 6-103.1965b Jress,'New York.1968' . Fung1 Populations in. Relation Stokes, J.L.Studies on thetFilamentous to Pollution ofhe Bear River,:ldaho-Utah. 1967, Sheathed Iron BaCterium Sphaerotilus Utah Acad. Prc. 44(1):298-315. natans.J. Bacterio1.67:278-291.1954 and Matsuura, George S. A Study van'Uden, N. a,rid Fell, J.W. Marine Yeasts. of Yeast Populations in a Waste Stabilizatipn 167 -201.In Droop, M.R. and Wood, Pond System. protoplasm 57:163-187.. E. J_. F. Advan-cealn-Microbiology of 19%3 the Sea, Academic Press, New York. 1968- , Phaff, Miller, M.W., Shifrine, M., and Knapp, E.Yeasts Yerkes, D. Observations on an Occurrence in Pollutedater and Sewage: of-Leptomitus lacteus in Wisconsin. 1960 Mycologia5:210-230. Mycorogis. 58:976-978.1966 EmersonTlialpTrandVe'ston,. W; H. 4.,Cooke-,---William B. and Ludzack, F. J. :Aqualinderella fermentans Gen. et Sp. Predac s Behavior in Nov., A PhyComyceie Adapted to c hated Sludge Systems. Jour. Water Poll.Cont.Fed.s30(12):1490-1495. 1958. .Fungi and the Sewage Fungus Cornpity

SELECTED REFERENCES (t ontinued)

Curtis, E. J. C. Review Paper'Sewage Fungus:_ Its Nature and Effects. Wet. Res. B:289.-311 1969.

.Curtis, E. J. C. and, Curds, C. R. Sewage Fungus in Rivers in the United Kingdom: The Slime Community and It&Consituent Organisms. Wafer Res,' O 5:1147-1159. 1971. . 'PE;r1).1

-PROTOZOA, NEMATODES, AND ROTIFERS

A

IGENERAL CONSLDE4ATIONS considered as indigenous to natural waters. Sulfur and iron bacteria are A Microbial quality ?onstitutes only one more common in the bottom mud. aspect of water sanitation; microchemicals and radionuclides are attracting increasing C,Actinomycetes,' Bacillus sp. Aeroares amount of attenticn sp., and nitrogen-fixation bacteria are primarily soil dwellers and may be washed bi Microbes considered:here include bacteria, into the 'water by runoffs. protozoa, and microscopic tnetazoa; algae a and fungi excluded.. E Nematodes are usually of aerobic sewage treatment origin.. C Of the free-living fOrmi, some are members of the flora and fauna, of surface D E. coli, streptococci, andCl..perfr iugea waters; others washed into the water from are True indicators offecarpollyon.. air and soil; still others of wastewater origin; nematodes most commonly from III PROTOZOA sewage effluent. A Classification D Hard to separate "native" from "foreign" free-litring 'microbes, due to close 1 Single- cell animals in the most association of water with soil and other primitive phylum (Protozoa) in the environments; generally speaking, bacteria animal kingdom.i adapted to water are those that can grow on very low concentrations of nutrient 2 A separate kingdom, Protista, Aci in- and zoomicrobes adapted to water are elide protozoa, algae, 'fungi, and' those that feed on algae, and nematodes, bacteria proposed in thp 2nd edition' especially bacteria eaters, are uncommon ofWard-Whipple's Fresh-Water water but in large numbers in sewage 2.3.191210,) 10) 14; yineffluent. 3Four subphyla or classes: E More species and lower densities of MattigdPhora (flagellates)- Subclass .mic`robes in clean water and fewer species Phytomastigina dealt with under and higher densities in polluted water. algae; only subclass Zoomastig a F Pollution-tolerance or nontolerance of included here; 4 orders:' microbes closely related to the DO level, 1) Rhizdmastigina - with flagell m required in respiration. or flagella and psetidopodia G From pollution viewpoint, the following 2) Protomonadine. -. With 1 to 2 groups of microbes ate of importance: ostly free-living man Bacteria, Protozoa, Nemateda, and apamstticflagelm Rotifera; 3) 'Polymastigina - with 3.to 8 flagella;..mostly parasitic In II BACTERIA , elementary tract 9f animals A No ideal,method for studying distribution and man and ecology of bacteria in freshwater, . _ 4) Hyperinaktigina - 1 inhitants ,of-alimentary 'tract o sects. B According to Collins,(9)Pseudomonas, Achrombacter, Alcaliguies Chromobac- Tterium, Fla..7/212usteriurn, and,Micrococcus . - '", are the most widely distributed and may be 9: . W. BA. 45c. 5.80 10-1 4 119 0

Protozoa, Nematodes) anti Rotifers

b Ciliophora-or Infusoria (ciliates) present in many for feeding. . no pigmented members; 2 classes;

1) Ciliate - cilia present during they_, 2Ciliophora: whole trophic life; containing majority of the ciliates Most highly' developed protozoa; with few exceptions, 'a macro and a micro- 2) Suctoria - cilia present while ._ nucleus; adoral zone of membranellae, young and tentacles during p-ophic -F mouth, .and groove usually present in swimmingand crawling forms, some with conspicuous ciliation of a disc-like Anterior region and little or no body cSarcodina (amoebae) - Pseudopodia cilia (stalked and shelled forMs); (faise feet) for locomotion andfood- Suctoria nonmotile (attached) and with- capturingi 2 subclasses: out cytostome cysts formed in most. 4 1) Rhizopoda - Pseudopodia without axial filaments; 5 orders: Sarcodine: a) Proteornyxa - with radiating Cytoplasmic membrane but no cell wall; pseudopodia; without test or endoplasm and ectoplasm distinct or i-, shell distinct; nucleus with small or farge nucleolus; some with test or shell; b) Mycetozo fming plasmodium; eying by protruding pseudopodia; few resembilin in sporangium apabIe of flagella transformation; fresh- formatIon water actinopods usually sperical with many radiating axopodia; some Testacea c) Amoebina - true amoeba containing symbiotic algae and mistaken. forming lobopodis2 N. for pigmented amoebae; cysts with single \or double wall and 1 or 2 nuclei. d) Testaceaamoeba with single test or shell of chitinaus material 4 Sporozoa: to be mentionedlater'.

e) Foraminiferaamoeba with 1 ... or more shells of calcareous C General Physiology nature; practically all marine 1 Zoomastigina: forms Free-living forms normally holozic; .food supply 'mostly-bacteria in growth 's d Sporozoa - no organ of locomotion; film on surfaces'or clumps relatively amoeboid in asexual-ohase; all aerobic, th -refore the first protozoa to parasitic disappear -innaerobic conditions and re-appearing at recovery; reproduction . by sim-Aelissions,r,--,_ or occasionally by B General' Morphology budding. ',/,

1 Zoomastigina:' 2 Ciliophora: .Relatively small size (5 to 40 p);*with - theexceptionOUthizomastigina, the Holozoic; trt# ciliate's concentrating body has a definite shape (oval, leaf- food particles by ciliary movement. like, pear-like, .etc.); common members around the mouth part; suctoria sucking with 1 or 2 flagella and some ,with 3, 4, through teycles; bacteria and small - or more; few formpit colonies; ..;:torne

120 1 4 Protozoa, Nematodes, and Rotifers

algae and protozoecon titute main 3 Tylenchida - Stylet in-mouth; mostly food under natural condi 'ons; so plant parasites; some feed on shown in laboratory to th ixe otyflead nematodes, such as Aphelenchoides. organic matter and serum protein; not as aerobic as flagellates -.some surviving 4Rhabditida - No stylet in Mouth or caudal under highly, anaerobic conditions, such glands in tail; mostly bacteria-feeders; as Metopus; reproduction by simple common genera: Diplogaster, fission, conjugation or encystment. DiplospAte...:2ALestMRhabiditistMT.hoides Peisferat 1.fa.12..a...... ellus, and Turbatrix.

4%. ( 3Sarcodina: 5' Dorylaimida - 'Relatively large nematodes; stylet in mouth; feeding on other nematodes, Holozoic; feeding through engulfing by algae and probably zoomicrobes; Dorylaimus pseudopodia; food essentially same as common genus. for ciliates; DO requirement somewhat similar to Ciliates - the small amoebae 6 Chrom dorida-Many marine forms; and Testacea frequently present in large some f, e'shwater dwellers feeding on numbers in sewage effluent and polluted algae; chacterized b strong orna- water; reproduction by simple fission tioof knobs, bristles or and encystation. !punc1 atio s in cuticle. 1 M nhysteridi. - Freshwater dwellers; IV NEMATODES e ophago-intestinal valve spherical to elongated; ovaries, single or paired, A Classification usuallystraight;common genus in water - Monhystera. 1 All in'the phylumffiemata (non'egment- ed round worms); subdivided by some 8Enoplida - Head usually with a number authors into two classes: of setae; Cobb reported one genus, . Mononchulus, in sand filters in Secernentea - 3 orders: Washington, D. C.. (phasmids) . Tylenchida, Rhabditida, Strongylida, B General Morphology a Teratocephalida; with papillae on male tail, caiudal glands absent. Round, slender, n nsegmented (transverse markings. in.cuticle of some) worms; some small (about a mm long, as Tri- o cephalobus), ma 1 to 2 mm long (Rhabditis, Dinlogaster, and tiplogasteriodee Adenophora - 6 orders: for instance), and some large (2 to 7. nnn, ( aphasmids) such as Dorylaimus)rsex separated but few Araeolaimida, Dorylaimida, parthenogeristic complete alimentary; canal; - Chromdorida, Monhysterida, Enoplida, with elaborate mouth parts with or without__ and Trichosyringida no papillae on stylet; complete. reproductive system in male caudal glands absent. each sex; no circulatory or restiratory system; complex nervous system with conspicuous nerve ring across oesophagus. 2 Orders encountered in water and sewage treatment - Free-likring fot6s inhabitat- AK, ing. sewage treatment plants are usually C General Physiology bacteria-feeders and those,feeding on other nematodes; those inhabitating clean 1 Feeding - Most sewage treatment plant 'waters feeding on plant matters; they dwellers feeding bn bacteria; others fall into the following orders: preying on protozoa, nematodes, .rotifers,

10-3 Protozoa, Nematodes,and Rotifers

. etc., clean-water species apgarently Keratella, Monostyla, richocerca, vegetarians; those with stylet in mouth Asplanchna, Polyarthrta, Synchaeta, use the latter to pierce the body of/anithal- Midrocodon; common genera under the or plant and suck contents; metabolic order Ffosculariaceae: Floscularia, wadte mostly liquid containing ammoniiun and Atrochus. Common genera under c rbonate or bicarbonate; enteric order Melicertida: Limnias and pa ogens swallowed randomly with Conochilus. suspending fluid, hence remote possix 5 Unfortunately orders and families( of taffy of sewage effluent-borne nematodes A rotifers partly based on character of - being pathogen-carriers. corona and trophi (chewing organ), which are difficult to study, -esp. the latter; the foot and cuticle much ea.4ier 2 ,OxYge n requirement- DO, amiarently ,to study. diffused through cuticle into body; DO requirement somewhat simgarto General Morphology and Physiology protozoa; Rhabditis tolerating reduced DO better than other Rhabditida members;, 1 Body weakly differentiated into head,. all disappear under sepsis in liquid; some neck, trunk,- and foot, separated by thrive in drying sludge. , folds; in some, these regions are 3 ReproductionNormal life cycle requires merely gradual changes in diameter mating, egg with embryo formation, of body and without a separate neck; - hatching of eggs inside or outside femals, Segmentation external only. 4-larval Stages, and adult; fewrepro- duce in the Operice of males., 2 Head withccirona, dosal antenna, and ventral mouth;mastax,a chewing organ, located in head and neck, connected to/ ,-V ROTIFERS - mouttLanteriorly by a ciliated/gullet an posteriorly to a large stoma.01 occupyg A Classificatiolii ,mucli of the trunk..

- 1 Classified either as a crass of the 'phylum 3 Common rotifers reproducing partheno- Aschelminthes (various forms of worms) genetically by diploid eggs; eggs laid in or as a separatel phylum (fiotifera); cbm-, water, cemented to plants, or carried monly called wfirelanimalcules, on on female until hatching. b account df apparent circular movement of cilia arbund head (corona); corona con- 4 Foot, a prolongation of body, usually tracted when 244a4ing or swimming and 'with 2 toes; some with one toe; some ° expan. dedwithenched to catch food. with one toe and an extra toe-like _structure (dorsal spur). 2 Of the 3 classes,-.2 (Selsooidea and Bdelloidea)rollped by some authors ome, like Phlilodina, concentrating under Digon8nta (i °Varies) and the bacteria ariciother microbes and minute other beibOlonogimont (1 ovary); particulate organic matter by ciliary eisonideacontaining rn stly marine movement on corona larger-microbes forms./- chewed by mastax; some such ors Monostyla feeding on clumped matter, 3Class Digononta containing 1 order such as Cacterial growth, fungal massed, (Bdelloida) with 4. families, 'Philodinedae- -- etc. at bottom; virus generally not being the most important. ingested - apparently undetected by cilia. 4 °Class Monogononta comprising 3 ordetst-:' Notommatida (mouth not near cente -Of 6 DO requirement somewhat simil ar to corona) with 14 families, Floscularida protozoailome disappe ring under Melicertida (corona with two wreaths of reduced DO, others, likPhilodina, surviving at as little as cilia and furrow between them) with 3 . ppm DO, families; most import .genera included in the order Notommatida: Brachionus, 10.4_ 122 _ - eia0111=0. ProtozolLIImatodes.1_and Rotifers °

VI SANITARY SIGNIFICANCE B Protozoa and rotifers 4 should be included in examination for planktonic microbes. A Pollution tolerant and pollution non- toledigat species - hard to differentiate - C Nematodes re/411Eing specialist training in protozoa, - nematodes, and rotifers. D Laboratory Apparatus(3)

B Significant quantitative difference in clean 1 Sample Battles - One-galron glass or and polluted waters - clean waters con- plastic bottles with metalor plastic taining large variety of genera and species screw caps, thoroughly washed and but quite low in densities. rinsed three times with distilled water.

C Aerobic sewage treatment processes 2Capiplor Pipettes and Rubber Bulbs - (trickling filters and activated sludge Long (9 in.) Paaeur capillary pipettes 'processes, even primary settling) ideal and rubber bulbs of 2 ml capacity. breeding grounds for those that feed on bacteria, fungi, and mintztelLotozoa and 3Filtration Unit - Any filter holder present in very large numbers; effluents assembly usetd bacteriological from-sticifiCocesses carrying largenum- examination. 'The funnel should be bers of the-fie zoomicrobes; natural waters at least 650 ml and the filter flask at receiving such effluents show.ing significant least 2 liter capacity. increase in all 3 categories. j 4. Filter Membranes - Millepore SS (SS D Possible Pathogen and Pathogeri Carriers iiirgiM) type membranes or equivalent.

1 Naeleria causing swimming associated 5MicrOsc..._me - Binocular microscope, meningoencephalitis and Acanthameoba with 10X eyepiece, 4X, 10X, and 43X causing,nonswimming associated cases. objectives, and mechanical stage.

2Amoebae. and nematodes grown E Collection of Water Samples pathogenic enteric bacteria in lab;.tione alive in amoebic cysts; very few alive Samples are collected in the same mannero in nematodes after 2 days after ingestion; as those for bacteriological examination, ,virus demonstrated in nematodes only except that a dechlorinating agent is not: when very high virus concentrations... 41* needed. One-half to one gallon samples are present; some"TreeliNiing amoebae collected from raw water and one-gallon palasitizing humans. samples from lap water. Refrigeration is not essential and samples may be transported 5\ Swimming ciliates and some rotifers without it unless examination is to be delayed (Concentrating food by corona) ingesting for more than'five days. .1, large numbers of pathogenic enertic bacteria, but digestioh rapid; no. F Concentration of Sainples evidence of concentrating virus; crawling \ ciliates and flagellateei feeding on- clumped 1 One gallon of tap water can usually be organisms,', filtered through a single 8-u membrane within 15 minutes unless the water has ,4 Nematodes concentrated from sewage high turbidity: -"At least one gallon of effluent in Cincinnati area showing sample should be used in 'a single examina- live E.coli and streptococci, but not tion.Immediately after the last of the human enertic pathogens. water is disappearing from the membrane, the suction line is disconnected and the VII EXAMINATION OF WATER FOR MICROBES membrane placed on the wall of a clean 50 to 100 ml beaker and flushed repeatedly A Bacteria - not dealt here. with about 2-5 ml of sterile distilled water

10-5 Pr'otozoa, Nemato4s and Rotifers

with the aid of acapillary pipette and a sluggishly motile nematodes may be rubber bulb. The concentrate is then confused with root fibers, plant fila- pipetted into a clean Sedgewick-Rafter ments of various types,' elongated Counting Cell and is ready for examina- ciliates such as Homalozooji vernii- tion. culare, or segments of appendages_of small crustacea. To facilitate a, 21p concentration of raw water samples geAral identification of nematodes, the having visible turbidity, tem to :our gross morphology of three of the free- 87rriicron membrapes. may be required living nematodes that are frequently per sample, with filtration through each found in water supplies is sh9wn in the mellibrane being limited to not more attached drawing. The drawing provides than 30 minutes. Samples ranging from not only the gene ral anatomy for recogni- 500 ml to 21iters may be filtered With tion of nematodes but also most of the one membrane, depending on degree of essential structures for guidance to those turbidity.After filtration the membranes who want to use the "Key to Genera" in are placed M the walls of separated chapter No. 15 on Nemata by B. G. beakers and washed as above. To Chitwood and M. W. Allen in the book, prevent the particulates from obscuring Fresh Water Biorogy. (10) the nematodes, the washing from each filter is examinedsin a separate counting 2Under normal conditions, practically chamber. all nematodes seen in samples of finished water are in various larval G 'Direct Microscopic Examination, stages and will range from 100 to 500 L, microneiri length and 10 to 40 microns Each counting chamber containing the in width.Except in the fourth (last) filter concentrate is first examined under stage, the larvae have no sexual organs a 4X objective.Unless the concentrate but show other structural characteristics. contains more than 100 wormso they whole Fell area is surveyed for nematodes, with 3If identification of genera -i's desired, respect to number, develbpmental stage, the filter washings are 'centrifuged at and motility.When an object having an 500 rpm for a few minutes. The outline. resembling that of a nematode is supernate id-discarded, excapt a few observe it is re; examined under a ipx drops, and the sediment is resuspended objective for anatomical structures, ukless in the remaining water. A drop of the the object exhibitg typical nematode move- final suspension is examined under both ment, which is sufficient' for identifying the 10X and 43X objectives for anatomical object as a nematode. When the concentrate characteristics, without staining, and for contains more than 100 worms, the worm supplementary study of structures the density can.be estimated by counting the rest is fixed in 5% formarin or other number ofworms in representative Micro- fixation fluid and stained according to scopic fields and multiplying the average instructions given in Chitwood and number of worms per field by the number ,Chapter 'on Nemata, (7): of fields in the cell'trea. The nematode Goodey's Soil and Freshwater Nema- density may be expressed as number of todes(11) or other bboks on nematology. worms, per gallon with or without differenti- ation as to adult or larval stages or as to viability. VIIIUSE OF ZOOMICROBES AS POLLUTION INDEX

H' General Identificatiotrof Nernt.todes A Idea not new, protozoa suggested long ago; many considered impractical because of 1 While actively motile nematodes can be the need of identifying pollution - intolerant readily recognized by any person who and poIlution-tolerant specie's.- proto- has some general concept of micro- zoologist required. Method'also time scopic animals, the nonmotile or consuming. . .

10-6 r

Protozoa; Nematodes, and Rotifers

B Can use them on a quantitative basis FLAGELLATA nematodes,and nonpigmented protozoa present in small numbers in Bodo caudatus clean water. Numbers greatly increased Pleuromonas jaculans when polluted with effluent from aerobic treatment -plant or recovering from sewage Oikomonas termo pollution; no significanrerror introduced Cercomonas longicauda "when clean-water members included in the enumeration if a suitable method of com- Peranema trichophorum puting the pollution index developed. Swimming type C Most practical methodinvolves the equatibn; A + B + 1000 C = Z. P. I. , Ciliophora: A Colpidium colpoda where A = number of pigmented protozoa, Colpoda cuculus B = non pigmented protozoa, and Glaucoma pyriformis ,r0 C = nematodes in a unit volumeof sample, azd Z, P. I. = zoological pollution index. Paramecium candatum; P bursaria For relatively clean water, the valueof Stalked type Z.P.I. close to 1; the larger the value above 1, the greater the pollution byaerobic a2rcularia sp. (short stalk dichotomous) 'effluent (see attached report on zoomicrobial

M indicator of water pollution). Vorticella sp. (stalk single and contractile)

CONTROL aistylis plicatilis (like opercularia, more ; colonial, stalk not contractile) A _-Chlorination of effluent Carchesium sp.(like vorticella bat colonial, B Prolongation of detention time of of individual zooids contractile) C Elimination of slow sand filters in foothamnium sp., I entire colony contracts) nematode control. Crawling type LIST OF COMMON ZOOLOGICAL ORGANISMS FOUND IN AEROBIC SEWAGE TREATMENT Aspicisca costata PROCESS Euplotes patella Stylonychia mylitus PROTOZOA UrOstyla sp. Sarcodina - Amoebae Oxytricha sp. Amoeba proteus; A radiosa NEMATODA Hartmanne lla Amelia Vulgaris Dipluaster sp. Doryhrr...itis sp. Noegleria gruberi Manochoides sp. Chlindrocorpus sp. A ctinophrys 2klorosteroides sp. agtloblis sp. Rhabditis sp. Rhabditolaimus sp. Pelodera,sp. NIonhystera.sp.

AphelelichoideS sp. r-..24lolaus sp. 125 imp*, 10-1 Protozoa,.Nematodes, °s.nd Rotifers

ROTATORIA 3 Chang, S. L.Interactions between Animal Viruses and Higher Forins of Microbes. 21..glena Proc. Am. Soc. Civ. Eng. J1. San. Eng. 96:151.1970

, 4Chang, S. L.Zoomicrobial Indicators ° of Water Pollution presented at the --- Polarthra Annual Meeting of Am. Soc. Microbial, Philadelphia, April 23 -28, 1972. Philodina 5 Chang, S. L. Pathogenic Free-Living Ke rate lla Amoebae and Recreational Waters. Presented at 6th International Confer- Brachionus , en%e of Water Pollution Research OLIGOCHAETA (bristle worms) Association, Jerusalem, Israel,. June 19-24, 1972. Aelosomh hempachi.. > 6 Chang, S. L. Proposed Method for Examination of Water for Free-Living ,Aulophonts limosa Nematodes. 5.A.W.W.A. 52:695-698. 1960. 7Chang, S. L., et al.Survival and Protection 17=11DLumbricillus 111 lineatus all.a Against Chlorination of Human Enteric Pathogens in Free-LivingNematodes DiISEC LARVAE Isolated from Water Supplies. Am. Jour. Chironomus e Trop. Med. and Hyg. 9:136-142. 1960. 8° Chang, S. L.Growth of Small Free-Liiiing Psychoda sp. (trickling filter fly) Amoebae in Bacterial and Bacteria-Free ARTHRoPODA Cultiires4. Can. J. Microbial. 6:397-40s'1960. ' 'f- Lessertia sp , 9 ChaneSs. L. and fabler, /1. W. tree- Living Nematodes in Aerobic Aeatment Plant Porrhomma sp. Effluents. J.W.P.O.F. 34:1256-2161. 4 Achoratua subuiata s ('collembola). 1963. 10Chitwood, B. G. and Chitwood, M. B. An Folsomia sp. (collerhhola) Introduction to'Nernatolozz. Section I Anatomy:1st e71. Monumental Printing Tomocerus sp. (collembola) Co. Baltimore.1950. pp 8-9. REFERENCES 11Cobb, N.A. Contributions to the Science of 1 American Public Health Association, ' Nematsology VIt. "Williams and Wilkins Co. 4merican Water Work's Association and Baltimore.1918. Water Pollution Control Federation. 12Collins, V. G. The Distribution and Ecology Standard Methods for the Examination of Bacteria in Freshwater, Pts. I & of Water aWarftewater, 13th ed. Proc: Soc. for Water Treatment and Exam. New York. 1971. 12:40-73.1963: (England) 2Chang, S. L., et al. SurveY of Free-, 13Edmondson, W. T., et al..Ward-Whipple's Living Nematodes and Amoeba in Fresh Water Biolm. 2nd ed. John Muicipal Supplies.J. A. W. W. A.. 52: Wiley & Sons, New York.1959. pp 368-401. 613 -618. . Protozoa, Nematodes, and Rotifers

14 Goodey, T.Soil \and Freshwater This outline wa prepared by S. L. Chang, Nematodes.' (A Monograph) 1st ed. Chief, Etiology, Criteria Development Methuen and Co.Ltd.' London. 1951. Branch, Water Supply Research Laboratory, NERC, USEPA, Cincinnati, Ohio 45268. Descriptors: Protozoa, Ne atodes, Rotifers

:%"=.!

9

k

.1

tI AV

127 109 Piotozoa, Nematodes, andtifers j

Effluent 1-7-4-Raw Sewage 01=11 Insects A . ;1 TR RR \ \ FILTERS OligochaeteS-& Suspe)edoank matter insect larvae- (by h crolysis)

Nematodes & rotifers DisSolved organic matter

AERATION Nonpimented TANKS protozoa

I It A (respiration, de amination, Heterotrophic decarboxylation, etc.) bacteria

Inorganic C, P, N, Fungi S comp. TIT FILTERS Algae

Autott'ophic bacteria (Nitrification, sulfur & iron bacteria) Pathogenic organiSms

Food Chain in Aerobic.Sewage Treatnient Processes

The same sequences and processes occur in polluted streams and streami'receiving treated wastes.

1 28 .

IMO

ACTIVATED SLUDGE PROTOZOA.

Larggr animals (worms, snails, fly larvae, 1 etc. ) dominate trickling filters. Why are these always absent from the activated sludge process? Why are there numerous micro- species "2 common to both trickling filters and activated sludge?

What Organisms besides protozoans and 3 animals are present in activated sludge?

What is (he advantage(s) of microscopic 4 examination of activated sludge?

One sampling site would be the one of choice 5 . in sampling Ni activated sludge plant for microscopic analysis. Why? Where?

What organisms predominate in activated 6 sludge? a 4.

Why are photosynthetic green plants (in 7 contrast to animals) basically absent from the activated sludge process in general and mixed liquor specifically?

hy are the same identical species of protozoa8 f d in activated sludge plants all over the world?

What is, the significance of a microscopic 9 examination of mixed..)liquor? Define and characterize: 10 Activated Sludge .Mixed Liquor Flocs

What is the relation between bacterial) . 11 protozoan populations in activated sludge and the proces,s itself?

At what total magnificatiori wereyou able to 12 believe the smallest cells observed.w.ere in fact bacteria?

Activated sludge is a dynamic (although 13 man-manipulated) ecosystem. How-does, it differ from a natural ecosystem? e

Activated-Sludge PiOtozoa -AO a

What is the greatest problem(s) with a wet 14 mount slide preparation? . I C- How do you overcome these disadvantages? 15

How do yoU slow down fast moving protozoans16 on a wet mount?

Why are quantitative counts of protozoa (like 17 number/ ml) generally meaningless?

What' is the significance of proportional 18 counts?

Scanning a slide (in making a count) should 19 generally be done at X.(Total magnification) .

The iris diaphrUm on thenlicroscope is 20 used to adjust light intensity (true-false). Why sample the surface film of the 21- settleometer? Why is the thinnest film most ideal for a 22 wet mount? 63

9 What did you learn from the microscopic 23 examination' of the activated sludge?

What is the physical nature of the flocs 24 obsakved?

What filamentous organisms were 25° observed?

Why are "rare" species of no practical 26 significance in microscopanalyses of activated sludge? o

Why are there no protozoan indicator species27 of process efficiency in activatedillludee? .0 4 oak Activated sludge-biological communities 28 are temporal in contrast to biological .communities in trickling filters which are spatial-(TRUE/FALSE). "Seeding" a newly started activated sludge 29 plant with cultures or material from other O plants is only a. wasted effort (TRUE/FALSE). Justify,y.our answer. .

13 co . . 4- .1 . ti

Activated Sludge Protozoa

if a wet mount slide` of mixed liquor is 30 prepared and placed in 'a petri dish with a wet blotter underneath and allowed to sit for sgveralhours, what will be the distri- bution of the protozoa under the cover slip?

In a mixed liquor sample nearly all of the 31 stalke,d ciliates have""broken off. the stalks and are frerswimmineas utelotrcichs." What does'this indicate? tto

What are Monags? And are they good, tad, 32 4or indifferent in activated sludge?

What are hypotrichs or crawling ciliates, 33 and are they good, bad or indifferent ii activated sludge? A %. Q

What are swimming ciliates, and are they 34 _ goodbad or indifferent in activated sludge?

Whatare flagellAtes, and are they good, bad'35 . or indifferent in activated sludge?o What are amoebae, and are they good, bad, 36

A or indifferent in activated sludge? What are the ideal characteristics of a wet't 37 mount slide preparation?

Why does total community givea- betfbr 38 indication of process efficiency in activated .. sludge?

O In observing and identifying protozoa one looks 39 for what, characteristics of an individual organism?

What is the role of bacteAa in activated a ;10 sludge?

Vihatsis the role or,protozoa in activated 41; sludge?

Microscopic analysisofAmbeedliquor 42 ot, 4&) . sample can be very quick, simple, and - . meaningful (TRUE /FALSE). .

11-3 0

Activated Sludge Protozoa f

Protozoan communities present in activated 43 'sludge reveal: a.Plant efficiency b.Settlfabil4 c.ROD removal d.Solids removal ei.; Plant ling o .. (Circle applicable .descriptions)

Pr.otozob.n-communities in activated sludge 44

c. °reveal complete-and instantaneous to,nditions; average of physical and_chemical-conditions; -egtremes of chemical and physical conditions. (Draw a line through phrases true.)

Rank in increasing plant efficiency the 45 °following ptotoioan group which would pre-, dominate. Rotifers Stalked ciliates , Amoebae Swimming ciliatt s 9 Crawling ciliates lagellates (For exaple, use number 1-6. One would be startup co ditiops or least efficient, and six would be t e.most efficient.) .. Identification is usually done at X and 46 sometimes requires X.

Immersion oil should be used sparingly at 47 what two points on a slide?

'''''''4:9 Which comes first in microscopic examina- 48 lion; s anning at low power to pick out. unkno ns or higher power to identify ?, 1< ° In making proportional count, which total. 49 number to count would be better; total of ten _organisms or a total of 100 organisms? Why?

Why not kill the organisms so you can 50 identify and count them'on the slide?

,What simple chemical solutions are useful 51 to immobiliie protozoa if methyl cellulose or polyvinyl alcohol is hot-available?' t 4

Activated Sludge Protozoa

Initially the° wet mount slide should be 52 racked up close to the low power objective: by your eye on the eyepiece through the scope; or by glancing at the actual distance . with the naked eye While you rotate the coarse adjustment knob. (Underline which)

What are par -focal objectives on the 53, microscope?

Why should water on the microscope and all54 lj its parts be carefUlly avoided? If activated sludge is a mah manipulaiied 55 system, 6re there comparable,na.tuPal, ecosystems? Exainple? . What is the "community ';concept in exami-56 nation of aptivated sludge?

What a're the appliCations of direct micro- 57 scopist examination of activated sludge? What are rotifers, and are they good, bad -58 * or indifferent in activated sludge? . o*. List the five kingdomS of organismsnd give 59 a specific example for each.

What techniques are most useful in 60 identifying an unknown Organism, and why is -correct identification Important?

Scanning and counting is done at * X 61 magnification.Identification of most PROTOZOA usually requires . X -magnification and occassionally X

- magnification. -5' , OBJ. TOTAL MAG. USE 62

43.5 X 10 X I.

40 X ".

boo° x

(1,0 X eyepiece's)

11-5 Activated Sladge Protozoa

hand is constantly operating the 63

hand issconstantly, operating the

The microscope is manually operated and requires skill and understanding on the part of the operator. A microscope, no matter how costly is only as good as the micro- scopist operating it.

List the basic skills required in utilizing the 64 .7 optimum capabNty of your microscope.

N`A h,

0 _ , This outlinerv/as prepared Isr,R. M. Sinclair, .fiational Training tenter, MOTD, OWPO, USEPA, Cincinnati, Ohio 45268. 4 Descriptors: Zlicroorganisms, Protozoa Rotifers Activated Sludge. Riota ;...... ? 1,

*I

A C

". 3

134 01 FREE- LIVING AMOEBAE AND -NEMATODES

IFREE - LIVING AMOEBAE C Morphological Characteristics of Small, Free-Living Amoebae Importance of Recognizing Small, Ftee- Living Amoebae in Water .1Morphology of Trophozoites'- Supplies Ectoplasm and endoplasm usually ,dIstot; mar:dens-with large nucleolus. 1 Commonly found in soil, aerobic is sewage effluent and natural, fresh 2Morphology of cysts - Single or waters - hence,- frequently en- double wall with or without pores ea. countered in examination of raw watek.' D Cultural Characte ristics of Small; Free - Living- Amoebae stenot infrequently found in wits]. al supplies - not pathogen 1 How to cultivate these amoebae -. arrie- s. plates with bacteria; cell cultures, 1 \:-.1 axenre culture. lage 41,e-a,moebae Naeolerla involved-140 some cases of 2 Growth characteristics on plate, meningoencephalitis, about half . cell, and axenic culture in the U:S.; associated with swimming in small warm lakes. ,3Complex growth requirements Acanthamoeba rhisodes parasitizing for most of these amoebae hyman throats and causing (3 cases) nonswimming-associated meningo- E Resistance of Amoebic Cysts to encephalitis. . Physical andChemical Agents 4Cysts not to be confused with those of Endamoeba histoljtica in water - .81 borne. epidemics. IIFREE- LIVING NEMATODES B Classification of Small, Free-Living A Classification of Those Commonly Amoebae Found in Water Supplies

42> 1Recognized classification based 1 Phasmidia (Secerneutes): on characteristics in mitosis. Genera Rhabditis, piploga.ster, Dipl.oacqeroidesi Cheilolms 2 k Common species fall into the Panalalgromus following families and genera: Aphasmidia (Adenophoro): Genera Family Schizopyrenidae: ,Genera moL2-il st1., Aehelenchusr. Turbatibc.. _ Naegleriaj Didascalusi and (vinegar eel), l2c2D22.imusj and Schizopyrenu.s.- first-two being Rhabdolaimus , flagellate amoebae. B "MorphOlogical F4tures Family Hartmannellidae: Genera Hartmanella (Acanthamoeba) 1Phasnaids: papilla on tail 'at males, mouth 'adapted to feed on bacteria, 3 H9w to prepare material's for few exceptions. studying mitosisFeulgen stain 2 Aphasmids: no papilla on male ts.- - glandular cells in male. V BI. AQ."14b. 6. 76 I 2-1 '!$ (2. Amoebae aid IsTematodes in Water Ues we, 4. 4 C Life Cycle 2 Chang, S. L., et al.IsSurvey of Free - LivingNematddes and Amoebas' in 1 MetIods Of mating ' Municipal Supplies".J. A. W . W. A. 52.613-618, 1960. , 2Stages of developthent 3 Chang, S. L., "Growth of Small Free- 3 Parthenogenesis Living Amoebae in Various Bacterial and in Bacteria-Free Cultures".., Can, b Cultivation Jour. -.Microbiol:. 6 :397 -405,1960.

1 Bacteria -fed, cultures Nematodes

2 Axenic' cultures 1 Goodey, T., "Freshwater Nematodes", ,,>; 1st. Edition, Methuen & Co., LondyA, E Occurren-ie in Water Supplies 1951.

1. Relationship between their_.--- 2 Edmondson, W. T.,. Ed., Ward &Whipple' s appearance in finished,waer '''Fresh-Water Biology" 10th Edition,

and that in raw water. S' page 397, 1955.

2 r Frequency of occurrence in f 3Chang, S. L., et al. , "Occurrence of a diffetent types of'raw water,,, 'Nematode Worm in a City-Water Supply ". and sources. J.A.-W.W;A., 51:671:676, 1959.

3Survival of human enteric path- 4Chang, S. L., et al., "Suzvival, 3-11A ogenic bac't'eria and viruses in Protection Against Chlorination, of ; '- nematodes. HumanEnteric Pathogens in Free-' Living Nematodes Isolated From. Water 4 Protection of human enteric Supplies". Am. Jour. Trop. Medicine pathogenic bacteria and viruses & Hygiene, 9:136-142, 1960. in lematode-carriers. , -- 5 Chang, S. L., et al., "Survey of Free- F Control. Living Nematodes and Amoebas in Municipal Supplies".J. A. W. W. A., 1 Chlorination of sitwa.ge--eltu-ent 52:613-618, 1980. A '11 Floqdulation and sedimentation 6Chang, Si L. , "Proposed Method for of A4ater Exa-nination'of Water for Free-Living Nematodes".J. A W. W. A.,52:695-638, 3Chlorination of water -1950->7 -4*

4 Other methods of destruction Ch,g, S. L.; "Viruses, Amoebas, a d Nematodes and Public Water S pplies". J.A9rW.W.A.,53 :288 -296,, 1961. REFERENCES 8Chang,\S.L.and Kabler, P. W.,"Free4 Amoebae Living Nematddes 4n Sewage Effluent f tom erobic Treatment Plants": To 1_ Sinkh, B. N.,"Nuclear Division in Nine ub shed. - Species of Small, Free-Living Amoe- This outlineas prepared by ShT,ii L7Charig, bae and. its Bearing on the Classifica- M. D:, Chief, iology, Criteria Lievelopment. tion of the -Order Amoebida", Branch, Water Supply Research Laboratory, Trans; RcrialSoc. London; Series- B, _NERC, EPA, C cinnati. OH 45268. 236:405-461;, 1952. , Descriptons; Amebae, Nematodes 1 3 SUGGESTED, CLASSIFICATION OF SMALL AMOEBAE

Subphylum: Sarcodina Hertwig and Lesser ss: ,Rhizopoda von Siebold Subclass: Amoebaea Butschli 'Order: Amoebida Calkins and Ehrenberg to Superfamiky: Amoebaceae - free-living (Endarooebaceaeparasitic in animals) Family:Schizopyrenidae- active limax form common; transcient ° flagellates present or absent; nucleonus-origin of polar masses; polar caps and interzonal bodip-present absent Genus: Schizopyrenus - no transcient flagellates; single-walled cysts; no polar caps or interzonal bodies in mitosis Species: S. erzthaenu,3a - reddish orange pigment formed In agar cultures with gram-negative bacillary bacteria S. russelli - no pigment produced in agarcultures Genus: Didascalus - morphology and cytology similar toSchizopresms buFsmall 'numbers' of transcient flagellates fdrmed at times Species: D. thorntoni - only species described by Singh (1952) Genus: %aerie. Alexeieff - double-walled cysts; transcient fla,gellatcs formed readily; polar caps and interzonal bodies present in mitosis Species: N. gr.utieri (Schardin-ger) - only species established; Singh (1952) disclaimed the N. soli he described in 1,951 Family:_Hartmiannellidae - no trans dent flagellate formed; motility sluggish; no limax form; nucleolus disappearing, probably Vh., forming spindle in mitosis; no polar caps or masses, aster, and centrosome not known Genus: Hartmannella - ectoplasm clear or less granular than

. endoplasm; single-walled cysti3; single vacuole Species: H. fasbae - clear ectoplasm, agricola.- ectoplasm less granular than endoplasm Genus: Acanthamoeba - filamentous processes from ecto- or endoplasm; growing axenically in fluid bacteriological a/Arne dta

137 12-3' Su p, estecassification of Small Amoebae..

Air

. , Species: A. rhLsodea , . Genus: Sinehella -,double-walled cysts; ecto- and endoplasm indistinguishable; many vacuoles

I Species: Singilella latocherrius

/ x

* ,... i tr, . ...

61> / 0 , z

C5 I.

s 0

t

.-' . .. : 13L 4

ANIMAL PLANKTON

I INTRODUCTION, (Note Figure: Nonpigmehted, Non - Oxygen Producing Protozoan Flagellates in the A Planktonic animals or zooplankton are outline Oxygen Relationships.) found in nearly every major group of animals. 1 Commonly encountered genera

1 Truly pla:hktonic species (euplankton) Bodo spend all or most of their active life ,Cycle- suspended in the ;water.Three .Peranema groups are predominantly involved in . fresh water; the protozoa, rotifers, 2Frequently associated with eutrophic "and microcru.stotea. conditions

2Transient planktonic phases such as C Class RhizopodaamoebOi.dprotozoans floating eggs and cysts, and larval stages occur in many Other groups.. 1 Forms commonly encountered as plankton: B Many forms are strictly seasonal in occurrence. Chaos fi4noeba) C Certain rare forms occur in great numbers Arceila Centropyxis at unpredictable intervals. 77 Difflusia Fieliozoa D Techniques of collection, prese'rvation, and identification strongly influence the Euglyphe. ,species reported. 2 Cysts of some types may be encountered E In oceanographic work, the zoop ?Acton is in water plants bedistributiori systems; considered to include many relath.vely large rarely in plahkton of open lakes or animals such as siphonophores, ctenoplores,p reservoirs. hepteropods, pteropods, arrowworms, and etiphausid shrimp, D Class Ciliophora

F The plant-like or phytoplankton on the 1 Certain "attached" forms often round other hand are essentially similar in all floating freely with plankton: waters, and are the nutritional foundation for the animal community.. Vorticella

II PHYLUM PROTOZOA 2Naked, unattached ciliates,Halteria A The three typically free living classes, one of commonest in this grouirTa7Rous Mastigophora, Rhizopoda, and Ciliophora, heavily ciliated forms (holotrichs) maY all have planktonic representatives. As occur from time to time ailch as a group however, the majority of the phylum Colpidium, Enchel, etc. is benthic or bottom-loving:, Nearly any ofothe,benthic forms may occasionally be 3 Ciliates protected by a shell or test w.ashe'd up into the overlying waters and (testaceous) are most often recorded thus be collected along with the euplankton. from preserved samples. Particularly common inthe experience of the National B Class mastigophora, the nonpigmented 'Water Quality Sampling Network are: zooflagellates. Codonella fluviatile a These have frequently been confused with the phytomastigina or plant-like flagellates. Codonella cratera The distinction-is made here on the basis of the presence or absence of chlorophyll Tintinnidium (usually with organic matter) as puggested.by Earner 'and Ingram 1955. '4.. - TintinnoRgis

13 -1 139. AnimalPlanktA

III PHYLUM ROTIFERA 2) As unfavorable conditions develop, ,, males- appear, -and- thick walled .i/N Some forms such as .Anuraea cochlearis sexual eggs are enclosed in egg and Asplanchna pridor 'cases called ephippia which can at all tames of the year. Others such as often endure freezing and drying. lc,Totholca striate, N. Ionspina and Poly- arthra platypTera are repor eto be essen- 3) Sexual reproduction may occur tially winter torms.. at different seasons in different species. B Species in approximate order of descending frequency currently recorded by National 4) Individuals of a great range of * Water. Quality Sampling Network are: sizes, and even ephippia, are thus encountered in the plankton, Keratella cochlearis but there is no "Larval" form. Polyarthra vulgaris b Seasonal variation - Considerable variation may occur between winter Synchaeta pectinate and'summer forms of the same species in some cases.Similar Brachionus quadridentata variation also occurs between arctic and tropical. situations. Trichocerca longiseta c Forms commonly encountered as Rotariesp. open water plankton include: Filinia longiseta. Bosmina longkostrisCand others Kellicottia longispina Daphnia galeata ai.14.-erthers

Pompholp sp. a Other less common genera are: C Benthic species almost without number may Diaphanosoma, Chydorus, Sida, be collected witb4e plankton from time to 7croperus, Creriodaphnia, Byt 'ho- time. trephes, analTeEataVorous reYEZ'ara and Polyphemus. IV PHYLUM ARTHROPOD.A d :Heavy blooms of Cladocerans may build up in eutrophic waters. A Claes Crustacea 3 The copepods (Order Copepoda) are the 1 The Class Crustacea includes the larger perennial microc'ustacea of open waters, common freshwater euplankton. They both fresh and marine. They are the are also the greatest planktonic consum- most ubiquitous of animal plankton. ers of basic nutrients in the form of phytoplankton, and are themselves the a Cyclomis the genus Most often greatest planktonic contribution to the rdiirarby the National Water Quality food of fishes.Most'of them are herb- Sampling Network activities.Eucr. ivorous. Two. groups, the cladocera clops, Paracyclops, Diaptomue, and the copepods are most'conspicuous. UrriffiocaraptirCiiiiiscTirra, an LimnocIlanirrare other forms 2Cladocera,(Subclass Branchiopoda, FFIWe7-1-5.-We planktonic. Order Cladocerarar Water Fleas 4 b Copepods 'hatch into a minute char- aLife History acteristic larvae called a nailius which, differs considefably from the 1) During most of the year, 'eggs adults. After fiver six moults, the which will develop without fertil- copepodid stage is j'eached, and after.- ization (parthenogenetic) are six more moults, the adult.These deposited by the female in a dorsal larval stages are often encountered brood chamber. Here they hatch arfd are difficult to identify. ir}to minature adults which escape and mini away., Animal Plankton

Insecta yluirt-Nymathelminthes 1 Only a few kinds of insect that can be 1 N atodeslbr nemas) or roundworms ranked as a truly planktonic.Mainly appr h the bacteria and the blue-green .the phantom midge fly Chaborus: algae in, iquity. Thevare found in the soil and in tke water, and in the air 2 The larva of this insect has hydrostatic as dust. 'In both marine and fresh waters organs that enable it to remain suspendcd andfrom,the Arctic tb the tropics. in the water, or make vertical asaens ii: 2 Although the majority are free living, thewate column. some occur as parasites of plants, animals, and Man, and some of these 3It is usually benthic .during the daytime, . parasites are among our most serious. but ascends to the surface at night. 3 With this dfstribution, it is obvious that they. will occasionallz be encountered as V. OCCASIONAL VLANKTERS 'plankton. A more complete discussion of nematodes and their public health A While the protozoa, rotifers, and micro- implications in water supplies will be crustacea make up the bulk of the plankton, found elsewhere (Chang, S. L. ). there are many other groups as mentioned above that may also occur.Locally or E Additions.' 1 crustacean groups ,sporadically periodically these may be of major impoit- met with in the plankton include the folloWing: - ance. Examples are given below. 4. 1 Or er Anostraca or fairy shrimps B Phylum Coelenterate , merly included with the two flowing orders in the Euphyllopoda) TPoltsof the genus Hydra may become primarily planktonic in nature. detached and float aHourhadiging from the surface film or floating detritus. a Extremely local and sporadic, but when present, may be dominatihg. . 2 The freshwater medusa Craspedacusta I- occasionally appears in Tale% or reser- b Artemia, the brine shrimp,can voirs in great numbers. tolerate very high salinitie. C Phylum Platyhelminthes cIiery widely distributed, poorly understood. 1 Minute Turbellaria (relatives of the , - well known Planaria) are sometimes 2 Order Notostraca, the tadpole shrimps. taken with tbe 'plankton in eutrophic-- Essentially southern and western in conditions. They are often confused distribution. with ciliate protozoa. 3 Order Conchostraca, the clam shrimps: 2Cercaria larvae of Trematodes (flukes) Widely distributed, sporadic in occur-v parasitic on certain wild animals, rence.' Many local species. frequently appear in great numbers. When trapped in the droplets of water 4Subclass Ostracoda, the seed shrimps. i on a swimmer's skin,' they attempt to Up to 3 in. in length.Essentially bore in. Man not being their natural benthic but certain species of Cris, host, they fail.The.resultant irritation and Notodromas may occur in consi - is called "swimmer's itch". Some can erabTerniribiTs as plankton at certain beridentified, but many unidentifiable times of the year. species may be found. 5 Certain members of the large subclass 3In many areas of the world, _cerparia Malacostraca are limnetic, and thus, larvae of -human parasites such as the planktonic to some extent. blood fliAce-Schistosorna japonicum may Jive as planorkr7-'1-fT-"IpEngfiirf. Fia-humah a The scuds, (odder ) are skin,directly on contact. essentially benthic but are sometimes collected in plankton samples around

141 tr 13-3 ;0 'Animal Plankton

weed beds or near shore. Nekto- the uninitiated, they are sometimes planktonic forms include Pontoporeia mistaken for fungi or other organisms and some species of Gammarus. (and vice versa). b The mysid, or opossum-shrimps are In flowing waters, normally, benthic represented among the plankton by (bottom living) organisms are often found 41kP Mysis-relicta, which occurs in the drifting freely in the stream. This deeper waters,, large lakes, as far phenomenon may be constant or periodic. north as the Arctic Ocean. ' When included in plankton collections, _ . theyamust be reported, but recognized F The Class Archnoidea, Order Hydracarina for what they are. (or A cari) the mites. Frequent in plankton tows near shore although Unionicola crass- ipes has been reported to-be yirtually S,uria..t.films are especially rich in planktonic. micro "biological garbage" and these enrich the plankton. G The phylum Mollusca is but scantily represented in the freshwater plankton, REFERENCES in contrast to the marine situation. 1 Edmondson, W. F., ed.Ward and Glochidia (ciliated) larvae are occasion- Whipples'aFreshwater Biology, 2nd ally collected, and snails now and then . glide out bn a.,quiel surface film and are Edition, Wiley & Sons) Inc., New York. taken in a plankton net. An exotic 1959.- , bivalve Corbicula heave planktonic veliger stage. 2Hutchinson, G. Evelyn.A Treatise on Limnology.Vol. 2.Introduction to H Eggs and other reproductive structures . Lake Biology and the Limnopla.nkton. of many forms including fish, insects, and Wiley.1115 pp. 1967. rotifers may tpe found in plankton samples. Special-reproductive structures such as 3 Lackey, J. B.Quality and Quant y of the statoblasts of bryozoa and sponges, .Plankton in the South End of ke and the ephippia of cla.doCerans-may also Michigan in 1952. JAWW be ineltided. 36:669-74. 1944.

IAdveptitious and Accidental Plankters 4 McGauhey, P. H. , Eich, H. F. , Jackson, H. W., and Henderson, C.A Study ° Many shallow ,water benthic organisms of the Stream Pollution Problem in the may become accidentally and temporarily Roanoke, Virginia, Metropolitan , incorporated into the plankton. Many of District.Virginia Polytech. those in the preceding section might be Engr. Expt. Sta. listed here, in addition to such forms as certain free living nematodes' small 5Needham, J. G. and Lloyd, J, T.The oligochaetes, and tardigrades,. Collembola Life of Inland Waters.Ithaca, New and other surface film livers are also, York, Comstock Publishing Co., Inc. taken at times but should not be ,Mistaken- j 1937. for plankton. Fragments and molt. skins' , . from a variety of arthrOpods are usually 6Newell, G.E. and Newell, R. C. observed. Marine Plankton.Ilutchinsonduc. Ltd.London.221 pp. 1963. Pollen from terrestrial or aquatic plants is often unrecognized, or confused wit 7Palmer, C. M. and Irlgram, W M. one, of the above. Leaf hairs from Suggested Classification of Algae and texrestrial plants are also confusing to Protozoa in Sanitary Science. Sew; &''Ind. Wastes. 27:1183-88. 1955. ," , 142 Animal Plankton

8 Pennak, R. W.Freshwater Invertebrates 10 Welch, P. S.Limnology, McGraw-Hill of the United States. The Ronald Press, Book C'o., Inc., New York.1935. New York. 1953.

9Sverdrup, IL W. , Johnson, M. W. , and Fleming, R. H.The Oceans, Their Physics, Chemistry and General This outline was prepared by-H. W. Jackson, ,Biology.Prentice-Hall, Inc., New York. former Chief Biologist, National Training

1 1942. Center, and revised by R. M. Sinclair, Aquatic Biologist, National Training Center MOTD. OWPO, ,USEPA,_ Cincinnati, Ohio 45268. Descriptor: Zooplankton

re

1 A t

Anim1 Plankton

PhylumPROTOZOL 3/4 - Free Living Representative.'

I. Flagellated Protozoa, -C sae Mistigophora

6

Antholihysis. L Pollution tollerant Pollution tollerant 6,u 19 A.. Colony of Poteriodeidron Pollution tollerant, 35jz .t;$

II. Amsboid Protozoa; Class Saroodina

to

Dimastizamoeha tiuolearia,reported Diffluxia Pollution tollerant to be intollerant of Pollution tollerant 1067,50)u pollution,-451u. 601563.14 _III. Ciyied-Protozoa, Class'CiliophoraI

Coliod; Nolovhrva,reportod Aaitvlis, pollution Pollution tollerant to'be intollerant of tollerant. Colonies often, 20-120)4 pollution, 35,E maprosoopio.

H.W.Jaokson

6/' 4. Ahimal Plankton

PLA NKTONIC PROTO ZOA

Peranema trichophorum

, A r cella vu garis A ctinosphad r him

."11.%:..

J, A

; Godonella _Tintnidium Vorticeila cratera fluviatile

(., Animal Plankton

PLANKTONIC ROTIFERS *

. $ 'Various Fornof Keratella cechlearis 4,

a 11.4' a

a

b s Synchaeta PolyarthrA Brachionus pectknata vulgaris quadridentata I

0 Rotaria sp

4 Animal Plankton 1

SOME PLANKTONIC CRUSTACEANS a

CRUSTACEA

4

_A NatiPtiis larva of a Copepod 1-5 mm

, 14, Copepod; Cyclops, Order Copepoda

2=.3 mm .

Water Flea; -At Order Cladocera Daphnia 2-3 mm'

OSTRA CODE

Left: Shell closed Right: Appendages extended 1-2 mm

147 - Aninial Plankton

st

PLANKTONIC 4FiTHR OP ODA

A mysid shrimp - crustacean A water mite - arachnid

.

Chaoborus midge larva - Insect 174,"

...

a

/- Aspects in the life cycle of the human tapeworm, Diphyllobothrium Tatum, Class Cestoda. A.adult as in human/ intestine; B. procercoid larva in copepod;C. plerocercoid larva in flesh of pickerel (X-ray view).

. 9 H. W. Jackson

S. O

10 143 D

PREPARATION AND ENUMERATION OF PLANKTON IN THE LABORATORY

9

`I RECEPTION AND PREPARATION OF, The amount of sodium borate and SAMPLES merthiolate may be vAried slightly to adjust to different waiters, climates, A Preliminary-sampling arid analysis is an. ana organic, contents.' essential preliminarS, to the establish- ment of a permanent or- semi-permanent' 3 "Prepae a saturated aqueous. Lugol''S 'program. solution- as follows:

B Concentration or sedimentation of pre- aAdd 60 grams of potassium iodide served samples 'may precede analysis. (IP) and 40 grams of iodine crystals to 1 liter of distilled water. 1 Batch centrifuge , 4 Prepare the preservative solution by1 2 Continuous centrifuge adding approximately 1. 0 ml of the Lugol's solution to 1 liter of merthi-. 3 Sedimentation olate stock.

CUnpreserved (living) samples should be analyzed at once or refrigerated fo IIISAMPLE ANALYSIS future analysis.

II PREPARATION OF MERTHIOLATE A Microsc'opic examination is most frequently PRESERVATIVE employed in the laboratory to determine 7 what plankton organisms arepresent and -NI_ A The Vater Pollution Surveillance System bow many there .are: - of the FWPCA haS developed a, modified merthiolate preservative.(Williams, 1-Optical equipment need not be elaborate 1967)Sufficient stook to "make an approx- but should include. imately 3. 5% solution in the bottle when filled is placed in the sample bottle in aCompound microscope with the the laboratory.`The bottle is then_ filled' following equipment: # witli water in the \isteld and returned to the laboratory fornalysis,. 1) Mechanical stage 2)- Ocular: 10X, with :Whipple type B Preparation of Mertbiolare Preservative counting eyepiece or reticule 3) Objectives: Merthiolate isavailablelfrommany approx. 10X( 16mm) chemical laboratorsupply houses; aaprox,20X(8 mm). one should specify he water 'soluble approx.40X(4 mm) sodium salt. approx.95X( 1. 8 mm)( optional).

2 Merthiolate stock:d, -solve approxi-- A 40X objective with a working mately 1.5 dram of sodium borate distance of 12,8 mm and an erect. (borax atd approximat ly 1 gram of image may be obtained as special merthiolate in 1 liter odistilled water. eqUipment.A water, immersion objective (in addition to oil) .might be considered for use with water mounts. k S Binocular eyepieces are 'optional.

MIC. enu. 15f. 6.76 14-4_ 0 Z

Preparation and Enumeration40 of Plankton t, -A r

Stage micrometer (this may be 3 Direct 'counting of the unconcentrated' borrowed,if necessary, as it is sample eliminates manipulation, saves usually used only once, when the time, and reduces error.If frequency -equipment .is calibrated) . of organis is low, more area may

. need to bexamined or concentration Inverted microscopes offer certain, of the, sampmay be in ortfe'r. advantages but a r efoot widely available.The same is true of 4 Conventional techniques emplpying some of the newer optical systems, concentration of the sample provide more such as phase contrast microscopy. organisms for observation, but because These are often excellent but ex- they involve more m nipulations, pensivefor routine plant use. additional errorand fake moremore time. 2 Precision made counting chambers 4-- are required for quantitative -work with liquid mounts. CSeveral methods of counting plankton are in 'general use. aSedgwick- Rafter cells' (hereafter referred to as S-R 'cell) are used 1 The anumerical or clump count is for routine counts of medium and regarded as tile simplest. larger forms. aEvery organism observad must be' bExtremely small forms or "nanno- enumerated.If it cannot be identi- plankton" may be counted by use of fied, assign a symbol or number the nannOplankton (or Palmer) cell, and make a sketch of it on the back . a Fisher- Littman cell,a hemacytp- of the record sheet. meter, the Lackey drop method, or by use of an inverted microscope. bFilaments, colonies and other associations' Of cells are counted g Previous to starting serious analytical as units., equVr to single isolated work, the microscope should be cali- cells._Their identity as indicated brated as described elsewhere. Di- on the record sheet is the key to mensions of the S-R cell should also the significance' of such a count. be checked, -eecially thdepth/ Individual cell 'count.In this method, 4 Autom isrticle cou ters may be every cell of every colony or clump . useful for c9ccoid organisms, of organisms is- counted, as well as each individual single-lled organism. B Quantitative Plank-Ion Counts The_ areal standard unit method rs L certain technical advantages, but als 1 All quantitative eounting techniques involves certaininherrent difficultie involve the filling of a standard cell of known dimension's with either aAn areal standard unit is 400 square straight -sample or a concentrate or microns.This is the area of one dilution .theieof. of the smallest subdivided squares in the center of the Shipple eyepiece 2 The organisms in a predetermined at a magnification of 100:X. number. of microscope fields or other known area are then observed, and by bIn .operation, the nurnberf areal means of a' suitable series of multi- units of each species is recordedr on plier factors, projected to a number or the record. sheet rather than'the quantity per ml gallon,etc.' number of indiviiduals.Average ar=eas of the common spe'cies are

14 -2 t Preparation And Epuinberation of Plankton

k

are sometimes printed on record after each group of fields are counted sheets for a particular plant to and making up to 5 additional such obviate the necessity of estimating counts.This may not be practical with the area .of each cell- observed high counts. individually. 7 The strip count.When a rectangular cThe advantage of they method lies in slide such as the S-R cell is used,a,. the cognizance taken of the relative strip (or strips) the entire length (or masses of the various species as- known portion Thereof) of the cell may indicated by the area presented to be counted instead of separate isolated the viewer.These arel.s.,however, fields.Markinahe bottom of the cell. are often very difficult to estimate. by evenly, spaced cross lines as ex- .... plained elsewhere greatly facilitates 4The ,cubia standard unit method is counting. . logical extension of the areal met od, JJ but ha§ achievedness acceptance. a When the count obtained is multi- plied by the ratio of the width of 5 Separate field "count the strip counted to the width of the cell, the product is the esti- aIn counting separate fields,the mated number of organisms in the -'question always .arisee as to how cell, or per ml. to count Organisms touching or crossed by the edge of the Whipple bWhenthe material in the cell is field. Some 'workers *estimate the unconcentrated sample water, this eproportion of, the organism lying count- represents the condition of inside the, field as compared to -that the water being evaluated withobt outside.°nit), those which are over further calculation, half way inside are counted.'Y./ 8 Survey count.A survey count is an bAnother system is to select two examination of the entire area of a adjacent sides of the square for volumetric cell .using a wide field low The objective is O reference,. such as the top and left power microscope. boundaries.Organisms touching to locate and record the larger ,forms, these lines in any degree, from especially zooPlankton such as copep outsid e or inside, are then counted, or large rotifers whi2h may be present while organisms touching the opposite in size.Special large capacity cells sides fire ignored.It is important are often employed for this purpose. to adopt some such system and For still larger marine forms; numerous adhere to it consistently. special devices have been created. .

cIt is suggested that if separate 9 Once a procedure for "concentration 101P 'microscopic fields are examined,, and/or counting is adopted by a plant a standard number of ten be adopted. or other organization it should be These should be evenly spaced in two used consistently from then on so rows* about one-third of the distance . that results from year to year can be down from the top and one-third of compared. the distance lip frc he bottom of the S-R cell. rzDifferential or qualitative "counts" are 6 Multiple area count.,This is an ex- ' essentially lists of the kinds of organisms. tension of the separate field count. found. A Considerable increase in accuracy has recently been shown to accrue by entptying and refining the S-R sell

ti Preparation and. Enumeration of Plankton 1. Jr

E It9portional or relative counts of special eDifficulties include a predilec- groups are often very us%itilth .For ex- tion of extremely fine membranes- ample, diatoms'. is b always to clog rapidly with silt or . count a standard numbers of cells. increase in plankton counts, and the difficulty of making observations F Plankton are sometimes measured by on individual cells when the means other than microscopic counts. organisms are piled on top of each other.It is sometimes necessary 1 Settled volume of killed plankton in an to dilute a sample to obtain suitable Irnhoff-,cone may be 'observed after a distribution. standard length of time.This will evaluate primarily only the llsher forms.IV SUMMARY AND CONCLUSIONS

2 A gravimetric-method employs drying A The field' sampling program should be at 60° C for 24 hours followed by ep carefully planned to evaluate all significant ashing at 6000 C for 30 minutes.This locationin the reservoir or stream, is particularly useful for chemical giving e consideration to the capacity and radiochemical analysis. of the laratory.

3 Chemical and phySical evaluation of. B Adequate records and notes- should be plankton pdpulations employing various made of field conditions and associated instrumental techniques are coming - to lit h the laboratory analyses in a be widely used.Both biomass and productivity rates can be measured. permanent file. Such determinations probably achieve C Optical equipmentin the -laboratory should their greatest utility when coordinated be calibrated. with microscopic examination. D Once a procedure for processing plankton 4The membrane (molecular) filter has is adopted,it should be used exclusively a great- potential, but a generally by all workers at the plant.. acceptable technique has yet to be perfected. ESuch a procedure should enable the water plant operator to prevent plankton troubles aBacteriological techniques for or at least to anticipate them and have coliform determination are corrective materials or equipment stockpiled. widely accepted. 'bNematodes and larger organisms can readily be washed off of the REFERENCES membrane after filtration. 1-Ely Lilly Company.Merthiolate as a Preservative.Ely Lilly & Co. c 'els also being used to measure ultraplankton that pass treatment Indianapolis 6,Indiana. plant operations. - 2 Gardiner, A. C!Measurement of, . Phytoplankton PopulationWY-the dMembranes can -be clearedand .Pigment Extraction Method.Jour. organisms depbsited thereoni'; observed directly, although 4.3 Marine Biol. Assoc. 25(4):739-744. 1943. accessory staining is desirable. 3 Goldberg,E. D. ,Baker, 1VI. ,and Fox, D. L. Microfiltration in Oceanographic Research Sears. Foundation.'Jour: Mar. Res.11:194-204. 1952.- 'D

152

C.- Preparation and Enumeration of Plankton

4' Ingram, W. M., and Palmer, C. M. '7 Weber, C.I.The Preservation of Simplified Procedures for Collecting, Plankton Grab Samples.-Water Examining, and Recording Plankton Pollution Surveillance Systems, in Water.Jour. AWWA; 44(7): Applications and Develppment Report 617-6'24. 1952. No. 26, USDI, FWPCA, Cincinnati, ° Ohio. 1967, 5 Jacksti, H.W Biological Examination (of plankton) Part III in Simplified 8 Williams, L. G. Plankton Population Procedures for Water Examination. Dynamics.National Water Quality ,AWWA Manual M 12.Am. Water Network Supplement 2. U.S. Public Works Assoc. N.Y. 1964. Health Sy.vice Publ. No. .663.1962

6 Lund,J. W. G., and Talling,J.F. 9 Wohlschag, D.- D., and Hasler, As'.D. Botanical Limnological Methods with Some Quantitative ASpects of Algal Special References to the Algae Growth in Lake Mendota. Ecology. Botanical Teview.23(8&9):489-583; 32(4):581-593. 1951 October,1957.

This outline was prepared by W. Jackson, former Chief Biologist, National Training Center, and revised by R. M. Sinclair, Aquatic IJologist, National Training.Center,, MOTD,OWPO, USEPA-, Cincinnati, Ohio 45268 Descriptor: Plankton

153 14 -5 .r.A-T-TACHED GROWTHS (Periphyton or AUfwuchs)

I The cOmmunitS of attached microscopic IITERMINOLOGY plants and animals is frequently investigated. durtng water quality studies.The attached A Two terms are equally valid and commonly groWth community (periphyton) and suspended in use to describe the attached community growth community. (plankton) are the principal of organisms. Periphyton literally ,means primary producers in waterwaysthey con- "around plants" such as the growths over- vert nutrients to organic living materials and growing pond-weeds; through usage this store light originating energy through the term means the attached film of growths processes of photosynthesis.. In extensive that rely on substrates as a "place-to- deep waters, plankton is probably the pre- .,grow" within a waterway. The components dominant primary- producer.In shallow lakes, of this growth assemblage consists of ponds, and rivers, periphyton is the predominant plant's, animals, bacteria, etc. Aufwuchs primary producer.During the past two is an equally acceptable term (probably decades, 'investigators of microscopic originally proposed by Seligo (1905)]. organisms have increasingly placed emphasis Aufwuchs is a German noun w ithout on periph)tic growths because of inherent equivalent english translation; it is advantages over the plankton when interpreting essentiall) a ,collective term equivalent data from surveys on flowing waters: to the above American (Latin root) term Periphyton.(For convenience, only, A Blum (1956) ". 4 workers are geninally PERIPHYTON, with its liberal modern agreed that no distipctive association of meaning will be used in this outline,. ) phytoplanktdn is found in streams, although there is some e% idence of this for individual 13 Other terms, some rarely encountered in zooplankters (animals) and for a few the literature, that are essentially ^ individual algae and bacteria.Plankton, synonymous with periphyton or describe organisms are often introduced into the important and dominant components of the current from impoundments, backWater periphytk community are: Ne.eiden, areas or stagnanf arms of the streain.... Bewuehs, Laison, Belag, Besatz, attached, Rivers whose plankton is not dominated by sessile, sessile-attached, sedentary, species from upstream lakes or ponds are seeded-bn, attached mterials, slimes, likely .to exhibit a majority; of forms which slime-growths, and coatings... have been derived from the stream bottom directly and which are thus merely The academic community occasionally facultative or opportunistic plankters, " employs terminology based on the nature of the substrates the periphyton grows on i"B "The transitory nature of stream plankton (Table 1). makes it nearly impossible to ascertat which point upstream agents producingl TABLE 1 changes in the algal population were Periphyton Terminology Based introduced, and whether the changes on Substrate Occupied occurred at the sampling site or at some Substrate unknown point upgtream.In contrast,i Adjective bottom algae (periphyton) are true com- various epiholitic, nereiditic, sessile plants epiphytic ponents of the stream biota.Their , sessile-attached mode of life subjects animals epizooic them to the quality of water continuously wood epidenditic, epixylonic flowing,,over them. -By.observing the rock epilithic longitudinal distribution of bottom algae [ After Srarneck-Husek (1946) andis Slacleckuva within a stream, the sources of the agents (1962)j Most above listed latin-root adjectives producing the change can be traced arse derivatives of nouns Such as ethihola, (back- 'tracked)" (Keup, 1966). epiphyton, BI. IVIIC. enu. 19b. 5.80 154. 111 Attached Growths (Periphyton or Aufwuchs)

IIIPeriphyton, as with all other components ARTIFICIAL SUBSTRATETLACEMENT o'f the environment, can be sampled quell.- tatively (what is pregent) and quantitatively A Position or Orientation (how much or many are present). 1 Horizontal - Includes effects of%'-ettled A Qualitative sampling can be performed by materials. many methods and may extend from direct examination of the growths attached to a 2 Vertical - Eliminates many effects of substrate to unique "cuttings" or scrapings. settled materials. It may also be a portion of quantitative sampling. B Depth (light) - A substrate placed in lighted waters may not -reflect conditions in a B Quantitative sampling is difficult because waterway if much of the na ural substrate it is nearly impossible to remove the (bottom) does not receive li receives entire community from a standardized or light at reduced intensity.(Both excessive unit area of substrate. light and a ,shortage of light can inhibit growths and influence the kinds of organisms 1 Areas scraped cannot be determined present. ) precisely enough when the areas ape amorphous plants, rocks or logs that C Current is Important 4 serve as the principal periphyton substrates. 1It can prevent the settling of smothering materials. **2 Collection of the entire community within a standard area usually destroys individual 2 flushes metabolic wastes away and specimens thereby making identification introduces nutrient s.to the colony. difficult (careful'scraping can provide sufficient intact individuals of the species present to make qualitative determinations); VITHE LENGTH OF TIME THE SUBSTRATE or the process of collection adds sufficient IS EXPOSED IS IMPORTANT. 'foreign materials (i. e. detritus, sub- strate, etc. ) so that some commonly AThe growths need time to colonize and employed quantitative procedures are develop on the recently introduced not applicable. substrate. dsa, a- Established growths may intermittently

IVArtificial substrates are a technique break-away from the substrate because . ,designed to overcome the problems of direct of current or weight induced stresses, or sampling. They serve their purpose, but "over-growth" may "choke" the attachment cannot be used without discretion.They are layers (nutrient, light, etc. restrictions) objects standardized as to surface area, which then weaken or die allowing release texture, position, etc. that are plaCed in the of the mass. waterway for pre-selected time periods during which periphytic growths accumulate. They- _C A minimum of about ten days is required are usually made of inert materials, glass to produce ,sufficient growths on an being most common withzlastiesisecond in artificial substrate; exposures exceeding freqUency. Over fifty various devices and a longer time than 4-6 weelss,may produce methods of suppoil or suspension of the "erratic result" because of:sloughing or ., A substrates have been devised (Sladeokova,:--.-,," the accumulation of senile growths in 1962) (Weber, 1966) (Thomas, 1968). situations where the substrate is artificially protected from predation and other environmental stresses.

155 Attached Growths (Periphyton or Aufwuchs)

r

VIIDetermining the variety of growths present: 4 Nutrient analyses serve as indices of is presently only practical with microscopic 4" the biomass by measuring the quantity examinatioh. ,(A few,micro-chemical pro- of nutrient incorporated. . ,cedures for differentiatiOn'show promise, but, are only irrthe early, stages of development. ) a Carbon 1) Total organic carbon VIIIDETERMINING THE QUANTITY OF. GROWTH(S) 2) Carbon equivalents ("' A Direct enumeration of the growths while b Organic nitrogen attached to the substrate can be used, but

A is-restricted to the larger organisms c Phosphorus - Has limitations because (1) the problem' of maintaining because cells can stare excess material in-an acceptable condition under above immediate needs. the short working distances of thecobjectivd lenses on' compound microscopes, and d Other (2) transmitted iight is not adequate, because of either opaque substrates a/or 5 Chlolophyll and other bio -pigment the density of the colonial growths. extractions.

B Most frequently, periphyton is scraped -_ Carbon-14 uptake from the substrate and then processed according to several available procedures, Oxygen production, or respiratory the selection being based on theneecneed, 1 oxygen demand use of the dttta. 4 .

1 Aliquots of the sample maybe counted IX EXPRESSION OF 11:OULTS using methods frequently. employed iri `plankton analysis. A Qualitative

4 Number of organisms 1 Forms found b Standardized units 2 Ratios of number per group found 3 Frequency distribution of varieties c V.olumetric units ACM found 4 Autotrophic index (Weber) 2 Gravimetric 5 Pigment diversity index (Odum) a Total dry weight of scrapings B Quantitative,', b Ash-free dry weight (eliminates 1Areal basisquantity per square inorganic sediment) inchrfoot, centimeter, or meter. Fcir example: c A comparison of total and ash-free a16 mgs/sq.',inch dry, weights b16, 000 cells/sq. inch.. , 3 Volumetric, involVing centrifugation of the scrapings to det(ermirie a packed 2Rate basis. For example:/Z.. biomass volume. a .2 mgs/day, of biom accumulation b1 mg 02/mg ofowth /hour

*4 15G 15,3 Attached Growths (Perinhyton or Aufwuchs)

REFERENCES 7Thomas, N.A.Method for Slide Attachment in Periphyton Studies. 1 Blum, J. L.The Ecology of River Algae. -..e\slylanuscript.1968, Botanical Review. 22:5:291. 1956. 8 Weber, C.I. Methods of Collection and 2 Dumont, H. J.A Quantitative Method for Analysis of Plankton and Periphyton the Study' of Periphyton.Limnol. Samples in the Water Pollution Oceanogr.14(2):584-595. Surveillance System. Water Pollution Surveillance System Applications and 3Keup, L. E. Stream Biology for Assessing. Development Report No. 19; FWPCA, Sewage Treatment Plant EfficiencY. - Cincinnati.19 +pp. (multilith).1966. Water and Sewage Works. 113:11-411. 1966. 9 Weber, C. I. "Annual Bibliography Midwest Benthological Society. 4Seligo, A.Uberden Ursprung der Periphyton. ,1014 Broadway, Fischnahrung.Mitt. d. Westpr. Cincinnati, OH 4520'2. Fisch.V. 17:4:52.1905. 10 Hynes, H. B,N. The Ecology of Running 5 Sladeckova,. A. Limnological Investigation Waters. Univ. Toronto Press. -Methods for the Periphytoh Community. 555 pp. 1870: .0 Botanical Review.28:2:286. 1962. 11Spoon, Donald M.Microbial Communities o the Upper Potomac Estuary: The 6 ,Srameck-Husek. (On the Uniform Aqfwuchs in: The Potomac Estuary, Classification-of Animal and Plant Biological Resources, Trends and ,Communities in our Waters). Options.1CP1113 Tech Pub. No. 76-2, Sbornik MAP.20:3:2,13. Orig. in Czech. 046. 1976.

4. 12 Whitton, B.A."Plants as Indicators of River Water Quality. "(Chapter 5 in Biological Indicators of Water I Quality.Ed. 5, James, A. and Evison, Lilian Wiley.1979

This outline was prepared by Lowell Keup, Chief, Technical Studies Branch, Division of Technical Support, EPA, Washington, DC 20242 Descriptor: Periphyton I.

.157 J

EFFECT OF WASTEWATER TREATMENT, PLANTEFFLUENT ON SMALL STREAMS

I-*BENTHOS A,iORGANISMS GROWING 5 .Meioiauna ON Oh ASS° ATED PRINCIPALLY WITH THE OTTOM OF WATERWAYS Meiofauna occupy the interstitial zone (like between sand grains) in benthic Benthos is the noun. anti hyporheic habitats'.They are inter- mediate in size between the micrOfauna Benthonic, benthal and benthic are (protozoa and rotifers) and the macro - adjectives. fauna (insects, etc. ).They passa No. 30 / sieve (0.-5 mm approximately).In fresh- IITHE BENTHIC COMMUNITY water they include nematodes, copepods, tardigrades, naiad worms, and some flat A Composed of a wide variety of life worms. -They are usually ignores in fresh- forms that are related because they water studies, since they pass the standard occupy "common ground"--substrates sieve and/or sampling devices. of oceans, lakes, streams, etc. They may be attached, burrowing, or 6 Macrofauna (macroinvertebrates) move on theinterface. t Animals that aret retained on a No. 30 mesh 1 Bacteria sieve (0. 5 mm approximately).This group includes the insects, worms, molluscs, and A wide .variety of decomposers work occasionally fish.Fish are not normally on organic materials, breaking them considered as benthos, though there are bottom down to elemental or simple corn- dwellers such as sculpins, settles pounds. darters, and madtotns. 2 Algae B;The ,benthos is a self-contained community, though there is interchange with other Photosynthetic 'plants having no true communities. For example: Plankton roots. stems, and leaves.The basic settles to it, fish prey on it and lay their producers of food that nurtures the eggs there, terrestrial detritus and eaves animal components'of the community. are added to it, and many aquatic insects migrate from it to the terrestrial environ- ment for their mating cycles. 3 Flowering Aquatic Plants, (Riverweeds, yondweeds) CIt is an in-situ water quality monitor. The low mobility of the bioticcomponents requires The largest flora, composed.of . that they "live with" they:qualitychanges of the complex and differentiated tissues. -overrpassing waters. ChanOsimposed in the May be emersed, floating, or sub- long-lived componentsremain visible for mersed according to habit. extended periods, even after thecause has 4 Microfauna been eliminated. Only timewill allow a cure for the community by drift,reproduction, and re- J cruitment from the hyporheic Animals that pass through a U. S. zone; . Standard Series No. 30 sieve, but are retainedon a No. 100 sieve. D Between the benthic zone (substrate/water E'Xamples are rotiferS.and micro- interface) and the underground water table crustaceans. Some forms have is the hyporheic zone. Thereis considerable organs for attachment to substrates, interchange from onezone ,to another. , while others burrow into soft-niateri.I or occupy the interstices between rocks,III HISTORY OF BENTHIC OBSERVATIONS flfral or faunal materials. A Ancient literature recordsthe vermin associ-, ated with fouled waters. 16-1 BI. MET. fm. 8j.. 5.80. 153 Effect of Wastewater Treatment Plant Effluent on Small Streams

- . B 500 -year- oldlishing lit rature refers 3 Consumers to animal forms that are fish food and used as bait. a Detritivores and bacterial feeders C The scientific literature associating b Herbivores biota:to water pollution problems is over 100 years old (Mackenthun and c Predators Ingram, 1964). CEconomy of Survey , D-Early this century, applied biological investigations were initiated. 1 Manpower

1. The entrance of state boards of health 2 Time into water pollution control activities. - 3 Equipment 2 Creation of state conservation agencies. DExtensive Supporting Literature 3 Industrialization and urbanization. E Advaniages of the 4 Growth of limnological programs, at universities. .1 Relatively sessile E A decided increase in benthic studies 2 Life history length occurred in the 1950's anifinuch of today's activities are strongly influenced 3 Fish food organisms by .1evelopmental work conducted during this period. Some of the reasopS for this 4 Reliability'of Sampling are: . 5 Dollars/information 1 Movement of the universities from "academic biology" to applikd 6 Predictability pollution programs. , 17 Universality 2 Entrance of the federal government 8 Sensitivity to.perturbation into enforcement aspectS of water - pollution control. .F "For subtle chemicalchanges, unequivocal_,data, and observations 3 A rising economy and the development suited,th some statistical4evaluatipn will of federal grant systems. be needed.This requirement favors the macrofauna as' a parameter. Macro- 4 Environmental Protection Programs invertebrateiare easier to sample are a current stimulus. reproductively that) other organisms, numerical estimates are possible and IV WHY THE BENTHOS?.. taxonomy needed,for synoptic invest- gatioris is withirithe reach of a non- A t is a natural monitor . specialist." (Wuhrmann) BThe' community contains all of the components of an ecosystem. G "It is self-evident that for a multitude of non-identifiable.though biologically active 1 Reducers changes of chemical conditions in rivers, a bacteria small organisms.with high physiological b fungi - differentiation are most responsive. 2 Producers (plants) Thus. the small macroinvertebrates (e. g. insects) are doubtlessly the most sensitive organisms for demonstrating

vot 259 , 4

Af r, Effect of Wastewater-Treatment Plant Effluent on S 11 Streams

unspecified changes of water As water quality improves, these chemistry called 'pollution' reappear inlhe same order. Progress in knowledge on useful, autecological properties of B The Number of Survivors Increase organisms or of transfer of such knowledge 'into bioassay:practice 1Competition and predation are reduced hasbeen very small in the past. betw.pen different species. Thus, the bioassay concept (relation of organisms in a 2 When the pollutant is a food (plant's, stream-to water quality) in fertilizers, animals, organic 'materials). Water chemistry has'brought not much more than visual demon- C The Number of Survivors Decrease stration of a few overall chemical effects. Our capability to derive The m tevial added is toxic or has no chemical conditions- from biOlogical food va ue. observations is, therefore, almost on the same level as fifty years ago. 2 The material added p'roduces toxic In the author's opinion it is idle to conditions as a byproduct of decom- expect much more in the future because position (e.g., large organic loadings of the limitations inherent to natural bio- produce angnaerobicevironment assay systems (relation of organisms resulting in the production of toxic in a stream to water quality)." (Wuhrmann) sulfides, methanes, etc. ) 4, D The Effects May be Manifest in Com- V REACTIONS OF-THE BENTHIC NINCRO- binations COMMUNITY TO PERTURBATION 1 Of pollutants and their effects. A Destruction of Orgariism Types 2 Vary with longitudinal distribution in a stream. (Figure 1) _1Beginning, with the most sensitive forms, pollutantskill in order of E Tolerance to Enrichment Grouping sensitivity until the most tolerant form (Figure 2) - is the last survivor. This results in a reduction of variety or diversity of Flexibility must be maintained in the organisms. establishment of tolerance lists based on the response of organisms to the 2 The generalized order of macro- environment because of complex relation- inyertebra- te disappearance on a ships among varying environmental sensitivity soale,below pollution conditions., some general tolerance=. sources is shoign in Figure 2. patterns can be established.Stonefly and mayfly nymphs, hellgrammites., Water Stoneflies ater and caddisfiy larvae represent a grouping Quality Mayflies ality (sensitive or intolerant)'that is generally DeterioratingCaddisflies mproving quite sensitive to environmental. Amphipods changes.Blackfly larvae, scuds, sow - -Isopods bugs, snails, fingernail clams, dragon- ; Midges flyskana damselfly naiads, and most kinds of midge larvae are facultative Oligochaete s (or intermediate) in tolerance. Sludge-worms, some-kinds of midge larvae (bloodworms)t.t and some leeches

160 16-3 Effect of Wastewater Treatment Plant Effluenton Small Streams

are tolerant to comparatively wavy loads DIRECTION Of FLOW 7* of organic pollutants. Sewagp mosquitoes C. and rat-tailed maggots are tolerant of anaerobic environments for they are essentially air-breathers.

FStructural Limitations e

cc 1 The morphological structure of a tu a) species limits the type of environment z it may occupy.

D. a Species with complex appendages and exposed complicated respiratory structures, such as stonefly I- nymphs, mayfly nymphs, and caddisfly larvae, that are subjected to a constant deluge of settep.ble particulate matter soon abandon the_polluted area because of the constant preening required to main- tain mobility or respiratory func- tions; otherwise, they are soon TIME OR DISTANCE smothered. .NUMBER OF KINDS ,NUMBER OF ORGANISMS b Benthic animals in depositing zones ,SLUDGE DEPOSITS , may also be burdened by "sewage Fciur basic responsei of bottom animals to pollution. fungus" growths including stalked A. Organic wastes eliminate the sensitive bottom animals protozoans. Many of,these stalked and provide; ood in the form of sliidges for the surviving toldr- protozoans are host specific. ant, forms. B. Large quantities of decomposing organic wastes eliminate sensitive bottom animals and the excessive qucmti ties of byproducts of organic decomposition inhibit the tolerant 2 Species without complicated external forms: in time, with natural stream purification, water quality structures, such as bloodworms and improves so that the tolerant forms can flourish, utilizing thii sludgeworms, are not so limited in sludges as food. C. Toxic materials eliminate the sensitive adaptability. A bottom animals; sludge is absent and food Is restricted to that naturally occurring in the stream. which linos the number of tolerant surviving forms. Very toxic materials mayeliminate a A sludgeworm, for example, can all organisms below a waste source. D. Organic sludges with barrow in a deluge of particulate toxic materials reduce the number Of kinds- by eliminating organic matter and flourish on the sensitive forms. Tolerant 'survivors do not utilize the organic sludges because the toxicity restricts their growth. abundance of "manna." Figure 1 b Morphology also determines the species that are found in riffles,on vegetation, on the bottom of pools, or in bottom deposits.

J

16 -4 Effect of Wastewater Treatmen$ Plant Effluent op Small Streams f

VI SAMPLING PROCEDURES O

A Faufta. The Surber sampler is designed for sampling riffle areas; it reqtkires 1 Qualitative 'sampling determines the moving water to transport dislodged variety of species occupying an area. organisms into its net and is limited Samples may betaken by any method to depths of two feet or less. that will capture representatives of the species present.Collections from Kick samples of one minute duration will such samplings indicate changes in the usually yield'around 1,000 macroinyert, environment, but generally ,do not ebrates per square meter L10.5 X a one accurately reflect the degree of change. minute kick= organisms/mh). Mayflies, for example, maybe re- duced from 100 to 1 per square meter. 3 Manipulated substrates (often referred to Qualitative data would indicate the as "artificial substrates") are. presence of both speciest .but might not placed in a stream and left for a specific necessarily .delineate the change in pre- time period.I3enthicjcroinvertebrates dominance from mayflies to sludge- readily colonize these forming a manipu- worms. The stop net or kick sampling lated community.. Substrates may be con- technique is often used. structed-of natural materials or synthetic; may be placed in a natural situation or 2Quantitative sampling is performed to unnatural; and may or may not resemble, I,' observe changes in predominanc-e. the normal stream community. The The most common quantitative sampling point being that a great number, of envi- tools are the Petersen, Ekrnan, and Potter ronmental variables are standardized and grabs and the Surber stream bottom or thus upstream and downstream stations square-foot sampler. Of these, the, may be legitimately compared in. terms of ( _ Petersengrab samples the Widest variety water quality of the moving . of substrates.The Ekman grab is limited They naturally do not evaluate wliat may - to fine-textured and soft substrates, such or may not !e happening to the substrate as silt and sludge, unless hydraulically beneath said monitor.The latter could operated:.- easily be the more important.

REPREStNTATIVE,BOTTOM-DWELLING MACROANIMALS Drawings from Geckler, J.K. M. Mackenthun and W.M. Ingram,1963. Glossary of Commonly Used Biologicaland Related Terms in Water and Waste Water Control, DHEW, PHS,Cincinnati, Ohio, pub. No. 999cWP -2. (Sphaeriidae) Stonefly nymph(Plecoptera) IFingernail Clam A (Zygoptera) B Mayfly nymph (Ephemeroptera) J Damselfly naiad (A nisopt e'ra ) v C Hellgrammite or K Dragonfly, naiad Dobsonfly larvae(Megaloptera) L..,Bloodworm or midge D "Caddisfly larvae(Trichoptera) f ly larvae. (Chironomidae) E Black fly larvae(Simuliidae) M LeeCh (Hirudinea) F Scud (A mphipetla) N Sludgeworm (Tubificidac) (Isopoda) O Sewage fly larvae(Psyc4odidae) G Aquatic sowbug (Tubifera-Eristali H Snail (Gastropocla) P Rat-tailed maggot

KEY TO FIGURE 2 \

SENSITIVE

ti

G INTERMEDIATE 1",` e,6 110 Effect op-Wastewater Treatment -Paant Effluent on Small, Streams

.rF Invertebrates which are=: part o B, Flora 3. : benthos, but under certain co - become Carried downStrearn in 1Direct quaptitative sampling of natu- appreciable numbers, are, known z2411 rally growing bottom algie is difficult. Drift. MOP,' Ills basically one of collecting algae 'from a standard or uniform area of the Groups which have members forming 'bottom substrates without disturbing a4conspicjous partvf the drift the delicate growths and thereby dis -' include the insect orders Ephemeropterab, tort the,eample. Indirect quantitative Trichoptera, Plecoptera and the sampling is the best available method.-- crustacean order Amphipoda. . 2'Manipulated substrates, such as Aood- Drift net studies are widely used and blocks,f, glass or plexiglass have a proven validity in stream bricks, etc., are placed in a stream. water quality studies. Bottom-attached _algae will grow on these artificial substrates. _After two or more weeks, the artificial sub- 5 The collected sample is screened th strates are removed fo? analysis. a standard sieve to-concentrate the Algal growths are scraped from, the organiSms;_these are separated from substrates,and the quantity measured. substrate and debris, and the number Since the exposed substrate area and of each kirisVot'Organism determined. exposure periods are equal at all ot Data area` adjusted to number per unit ar,4,7els!Vy to number- of bottom the sampling sites,. differences in the meten.- quantity of algae can be related to organisztil per ii:guar changes in the quality of water flcaving. over the substrates. 6Indendently74itheCqualitativeot' quanati,ye data-, sufftee for fhorgh anal ofAn'Viron6ental conditions. VII, ANALYSES OF MICROFLO_FiA '''eitarnifiaion to, detect damage a r be made4fth either method; but- ., A Enumeration a co bination of the two gives a more pi-ece determination. If a choice must 1 The quantity of:algae on, manipulated be de, Tiantitative sampling would substrates can be measured in several beest, because it incorporates a ways.Microscopic counts of algal partial qualitative sample. cells and dry weight of a algal mater- ,7 ' ial are.long-established-methods._ _ - 7 'Studies have shown-that a significant .4. number and variety of.ma'croinve0e- 2Mieroscopic counts involve thorough bl-ates inhabit the hyporheic zone in streams. scraping, mixing and suspension of As much r6: 80% of the macrolfiverte- the algal cells. From this mixture ' brdtts may be below 5 cm in tips an aliquot of-cells is withdrawn for hyporheic zone,Mos't samples and enumeration under a microscope. sampling techniques do not penetrate Dry - weight is determined by drying the substrate below the 5 cmdepth. and weighing the algal sample. then---N All quantitative studies must take this igniling the sample Ito burn offe -and otter substrate factord'into account gal materials, leaving iner inorganic hen absolute figures are presented on materials that are again weighed. anding crop and numbers per square The difference between initial dry weight ter, etc. and weight afterignilikion is attribilted to ,algae. r 3 Any organic sediments, however, that settle on the substrate along -- 0 with the algae are processed also.

.i..+.0 1 64

-. Effect of Wastewater Treatment_Plant'Effluent-on-Small-Streanis-- Y

A.* "" Thus, if organic wastes are present Autotrophic Index Aish-free Wgt (n m2 ) appreciable errors may enter into Chlorophyll a (mg/m2),_. this method.

13 Chlorophyll Analysis 1

iDuring the past decade, chlpro,phyll analysis has become a popular method viiiMACROINVERTEBRATE ANALYSES for estimating algal growth.' Chloro- phyll is extracted from the algae and A Taxonomic is used as. an index of the quantity of algae present.The advantages of The taxonomic level to whic.l'h animals.are chlorophyll analysis are rapidity, identified depends on the needs,experience, simplicity, and.ViVid pittoriat results.. and available resources.However; the taxonomic level to which identificationsare 9 The algae are scrubbed fr,om the carried in each majorgroup should be placed substrate samples, ground, constant throughouta, given study. then each ,sample is keened in equal volumes? 90% aqueous acetone, which liBiomass ex*acts the Chldrophyll from the algal cells.The chlorophyll extracts may Macroinvertebrate biomass (weight of be compared visually. organisms per unit area) ip °a. useful quantitative estimation of standingcrop. 3 Because the cholorophyll extracts fade with time, oolorimetry should be used C Reporting Units for permanent record For routine 'records, simple 'colorimeters will Data from quantitative samplesmay be used At high cholorOPhyll _ to- obtain: densities, interference Vvith colori- metry occurs, which must he corrected 1 Total standing crop of individuals,or through seiial dilution of the sample biomass, or both per unitarea or unit or with a nomograph. volume or sample unit, and aututrOpliic 2Nilmbers orbiomass,or both, of individual The ChlosoRhyll'content of the periphyton "'taxa per unit area or unit volumeor sample is used to estimate the alkdl Inomas-sand cis :In inditA;",e,t. tmr. Inelont.onfeent for trophic status) cir"to7i-Icity of the water Data, from devices sampling a unitarea unit the taxononne-comPosition of the : \,- , of bottom willbe reported in grams dry 0 .4 community:-.1. Periphytbn growing in sur-. weight or ash5free dry weightper square fa.ce water relatively free of organic- s meter (gm/ m ), or numbers ofindi- nonillion consists largely of algae, viduals per square meter, or both.- which contain approXimately1to 2 percent chlorophyll'a by dry 'Weight.If dissolved 44; Data from multipldte samplers will-be Or partiCulate organic matter present reported in terms of the total surface In high concentratione, large popula;tions area of the plates in grams dry weight .s of filamentOut-bacteria, stalked prOtozoa, ' or ash-free dry weight or numbers of and otlidrli.rliorophyll'beai-ing micro- irdividualper square. meter,or both. organisms,,yelop and the percentage of chlorophyll is'then reduced.. 4f the 5data fr'Orn rock-filled basket samplers hioniass-chlorophyll a relationship will be reported as grams dry weight expressed as a ratiolthe autotro- or numbers of individuals per sampler, index); yallies'greater than 100 or both. .may result from orgahad'pollution (Weber and 11/IeFarlan,dq 19169; Weber,

16 -$ 165

' r Effect of Wastewater Treatment Plant Effluent on Small Streams

IX FACTORS INVOLVED IN DATA INTER- reasons, the bottom fauna of a-lake or PRETATION , impoundment, or stream pool cannot be directly compared with that of a flowing,. Two very important factors in data evalua- stream riffle tibn are a thorough kriowledge of conditions under which the data were collected and a D Extrapolation' critical assessment of the reliability of the data's representation of the situation. How can bottom `-dwelling macrofauna data be extrapolated to other environmental A Maximum-Minimum Values components?It must be borne in mind that a component of the total environment The evaluation of physical and chemical is being sampled.1-114e sampled com- data to determine their effects on aquatic ponent exhibits cha.nges, then so must the organisms is primarily dependent on other interdependent components of the maximum and minimum observed values. environment.For example, a clpan stream The mean is useful only when the data are with a wide variety of desirable bottom relatively uniform. The minimum or organisms would'be expected to have a maximum values usually create acur wide variety of desirable bottom fishes; -conditions in the environment. when pollution reduces the number of bottom organisms, a comparable reduction would BIdentification be expectedin the number of fishes.More- over, it would be logicApto conclude that , Precise identification of organists to any factor that 'eliminates 'all bottom organ- species requires a specialist in that. isms,.would eliMinate most other aquatic taxonomic groups. Many immature forms of life. A clean- stream with a wide aquatic forms have not been, associated variety of desirable bottom organisms with the adult species.Thbrefore, one Would be expected to permit a variety of who is certain of th& genus but not the recreational, municipal and -industrial uses. species should utilize the neric name, not a potentially incorr,e Gies name. E Expression of Data The method of interpret logical data on the basis of numbers of kinds 1 Standing crop 'and taxonomic composition and numbera of organismavill typically 'Suffice. Standing crop and numbe-ts of taxa (q`pes or kinds) in a community are highly C Lake and Stream Influence sensitive to environmental perturbations resulting from the introduction of contam- Physical tharacteristics of a body of inants.These parameters, particularly water also affect animal populations. standing crop, may vary considerably in Likesor impounded bodies of water unpolluted habitats, where they may range support Afferent faunal associations from the typically high standing $rop of than rivers, The number of kinds littoral zones of glacial lakes to the present in a lake may be less than that sparse ,fauna of torrential soft-water found -in a stream. because of a more streams.Thus, it is important that uniform habitat. *A lake is all pool, comparisons areznade only between truly but a river is compdsed of'both pools comparable erOironments. and riffles.- The nonflowing water of lake exhibits a more complete set- tling of particulate arganic'matter that naturally supports a higher population of detritus consumers. For these

i6 16-9. )

Effect of Wastewater11)eatmentPlant Effluenton Small Streams

2Diversity If.adequate background data are avail- able to an experienced investigator, Diversity indices are an additional tool . these techniates can prove quite useful for measuring the quality of the environ- particularly for the purpose of demon: ment and the effect of perturbation on strating the effects of gross to moderate the structure of a community of macro- organic contamination on the macro - invertebrates,:Their use is based on invertebrate community. To detect the generally observed phenomenon that more subtle changes in the macroinver- relativelPtuidtsturbed environments tebrate community, collect quantitative support communities hiving large data on numbers pr biomass of organisms. numbers of species with no individual Data on the presence of tolerant and species 'present in overwhelming intolerant taxa and richness of species abundance.If the species in such a may be effectively summarized for evalu- community are ranked on the basis of ation and presentation by means of line their numerical abundance, there will , graphs, bar graphs, pie diagrams, --rbe relatively few species with large histograms, or pictoral diagrams. numbers of individuals and large numbers of species represented by only X IMPORTANT ASSOCIATED ANALYSES a few individuals.Perturbation tends 4 to reduce diversity by making the A The Chemical Environment environment unsuitable for some species or by giving other species a competitive 1 Dissolved oxygen. advantage.. 2 Nutrients Indicator-organism scheme ("rat-tailed maggot studies") 3 Toxic materiels...-' a-=-For-thi-s-teehnique, the individual 4 Acidity and alkalinity taxa are classified on the basis of their tolerance or intolerance to '- 5 Etc. various- levels of putrescible wastes. Taxa are classified acdortling to B The Physical Environment their presence or absence of different environment's as deter- 1 Suspended solids mined by field studies.- Some reduce data based on the presence 2 Temperature or absence of indicator organisms to a simple numerical form for ease 3 Light penetration irrkesentation. 4 Sediment composition b Biologiats are engagingin fruit- ., less exercise if they intend to make 5 Etc. any decikons about indicator organisms by operating atthe XI AREAS IN WHICH BENTHIC STUDIES generie level of macroinvertebrate CAN BEST BE APPLIED identifications." (Resh and Unzicker) A Damage Assessment or Stream Health 4 Reference station methods If a stream is suffering from abuse the Comparative'or control station methods biota will so indicate. A biologist can ,compare the qualitative characteristics determine damages by looking at the of the fauna in clean water habitats with "critter" assemblage in a matter of those of fauna in habitats subject to stress. minutes. Usually, if damages are not Stations are compared on the basis,of found, it will not be necessary to alert richnqss bf species. 'the remainder of the agency's staff, 167 yr1

Effect of Wastewatei TreatmentPlant Effluent on Small Strea.rris

pack all the equipment, pay travel and per 4 Mackenthun, K. M. The Practice of diem, and then wait five' days before Water Pollution Biology. FWQA. enough data can be assembled to begin. 281 pp.. 1969. evaluation. 5 Weber, Cornelius I.,Biological Field BBy determining what damages have been and Laboratory Methods for Measurift done, the potential cause "list" can be the Quality of Surface Waters and reduced to a few items for emphasis and Effluents.U. S. Environmental Pro- the entire "wonderful worlds" of science tection Agency, NERC, Cincinnati, and engineering need not be practiced with OH. Environmental Monitoring Series the result^that much data are discarded 670/4.73.001 July 1973 later because they were not applicable to the prOblem being investigated. 6 Keup, L. E. and Stewart, R. K.Effects C Good benthic data associated with chemi- of Pollution on Biota of the Pigeon River, cal, physical, and engineering data can NorthCarolina and Tennessee. U. S. EPA, be used to predict the direction of future National Field Investigations Center.35 pp. changes and to estimate the amount of 1966.(Reprinted 1973, National Training pollutants that need to be removed from -C enter) the waterways to make them productive 7 Wuhrmann, K., Some Problems and and useful once more. Perspectives irk-Applied Limnology Mitt. Internat.Verein Limol. 20:324-402. D The benthic rnacroinvertebrates are an 1974. easiled index to stream health that citizens may use in stream improve- 8Armitag, P. D. ,Macliale, Angelu M., and ment programs. "Adopt-a-stream" Crisp; Diane C. A Survey of Stream efforts have successfully used simple Invertebrates in the Cow Green Basin macroinvertebrate indices. (Upper Teesdale) Before Inundation. FreshwaterBiol. 4:369-398.197+. E The potential for restoring biological integrity in our flowing streams using 9Resh, Vincent H.., and Unzicker, John D. macroinvertebrates has barely been - Water Quality Monitoring and Aquatic touched. Organism's: the JWPCF 47:9-19.1975. REFERENCES 1 Freed, J. Randall and Slimak, Michael W. This outline was prepared by Lowell Keup, Biological Indices in Water Quality Chief, Technical Studies Branch, Assessment. - (in The Freshwater Division of Technical Support, EPA, Potomac Acidatic Communities and . Washington, D.C.20460, and revised by Environmenta.1 Stresses). Interstate R. M. Sinclair, National Training and Commission on the Potomac'River Operational Technology Center, OWPO, Basin.1978. USEPA, Cincinnati, /Ohio 45268. 2 Hellawell, J. M. ,Biblogical Surveillance Descriptors: Aquatic Life, Benthos, Water Of Rivers. Water Research Center. Quality, Degradation, Environmental Effects, Stevenage and Medenham. 333 pp. Trophic Level, Biological Communities. 1978. 4 Ecological Distributions

:3 Hynes, .11*B.N. The Ecology of Running Waters. Univ. Toronto Press.1970 .

+,

- 163 16-11 .

4. ECOLOGY OF WASTE STABILIZATION PROCESSES

IITT >RODUCTION IITREATMENT PLANT ORGANISMS . Living organisms will live where they can live. WaStewater is characterized by overfertili- This holds for treatment plant environments zation from the standpoint of nutritional just as it does for streams, impoundments, elements, by varying-amountof items that oceans, dry or weft lands. may not enter the metabolic Vattern but have some effect upon it, such as. silt, and by A Each species. has certain limits or toler- materials that will interfere with metabolic ances, growth, feeding habits and other patterns.Components vary in availability characteristics that determine its favored from those, that are readily acceptable to habitat. . those that persist for long periods of time. Each item has some effect upon the organism B The presence of certain organisms with response to the` mixture. well defined characteristics in a viable condition and in significant numbers also A Slime forming organisms including certain provides some inference with respect to bacteria, fungi, yeasts; protista monera the habitat. and alga tend to grow rapidly, on dissolved nutrients under favorable conditions.These C The indicator organism concept has certain grow rapidly enough to dominate the overall pitfalls.It is not sufficient to base an population during early stages of growth. opinion upon one or more critters which There may be tremendous numbers of may have been there as a result of gas relatively-few species until available liquid or solid transport.It is necessary nutrients have been converted to cell mass to observe growth patterns, associated or other limiting factors check the pop- organisms, environmental conditions, and ulationexplosion: nutritional characteristics to provide it. information on environmental acceptability. B Abundant slime growth favors production of predator organisms such as amoeba or D Organisms characteristic of wastewater flagellates. These field upon preformed treatment commonly are those found in cell mass. Amoebas-tend to flow_around_ nature under low DO. conditions. Perform- particulate materials; flagellates also ate ancecharacteristics are related to relatively inefficient food gatherers. They certain organism progressionS and assoc- tend to become numerous when the nutrient iations that are influenced by food to level is high. They are likely to be assoc- organism ratios and pertinent conditions. iated with floculated masses where food One single species is unlikely to perform is more ablirdant. all of the functions expected during waste treatment. Many associated organisms C Ciliated organisms are more efficient' compete in an ecological system bona food gatherers because they have the favored position. 'The combination includes ability to move more readily and may synergistic, antagonistic, competitive, set up currents in the water to bring food predative, and other relationships that may to them for ingestion.Stalked ciliates favor predominance of one group for a time are implicated with well stabiliied effluents and other groups under other conditions. because they are capable of sweeping the fine particulates from the water between E It is the responsibility of the treatment floc masses While their re§idues tend to plant control team to manage conditions become associated with the floc. oftreatMent to favor the best attainable performance during each hour of.the day D Larger organisms tend to become establish- each day of the year. This outline con- , ed later and serve as scavengers. These. siders certain biological characteristics include Oligocha:etes (worms), Chiobomids (bloodworms and insectilaryae), Isopods and their implications with respect to (Sow bugs and crustacea), Rotifera and treatment performance. others.

*PC 10.1.0.69 17-1. Ecolog_of Waste Stabilization ProoesAes

104, III TREATMENT OPERATIONAL CONTROL E Observations of the growth characteristics and populations do not provide quantitative An established treatment pla-nt is likely to information, but they do indicate trends *pntain representative organisms from all and stages of development that are useful groups of tolerant species. Trickling filters, to identify problems.It is not possible to activated, sludge, or ponds tend to retain identify most slime organisms by direct previously developed organisms in large observation.It is posSible to recognize numbers relative to the incoming feed. The growth and flocculation characteriStics. number and variety availrquiredie.determine the Certain larger organisms are recqgnizable nature, degree and time for partial and are useful as indicator organisms to oxidation and conversion of pollutants from suggest past or subsequent developments. liquid to, solid concentrates.

A Proliferation of slime forming organisms IVILLUSTRATIONS OF ECOLOGICAL characterize the new unit because they grow SIGNIFICANCE rapidly on soluble nutrients. Predators and scavengers may start growing as soon A The first group repreents initial devel- as cell mass particulates appear but growth opment of non-flocculent growth.Single rate is slower and numbers and mass lag celled and filamentous growth are shown. as compared with slime organisms. As Rapid growth shows little evidence of slime growth slows due to conversion of flocculation that is necessary to produce soluble nutrients to cell mass, the slime a stable, clear effluent. formers tend tO associate as agglomerates or clumps promoting floculation and liquid B The next group of slidesAndicate develop- solid separation. ment of floc forming tendencies from filamentous or non-filamentous growth. 13 Overfeeding an established unit encourages Clarification and compaction characteris- rapid growth of slime organisms as individ- tics are relatable to the nature and density ual cells rather than as flocculated masses. of floc masses. This results in certain characteristics resembling those of a young, rapidly . Organisms likely to be associated with growing system. more stabilized sludges are shown in the . third grntipScavengers-esseOn- Toxic feeds or unfavorable conditions sist of a large alimentary canal with materially reduce the population of exposed accessories. sensitive organisms. The net effect is a population selection requirihg rapid regrowth D The last two slides illustrate changes in - to reestablish desired operating character- appearance after a toxic load.Scavengers, istics.The system assumes 'new growth ciliates, etc. have been inactivated. New , characteristics to a degree depending upon growth at the edge ot the floc masses are the fraction remaining after the toxic effect not apparent. Physical structure indicates has been relieved;h-y dilution, degradation, dispesed residue rather than agglomera- sorption, or other means. tionndencies.The floc probably contains D Treatment units are characterized by -living organisms protected by the surround- changes in response to-feed sequence, ing organic material, but only time and load ratio,, and physical or chemical, regrowth will reestablish a working floc conditions.Response to accutectoxiCity with good stabilization and clarification 4.--." may be immediately apparent. Chronic tendenc %s. overloading or mild toxicity may not be apparent for)several days.It may be ACKNOWLEDGEMENTS , expected. that it will require 1 t ,t3 weeks to restore effective performae after any This,outline contains significant materials from major upset.' Performance . previous outlines by H. W. 1Jackson and R. M. iteria,may Sinclair.Slide illustrations were provided by not indicate a smooth progression toward Dr. Jackson. imprc:qed operation. .

Thisoutline was prepared by F. J. Ludzack, Chemist, National Training Center,. EPA, Cincinnati, OH 45268.

170 ECOLOGY PRIMER (from Aldo Leopold's A SAND COUNTY ALMANAC)

Ecology is a belated attempt to-con

II The outstanding scientific discovery of VII Every farm is a textbook on animal the twentieth century is not television or ecology; every stream is a textbook radio, but rather the complexity_of the on aquatic ecology; conservation is land organism. the translation of the book.

LEI One of the penalties Of an ecological edu- VIII There are three spiritual dangers in not cation is that one lives alone in a world owning a farm- of wounds. Much of the damage inflicted on land is quite invisible to laymen.An A One is the danger of supposing that break- ecologist must either harden his shell ,,fast comes from the grocery. and make believe that'the consequences of science are none of his business, or B Two is thZt heat comes from the furnace. he must be the doctor who sees the marks of death in a community that believes C' And three is that gas comes from the itself well and doe's not want to be told pump. Otherwise. IX in general, the trend of the evidence IVEcosysteins have been sketched out as indicates that in land, just as in the pyramids, cycle', and-energy circuits. fishes body, the symptoms'may lie in The concept of land as an energy circuit one organ and the cause in another.The-. conveys three basic-ideas: practices we now call conservation are, to a large extentlocal alleviations of A That land is not merely sail. biotic pain.They are necessary, but they must not be confused with cures. B That the native plants and animals kept the energy circuit open.; others may or X An Atom at large in the biota is too free may not. to knokw freedom; an atom back in the sea has folgotten it.For every atom lost to C That man-made changes are of a different the seaXthe prairie pulls another out of order than evolutionary changes, and have the decaying rocks.The only certain effects more comprehensive than is intend- truth is that its creatures must suck ed or foreseen (See figures 1-4). hard. live fast, and die often, lest its losses exceed its gains. D To keep every cog and wheel is the first precaution of intelligent tinkering. REFERENCES

V The process of alteringthepyramid for 1 Leopold, Luna 13. (ed.) Round River. hilman occupation releases Stored energy, Oxford University Press.1953. and. this Often gives rise, during-the pioneering period, to a deceptive exuber- 2Leopold,' Aldo. A Sand County Almanac.' ance of plant and animal lite,both wild Oxford University Press.1966. and tame. These releases of biotic capital tend to becloud or postpone the 3United States Environmental Protection Agency. The Integrity of Water. penalties of violence. . WashingtOn, D. C.1977.

BI. ECO. 26b. 9.79 18-1 .-171 Ecology Primer (from Aldo Leopold's A Sand County Almanac)

REFERENCES (Continued) Thisoudnewas prepared bly R. M. Sincla- 4. United States Geological Survey. ogational Training and Operd.tional Technology ographic Maps" Center, OWPO, USEPA, 'Cincinnati, Ohio . Reston, Virginia.1978. 45268. Descriptor: Ecology

04.

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figure 1. . Figure 2.

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tio 0 % / ,''. .; .61- -/,0\-?tt oll Course.7,' A ,;,Landing U. 15,:,i 1 D :;:s70, /0 0:1/ ,,,/..C' 1 .1 14 ig ' Xe;. I . .' 16. .-" . ii ...... y :1 Golf Course ;)err Of().,i//' Roll Course I"? : s I ' I .! ': OCean Reef - Ocean Reef' 00 '1 lub )

1 1 Q06 , I'

.641 it5"1.::44:1.Vtri.k51.15/Ve-t Photorevised In 1969' Phototevised In 1973 li,igure 3. Figure 4: 1'2

182 I

THE LAWS OF ECOLOGY

6 -Law of the continuum.The gamut I These so-called Laws of. Ecology have of ecological niches, in a regional been collected and reformulated by unit, permits a gradual' shift" in the Dr. Pierre Dansereau. -qualitative and quantitative compo- A They have a broad range of appli- sition and structure of communities. cation. in aquatic as well as terres- 7 Law of cornering.The environ- trial ecosystems. mental gradients upon which species and, communities are ordained either B Only the ones which have strict steepen or smoothen at various times application to fresh -water organisms and places, thereby reducing utterly will be discussed. or broadening greatly that part of th5 ecological spectrum which offers the best opportunity-to organisms of II The Laws Verbatim - -adequate valence. APhysiology of Ectopic Fitness (1-9) 8 Law of persistence.Many species, Law of the inoptimum.No species especially dominants of a community, encounters in any given habitat the are capable of surviving and main- optimum conditions for all of its taining their spatial position after - their habitat,and even the climate functions.. itself have ceased to favor full vitality. 2 Law of aphasy."Organid evolu-. tion is slower than environmental 9 Law of evolutionary opportunity. The change on the average,. and hence present ecological success of a migration occurs. " species is compounded of its geo- graphical and ecological breadth, its 3 Lavr of tolerance.. A species is confined, ecologically and geo- population structure, and the nature graphically, by the extra:ids of of its harboring communities. , environmental adversities that it can withstand. B Strategy of CommunityAdjustment (10-14) Law of valence:In each part of its area, a given species shows a 10 Law of ecesis.The resources of -an greater or lesser. amplitude in unoccupied environment will first be ranging through various 'habitats' exploited by organisms with high, (or communities); this is condi- tolerance and generally with low tioned by its. requirements sand , requirementS. -,- ,tolerances being satisfied, or - 11 Law of succession.The same site nearly bvercome. will not be indefinitely held by the sate plant community, because the 5 Law of competition- cooperation. Organisms' of one or more species physiographic agents and the plants occupying the same site over a themselves induce changes in the given period of time, use (and whole environment, and these. allow frequently reuse) the same re- other pants, heretofore .unable to sources thrOugh various sharing invade, but now more efficient, to processes, which allow a greater displace the present decupants. portion to the most efficient.

19*k BI. AQ. 35.5 80 171

40,101 fork; I The Laws of Ecology YI

Law of regional climax.The correlated to climatic conditions processes of succession go of the, present as well as of the through a, shift of controls but past. 4. are not indefinite, for they tend to an equilibrium that allows no 18Law -of vegetation regime.Under further relay; the climatic-topo- a similar climate,in different graphic-edaphic-biological bal- parts of the world, a similar ance of forces results in an structural- physiognomic- functional ultimate pattern which shirts response can be induced in the from region to region. vegetation, irrespective of floristic .affinities and/or historical con- 13 Law of factorial control. 'Al:- nections. though living beings react holo-, cenotically (to all ,factors of the / 19 Law of zonal eqUivalence.Where environent in their peculiar climatic gradients are essentially conjun tion ,there frequently similar, the latitudinal and altitudinal occurs a discrepant factor which zonation and cliseral shifts of plant has controlling power through formations also tend to be; where its excess or deficiency. floristic history is essentially iden- tical, plant communities will also 14 Law of association segregation. be similar. Association of reduced composi- tion and simplified structure have 20 Law of irreversibility.Some arisen during physiographic or resources Imineral, plant,or climatic change and migration animal) do not renew themselves, through the elimination of some because they are the result of a species and the loss of ecological process (physical or bilogiCal) which' status of others. has ceased to function in a particular habitat of landscape at the present C Regional Climatic Response (15-20) rtime.

.15 Law of geoecological distribution. 21 Law of specific integrity.Since the "The specific topographical dis- lower taxa (species and subordinate tribution (microdistribution) of an units) cannot be polyphyletic, their ecotypic plant species or of a presence in Widely separated areas plantrcorninUnity is a parallel can be explained o'nly by former function of its general g,eograpical continuity or by -migration. distribution (macrodistribution), since both are determined by the . 22 Lavi-Of phylogenetiC trends.The same ecological amplittides and relative geographical positions, ultimately by uniform physiological within species (but more often genera requirements." and families), of primitive and ad- vanced phylogenetic features are good 16/, Law of climatic stress.It is atI indicators of the trends of migration. the level of exchange ,,,,b.04-cen the organism and the. environment 23- Law -ofmigration.Ge'agraphical (microbiosphere) that the stress migration is determined by population is felt which eventually cannot be piessure and/or environmental change. overcome and which willAistablish a geographic boundary. 24 .Law -cif differential evolution. eo, graphic and ecological barriers 17 Law of biological spectra.Life-, favor independent evolution,biz tthe form distribution is a characteristic divergence of vicariant pairs is not of regional floras which can be necessarily proportionate to the 'gravity, of the barrier or the duration of 41, isolation.

174 Fes- The Laws of Ecology

. 25Law of availability.The geo- Iv THE LAW OF THE EQUIVALENCE 440 graphic distribution of plants OF WINDOWS (deAssis) and animals is limited in the first instance by their place "The wayto compensate for a'Cclosed and time of origin. window is to, open another window."

26Law of geological alternation. -41P Since the short revolutioriary periods have a strong selective REFERENCES force upon the biota, highly differentiated life forms are Dansereau, P.Ecological Impact and Human are more likely to develop 'Ecology, in the book Future 4nvironinentl dUring .those times than during of,North America, edited by F. Fraser equable normal periOds. Darling and John P. Milton;The Nalural His tory press, division of Doubleday, 27Law of domestication.Plants Company;New York,' 19661970. and animals whose selection has been more or:less domi- dassis, Machado. Epitaph of a Small Winner. nated by man are rarely, able Noonday Press.1966. to survive without his continued protection. Hynes, H. B. N. The Ecology of Running Waters. University of Toronto Press. 1970. III THIENEMANN'S ECOLOPICAhXRINCIPLES These three Principles apply to stream This !outline was prepared by Ralph M.,S,inclair, invertebratesand will be noted specif- Aquatic Biologist, National Training Center, ically during your stream examinations MP&ODWPO, USEPA, CinCinnati, OH 45268. as you compare aquatic communities. A The greater the diversity of Descriptor: Ecology. the conditions in a locality the larger is the number of species which make up the biotic community. The more the conditions in a locality deviate from normal, and hence from the normal optima of. most species, the, smaller is the number of species' which 'occur there and the greater the number of individuals of each of the species which do occur. C The longer a locality has been O in the same condition the . richer I§ its biotic community and the more stable it is.

'? * V

APPLICATION OF BIOLOGICAL DATA

IECOLOGICAL DATA HAS TRADITIONA 41.X. IIIQUANTITATIVE DATA: Typical - BEEN DIVIDED INTO TWO GENERAL , Parameters of thity,pe inblude: . CLASSES: / .. ° ACountsalgae / ml; benthos/J; A Qualitative - dealing with the taxonomic fish/net/day composition oP communities Volumemm3 algae/liter B Quantitative - dealing with the population' / densityyor rates of processes occurring CWeight - dry wgt; apt}-free .wgt.. in the communities DChemical contentchlorophyll; Each kind of data has been useful in its own carbohydrate;'ATP; DNA,; et6.

E caloric equivalents)

IIQUA LITA TIV E. DA TA FProcesses - productivity;0respiration A Certain species have been identified as: IVHistorically, the chief use of statistics .1 Clean water (sensitive) or oligotrophic in treating biological data has been in the. collection and analysis of sampreS-for-these 2 Facultative, or tolerant p4meters. Recently, many methods have been devised to convert taxonomic data into 3Preferring polluted regions numerical form to permit: (see: Fjerdinstad 1964, 1965; Ganfin. Tarzwell 1956;Palmer 1963, tt,69; A Better communication between,the 'laws-60056; Teiling 1955) * biologists and other scientific disciplines B Using our knowledge about ecolcigical B Statistical treatment of taxonomtc data requirements the bioLogist may compare the species present C In the field of pollution biology these methods include: At different stations in the same river (Gaufin 1958) or lake (Holland 1968) 1 Numerical ratings of organisms on .the basis of their pollution ,tolerance In different rivers orlakes (Robertson and. Powers 1967) (saprobic'valency: Zelinka Sladecek .1964) or changes in the species in arive?: or/lake over a period of several years. (Carr (pollution index: Palmer 1969) & Hiltunen 1965; Edmondson & Anderson 1956; Fruh, Stewart, Lee & Rohlich 1066; , 2 Use of quotients or ratios of species in Hasler 1947). different taxonomic groups (Nygaard 1949). C Untilcomparatively recent times taxonomic data were not subject to statistical treat-, ment..

20-1, 17G I AppUcdL± on of BLO1b 4

Q Simple indices of community diversity: jinforation theory: a Organisms are placed in taxonomic The basic equation used for groups wHichbehave similarly under information theory applications was. the. same ecological conditions. The deyeloped by Margalef (1957). numbe'r of species in these groups E 1 N! found at "healthy" stations is corn- /.0 = log parIed to that found at "experirhental" N 2 Na ! Nb !' ..Ns ! stations.(Patrick 1950) b A truncated log normal cuve where, Iinformation/individulal; plotted on the basis a the number N ...Ns are the number of of individuals per species. species a, b,.,. (Patrick Hohn, &.Wallace 1950°. sW4dN is their sum. 4740041e4; , Sequential comparison irldex. This equation has' also beefiliSed (Cairns, Albough, Busey & Chanay with: 1968)..In this te,chniqUe,' similar organism's enco.unteredosequentially 1) The fatty, acid content of algae (McIntire, Tinsley, and Lowry 6' are grouped intoeruns". -runs Jew: SCI=total Organisms examined 2) Algal productivity (Dickman 1968).

dR.tio-sof° caroterioidiJo chlorophyll 3) Benthic biomass (Wilhm 1968) in phytoplanktoq populations:

- OD430OD iNlargalef 1968) / 665 ADD OD (Tanaka} et al 1961) - 43.5/ 670 e.The number of diatOm species present at ac, station ,1s considered indicative Of water quality, or pollution level. REFERENCES (Williams' 1964) ' numbe'r of specieS (S)- 1 " Cairns, J.Jr., Albough, D. W. , Busey, F, and Chaney, M, D. f; number of individuals W) The sequential comparison index - ,--ivriber of- .species (S) - a simplified method fornon-biologists Stiar.irelOot. of nuinb&l.of indiYiduals ( N) to estimate relative differences in fF i° biological diversity 'in -stream pollution -log-4- (MenhinickN. 1964) studies.J. Water Poll. Contr. Fed. 40(9):1607-1613. 1968. 0 E n 1) (Simpson 1949) i Changes d 2 Carr, J. F. and Hiltunen, J. K. N (N 1) in the bottom fauna of Western Lake' Erie from 1930 to1981. Limnol. where n. = number of individuals OceanOgr. 10(4):551-569.. 1965. 1 belonging to the i-th species, >. and 3 Dickman, M. Soine indices of diversity. N = tot tuber of individuals Eco1ogy49(6):1191-1193. 1968.

s4) 1

1 r-1

20 -2 .A

Tt" Tto AliCation of`BiolOical Data

EStnondson,, Anderson, G. C. 15 Menhinick, E. F.A comparison of s ome Artificiai,EutA ation of Lake species- individualsdiver'sity indices Waahingtcin. Oceanogr; applied to samples of field,insects. 1(1):47-53: 195'6. Ecology 45:p59.1964. 5. Fjerdingstad, E. 'Pollution's-of Streams estsimateclby,;5berIthal phytomicro- 16 Nygaard, G.Hydrobiological studies in- organisms-. -''.4,%h\ saprobic.,system some ponds and lakes:II.The. based on commtinitios of organisms quotient hypothesis and some new or and ecological facto1:44, Internatil, little-known phytoplankton organisms., Rev. Ges.. Hydrobiol. 49P:63-M1.1964. Mg. Danske Vidensk.Selskl. Biol. J. D Skrifter 7:1-293.1949. 6Fjerdingstad, E.Taxonomy and saProbic valency of b'enthio phytomicroi,,,, 17Patten, B. C.Species diversity in net _organisms.Hydrobiol. 50 (4):05-604. plankton& Raritan Bay.,J. Mar. Res. 20:57-75. 1962. -

7 Fruh, E. G.,Stewart, K. M ,Lee, G.7t:. iB Palmer, C. M. The effect of polltifionon And Rohlich, G.A . Measurements-* river algae.Ann. New York A.Cad. eutrophication and trends.J. WatW-,.: Sci.'108:389-395. 1963. Pail, Contr. Fed. 38(8):1237-1258. '19Palmer, C. M.A compOsite rating of algae tolerating organic pollution., I 8. Gaufin, IVA.Effects Of Pollution on J. Phycol. 5(1):78-82. 1969. nkidAyeSthrnstream,Ohio J. Sci. 7 40i:197-208. 1958. 20 Patrick, R., Hohn, M. H. and Wallace, J.I. -4 A new method for determining 9Gaufin,1 A.R. and Tarewell, C. M. AquatiO the pattern of the diatom flora.Not.

,macroinvertebrate comunities as ,Acad. Sci., No.Natl. 259. indicators of organic plution,in Lytle ,-PhiladePphia'. 1954. Creek.Sew. Ind. Ira-Sts. 213(1):906- 924.1956. " 21 tRawson, D. S.Algal.indicators of trophic 'Jake types. 7 Limnol. Oceanogr, 10/ Hasler, :-g__titrophication of lakes by 1:18-?5. 1956. domeiti& ralOge.Ecology 28(4)1383- 395.1= 22: Robertson, §. and Powers, C.F. Cdinfijarison,of the distribution of 11Holland, R,E. Correlation of Melosira organie,Irnatitet..in the five Great Lakes. species with trOphic conditions in Lake in:J.. C. A y e s., an d D. C. Chandler, Michigan.Limnol.Oceanogr. . eds.Stiftlies on the environment and 13(3):555-557. 1968. eutrophigtion of:`:-.L.ake Michigan. Spec. Rpt, No. 364.:Great Lakes Res. / 12 Margalef, R,Information theory in Div., Inst. Sci; .8t:,%echn., Univ. a ecology.Gen. Syst. 3:3Q-71,1957. Michigan, AnKAOiar. 1967. .; galef, R.Perspectives in ecological 23Simpson, E. H.Measur ement ofiversity. ry.Univ. Chicago Presi, 1968. Nature (London-) 1949. --t.%,

0 14 McIntire, Tinsfey, I. J. and o . 24. Tanaka, .0.H., Irie, STlzuka, and Koga, F. Lowry, R. fkFatty acids in lotic Nndainent41 investigation on the periphyton: 'notheRipreasure of biological productivity*: the Northw est community, strritcture T Phycol. of Kyushu.I.The investigation of 5:26-32. 19.69. plankton.Rec. Oceanolr. W, Japan, Spec. Rpt. No. 5,

z.4 'Applicat riof aiologicarData

25 !Ta iling, E.Some mesOtroptric phyto- 28 Williams, L. G.Possible relationships plankton indicators.Proc. Intern. between diatom numbers and water - -'Assoc.Limnol. 12:212-215. 1955.... quality.2cology 45(4):810-823. 1964. . . 26Wilhm, .1, L.'Comparison of some 29Zelinka, M. and Sladecelt, V.Hydro-, diversity- indices applied to populations biology fog water management. of ben.thic,macroinvegtebOtes in a State Publ. House for Technical /.stream receiving organic wastes.J. Literature, Prague'. 122 p. 1964. _` Water Poll. Conti% .ked,39(10):1673-1,683, /967.

27 Wilhm, J. L.Use of biomass units in This outline was,prepared by C. I. Weber, . Shannon's formula. Etdogy 49 :153 -156. Chief, Biological Methods Section, Analytical 1968. Quality Control Laboratory, NER:C, EPA, Cincinnati, Ohio, 45268

i6 Descriptors: Analytical 'Techniques, Indicators

1'

t IS

1

4

o . -

.

.1 ,

4

a

L.

.

a.. . I

4

,<-. . e'

SIGNIFICANCE OF "LIMITING FACTORS" TO POPULATION VA RIA-TION

O

Q

.0 I INTROCUCTION A Liebig's-Jaw of the Minimum enunciates the first, basic concept.In orner for au A All aquatic organisms do not react uniformly organism to intiatAt a pain( u1aenvion- to the vario4chemical, -phy-ical and ment, specified levels of tiA ateriaks ' iolokal features in the,ir environment. necessary for growth and de elopment Through normal cvolutibliary processes (nutrients, respiratory gases, etc.) tnust various organisms,have.become adapted' _be present.If one_of these materials is

totccrtoln combinations of environmental abs'en"- m lb,.It\ ,runmen pt . I conditions. The successful development, in rnn ma, quantities, a given spe(Ies and maintenance of a population or community will oti..y sui rive in limited numbers, if depend uponitarmonious ecological oalance at all Ileigurr 2). between environmental conditions and ,Coppe, for example, tolerance of the organisms to variations ris essential in trace amounts to 1 in more of these conditions. mam, spet tes. . B Afactbr whose presence or absence exerts some restraininit, influence upon a population z OPTIMUM through incomPat;ibility with species o. requirements or tolerance 4-: said he a z factor. The principle of limiting factors is one of th'e major aspects of the I. environmental control of aquatic organisms 1 (FigurEl 1). 0 RITI AL RAN LOW - MAGNITUDE OF FACTOR - HIGIH

IIPRINCIPLE OF r:IMITING FACTORS' Figure-2'.Relationships of environmental This principle rests essentially upon two basic fa( tor:- and the abundam. c of organisms. concepts. One of these relates organisms to the environmenial supply of materials essential tor their growth and development. The second 1 Thesubsidiartvprinciple of fi, tor! -pertains to the tolerance which orgonisms aciton stals that high LootArratiott exhibit toward'. n . it uomental LynoitiOns. or availability of 'sornc sunstance, c. the action of some factor in the environ- 1 ment, may ti,o(lif:, of the minttnuri one.For example: a Tne uptake of-phospitous oy tile /UNLIMITED, GROWTH / DECREASE IN algal Nitzcnia losterium is influenced // .." -LTiarATIOMNS v. the 0- lati vtpia tit ities of nitrate', &GUI $RIUM WITH and phosphate in the elivirourptlitt;' N' ONMENt ,, now .:ver, nitrate ut iftZ'at ion,hppears 1"... INCREASE INs, to be unaffec:tecrby th,e phersphate TIMM/IONS (Reid, 1961), POPULATION DECLINE b Tbas..;imilatio,1 of sonic 'algfieis ' TIME ti 4 closely rateu to temperature. .. c The rate ofpxy.,eutilisation,by

'Figure f.The relationships- of lirnitin, factors may "e. affected try many19,1-ter U1) lei population,gro.:.th' and dcs,olopment. standes or factors ine the environment.

.

13 Eo0. 20a. 7.79 21 180 a a

a Significance of "Limiting Factors" tooputation Variation

d Where strontium is abundant, mollusks. a. are able to substitute it, to a partial extent, for ealcium in their shells (Odum, 1959).

2If a material is present in large amounts, but only a small amount is available.for pse/bv the organism', the amount available andnot-the -total amount present deter- S. :mines whether or not tke particular .1 material is liinkting.(calcium in the form of CaCO3

B Shelford pointed out in his Law.of Tolerance CONCENTRATION that there are maximum as well as minimum values of moss environmental factors which Figure 4.Relationship of purely harmful can be tolerated. Absence or failure of an factors and the abundance of organism can be 'controlled by the deficiency organi'sms. or excess of any factor which may approach the limits of tolerance for that organism 1 (Figure 3). 3 Tolerance to environmental factors" varies widely among aquatic organisms. A species may exhibit a wide range' of tokrance toward ote faCtor and a Minimum Limit of Range of Optimum ' Maximum Lunit of Toleratg,n ,_ of Factors Toleration narrow..Lnge toward anothem. Trout, V stO! --1- , ....e.^,.. for instance, have a wide range of Absent Decreasing Greatest Abundance i Decreasing Absent , Abunsance e, Abundance tolerance for salinity and a narrow . range lot; temperature. b All stages in the life,historiof an organism do not necessarily have the same ranges of tolerance. The Figure 3.Shelford's Law of Tolerance. period of reproduction is a critical time'in theIife cycle of most

organisms. . 1 Organisms have an ecological minimum 0 and maximum for each environmental c The range of tolerance toward one factor with a range inbetween called factor may be modified by another the 'critical rage which represents the factor.The toxicity of most sub- mange of tolerance (Figure2).. The stances .increases as 'the temperature actual range thru which an organism can increases. grow, develop and reproduce normally is ususWy much smaller than its total d The range of tolerance toward a given range aiK.t,olerance. factor may vary geographically within 'the same species.Organisms That 2Purely derZte*.ciOds factors (heavy metals, adjust to local conditions are called pesticides, etc.) have a maximum, ecotypes.' tolerable valut but no qptimuin (Figure 4). e. . V, *`, . - .

2

W 6

iD - .04

Significance of "Limiting Factors" to Population Variation

e The ran,p of tolerance toward a given C The law of the minimum as it pertains to factor may vary seasonally.In general factors affecting metabolism: and the law organistend to be more "sensitive of tolerance as it relates to density and to environdiental changes in summer- distribution, can be combined to form a than in other seasons.This is broad principle of limiting factors. primarily due to the higher surqmer / temperatures.' 1 The atundance, 'distribution, 'activity" and growth of a population aredeter- 4 A wide range of distribution of a species mined by a combination of factors, any is usually the result of awide range of one of which may through scarcity or tolerances. Organisms with a wide overabundance' be limiting. range Of tolerance for all factors are likely to be the most widely distributed, 2 The artificial introduction of various although their growth rate may vary substances into the environment tends greatly, ,A one-year old..carp, for to eliminate limiting minimums for instance, may vary in size'from less some species and create intolerable than an ounce to more than a poilnd maximums for others. depending on the habitat. 3 The biological productivity of any body 5. To express the relative degree of otwater is the end result of interaction tolerance for a particular environmental a of the organisms present with the factor the prefix eury (wide) or steno surrounding environment. (narrow) is added to a term for that feature (Figure 5). IIIVA LasND USE OF THE PRINCIPLE OF LIMITIK, FACTORS A The organism-environnt'ent relationship is apt to be so complex that not all factors 4, are of ecfual importance in.a given situation; some links of the chain guiding the organism a are weaker than others: Understanding the broad principle of limiting fa.ctors and 3. the 'subsidiary principles involved make the task of ferreting out tire weak link in STENOTHERMAL STENOTHERMAL (OLIGOTHERMAL)""ERRAL (POLYTHERMAL) a given situation much easier and possibly less time consuming and expensive.

1If an organism has aide range of tolerance for a fac or which is rela,tiVely constant in the environment that factor is not li ely to be limiting. The factor cannot bcompletely AihATEMPERATURE eliminated from conseration, however, bfa'ause of factor interaction. 2' If an organism is known to have narrow limits of tolerance for a factor which is Figure 5.Comparison cd'relativelimits o also vs/viable in the environnfent, that tolerance 'of stenothermal and .factor merits caref. study since it eurythermal organisms. .mht be limitirig. //' I Significance of "Limiting Factors" to Population Variation

,

B Because of the compleCity of the aquatic D Specific factors in the aquatic environment environment, it. is riot always easy to determine rather precisely what kinds of isolate the factor in the environment that orgt.nisms will be present In a particular is limiting a particular population. area.Therefore, organisms present or Prematfire conclusions may result from absent can be used to indicate envion- limited Observations of aparticular. mental conditions.Theeciiversitrof situations. Many important factors may organisms provides a better inc/cation of be overlooked unless a.sufficiently long Environmental conditansthan doWS any period-of-time is covered to permit the single-ape-cies.- Strong ph3)sio-chemical- factors to fluctuate within their ranges of . limiting factors tend to reduce the diversity possible variation. Much time and Money within a community; more tolerant species may be wasted on control measureswithout are then able to undergo population growth. the real limiting factor ever being(As"- covered or the situation being improved. REFERENCES C Knowledge of therinciple of limiting factors may be u ed to limit the number 1 Odum, Eugene P. :Fundamentals of of parameters tt need to be measure&or Ecology, W. B. 'Saunders Company, observed for a articular study.Not all Philadelphia. (1959) of the numero s physical, chemical and biological par meters need to be measured 2Peid, George K.Ecology of Inland -Waters or observed 7.r each study undertaken. and Estuaries.Reinhold Publishing The aims ofpollution survey are not to Corporation,' New YOrk.(1961) make and ob erve long lists of possible /40 Rosenthal H. and Alderdice, D. F. limiting factobut tip discovq which 3 factors are sign icant, how they bring Sublethal gffects of Environmental about their effects,e source or sources Stressors, Natural and Pollutional, of the problem, and w control measures. on Marine Fish Eggs and Larvae. should be takenif J. Fish. Res. Board Can. 33.2047-2065, 1976.

4Thorp, James H. and Qib>rke-r\J. Whitfield (editors), ,Energy and Environmental Stress in Aquatic I Systems. Tech Inform .Ctr, U. S. Dept of Energy. 71978.

O This outline was prepared by John E. 9 Matthews, Aquatic Bfologist, Robert S. Kerr Wateg Research Center, Ada,' Oklahoma. Descriptors: %Population, Limiting Factors .

21 -4 O \)

ALGAE AND CULTURAL EUTROPHICATION

4

-I,INTRODUCTION -' A 3, Nutrients This top% covers a wide spectrum_ of item A component or element essential to often depthiding upon the individual discussing sustain life or living organisms.This r. the subject and, the particllar situation or includes many different materials, objectives that fie .is trying to "prove ". some in gross quantities - others in _Since the -writer is not a-biologist, these minor quantities.Deficiency of any *viewpoints arev,"from the 'outside-looking in". one essential item make living Any impreSsion of bias is intentional. impossible. Nutrients needed in large quantities include carbon, hydrogen, A Some Definitions are in Order,to Clarify, oxygen, nitrogen, phosphorus, sulfur Terminology: and. silica. N andoP frequently are roosely considered as "the"nutrients Eutrophication - a process or action of because of certain solubility, con- becoming eutrophic, an enrichment. version and "known" behavior' t , To me, this,is a dynamic pr"ogression characteristics. Characterized by nutrient enrichment. , Like many deitiitions, this one is not' 4Algae 'precise; stages of eutrophication are classified as olig-, meso-, and eutrophic A group of nonvascular plants, capable depending upon increasing degree. Just of growth on mineralized nutrients with how a given body of water may be the aid of chlorophyll and light ener 'classified is open to question.It known as producer organisms, ,since depends upon whether you look at quiet the food chain is. based directly or 6.1; tip-bulent, water, top or'bottom °indirectly upon, the organic =material samples, season of the year, whether produced by algae. it is'a.. first impression or seasoned judgement.It, also-depends upon the, B Now that we havelalkacked Alto' itle water use in which you areintemated, words via 'definitions, some of the such as for fietting-tewaste discharge. ramifications of eutrophication, nutrient The tisnsitiOnaf'stageS are the major enrichment,, and cultural behavior are a problems -. it is loud and clear to a possible. , trout fisherman encountering carp and scum. . ,. II NUTRIENTS INTER'R'ELATIONSHIPS 2Culture I A nutrients are interchangeabletin form, Fostering of plant or animal growth; solubility, availability,. etc.There are cultivation. of living material and no "end" productg. We caa9 isolate, cover, prOducts of such cultivation, both fit. convert to gas liquid or-seid,. oxidize, t Some degree of control is implied but, reduce, complex, dilute, etc.some the -control may have limitations as . time, some place,- that nutrient may well as advantage sL Human cultural redycle p.'s part of cultural behavior. developnient has fogii-fr human num- a bers successfully, but, has promoted 1 Water contact is'a major factor in ., rapid degradation of his natural environ- recycle,dynamics just as water tient. e represents two-thirds or more of cell

3. e .

BI. ECO. hurn.3.6. "76 22-1 . . J

Algae and Cultural Eutrophicaan / mass and appears to be the medium in which living forms started.Waste disposal interrelationships (Figure 1) SGUIDLE ELEMENT- CUL:7. suggests physical interrelationships of p soil, air and water., The wet apex of ATLIOSPHERE this triangle is the basis for life.It's difficult to isolate water frog the soil 41:0 or atmosphere - water contact means 4k.o. solution of available nutrients. . ,/AS ".1Si'OSAL INTER:lc:LAI-IONS:11PS \ 0 4 0 1 .0 0 4 ATMOSPHERE BIOSPHERE are4i, SeAs SS. :-:"\ LITHOSPHERE \

NO pa0 # r . i$N, - FOG DUST 4 3 The nitrqgen cycle starts with ele- Mental nitro in the atmosphere. It candle 'converted to combinedform t by electrical discharge, certain WATER t > SOIL bacteria and algae, some plants and by industrial fixation. Nitrogen gas PAUD;k-:''' thus may go directly into plant form or be fixed before entry.Derlitrification 2 Figure 2 takes us Into the'biosphere (1) occurs mainly via saprophytes. via the sohible element cycle. This (Figure 3) Industrial fixation is a refers 'mainly tophosphorus interchange. . relatively new contribution to Phosphorus of geological origin'may be solubilized in water, used by plants or eutrophication.' - animals and returned to water. Natural rrfovement is toward the ocean. Less Of. phosphorus returns by water transport.. Phosphorus does not vaporize; hence, atmospheric transport occurs mainly A2S/IIIIC as windblown du t.Man and geological 1103113 upheaval, parti reverse the 'flow of phopshoerus tow rd the ocean sifik. . 11r3St1Uts ti

MI* MIISIllit 11111111 1111(11 c111:(1E .171--,M__--1.1 .---,4 M 11111Cit i 111(11$ MIS MIT° ial:"1l1(111133$.M4U)iIl1i nitIn

11113.-- NI 1** IA, AA 4

Ale and Cultural Eutto hication

4 Carbon Con versions (Figure 4) show 18 elements. More than 30 have been most of the carbon in the form of,. ' implicated as essential and they still 111% geological carbonate (1) but bi,carhonate Would not, "live", unless they were and-CO, readiily are c'onyerted to plant 'correctly assembled. As a nutrient Mnemonic H. COPKINS - - Mg(r)- ,cell maw -end into other life forms: Note the relatively small fractipn of CaFe-MoB does laitly well.It also carbon in liting mass. indicates Iodine 7 I, Iron-Fe, MolybdenAm-Mo, and Boron-B that .CA:13 01 c: ;c Were not included earlier. ZIOSPNEIIE, - _ 2 The Law of Distribution states that ..., ::tinsmit e4.4 IBS "Any given habitat tends to favor all

III SI suitable species - any given species Irlt2111'Vet tends to be present in all suitable

. It ES SEA IN LAIS,VITTUA141111 "S""1" habiiats." Selection tends to favor'the ...%-" ...... 411 . S SIII/Ct [4$11 LAMS most suitable species at a given place .--ks / SBA 2 IINSISII,1111111 1 $ and time. 4s. 71 TIII ,,A4 EICSAISt 3 Liebigs Law of the Minimum, states \,...1;111 ;BMX 1111 1111/114'S 411 VS 1131111 MTI, IASI i i III? that "The,essential material available 'SEA 14.311 in amounts most closely approaching CI the critical minimum will tend to be the limiting growthfactor." SIM ITS 10.11111 III 4Shelfords law recognizes that there will be some low concentration of any 4 nutrient that will not s nt growth. Some higher concert atio will stimulate B, Nutrient -.Growth Relationships growth. Each nutrient will have some still higher concentration that will be Nutrient cycles could go on, but, life bacteriostatic or toxic.This has been depends upon a mixture of essential diScussed earlier but was considered in nutrients under favorable conditions. a different manner. Too much of any significant item in the wrong place may be considered' as pollution.Since toxicity is related to HI BIOLOGICAL PROGRESSIONS chemical concentration, time of exposure and organism sensitivity, too-much The biological "balance" appears to bey. very becomes toxic.If it happens to 6e too transitory condition in cultural behavior, much growth, its a result of'eutrophication. Man favors 'production. A steady slate 'How much' is generally more important "balance" does not persist very long unless than op 'what'? Botli"natunal and manmade energy of the system is.too low to permit ..processes lead to biolbkical conversions, significant growth. A progression-of species to pollution, to eutrophicatiOn and to where each predominent form thrives for a 111 toxicifye Man is the only animal that can , time, then is displaced by another tempgrarily concentrate, speed up, invent, of otherwise favored group is usual. Yearly events in the alter these conversions to make a collossal -lawn start with chickweed, then dandelion, metes. plantain, crab grass, rag weed, etc., in . successive predominence. Occasionally, 1 Life forms hie been...fpirpulated,iri more 'desirable grasspg may appear on the terms of elemental or htitrient com- lawn. ,,prass is a selected unstable "culture". ponents 'many times. The simplest is; C irp N. A more complex formula?, is5 C8.2..0 NCa Cl P CuF 100 76 80 20 6 72 - 2 f SilftMn2IcNaS21Zzi. This includes

3 1 8C Algae and CulturalEutre?phication

.16 A Figure 3 shows a biological progression (2) following introduction of wastewater in an unnamed stream. Sewage or slime baCteria proliferate rapidly at first followed by ciliates, rotifers, etc.

THE BIOTA SEWAGE BACTERIA NO:PERIM:

CL 300 c:). CILIATES NO.PER ml.x1,000. ROTIFERS 3 .&CRUSTACEANS . NO. PER ml.x 25,000 2200.7

CC W

6. a. o foo COLIFORM (NO.rER MI.)

1 3 4 5 6 7 8 9 DAYS 24 12 0 12 2436486072 84 96108 MILES

qactetio thrive and finally become prey of the ciliates, which in turn arefood for theatifers and crustaceans. Figure 5. _ .

B Figure 4'shows another'progression of L bottom dwelling larva. Here the sequence of organisms changes after'wastewater introduction from aquatic insects to sludge worms; midges, sow bugs and then to re7eslishment of insects. THE BIOTA SLUDGE WORMS ,

TIC INSECTS . _.. DUOS (X 30) ttl

"...FA I: Se6. ( I I 014g I I 4 $ I .1 1 f 14 14 11 11 It /4 1MILESniS41 li 72 '

. . Figure 6.The gonfalon curee,,_44,,FIgure 7 is composed of a 'series of maxima Id, Individual species, each math:404 and, dying off as stream conditions spit.

. .t N.- . 4 . - A 87 ..' Algae and Cultural Eutrophication \CM

C Another progression afier waste . introduction changes the biota from an algal culture to sewage moulds with later return to algalpredominence. A ,

t

figure ?shortlyafter sewage discharge, the moulds attilin maximum growth ese ore assooted with sludge deposition shown in the fewer curve. The sludge i decomposed gradually; as conditions clear up, algae gain a foothold and multiply.

.Figures 5,6, and 7 are shown separately Since the algae also acq ire CO2 from only.,6ecause one visual would be Unreadabl% the atmosphere, f rbin ewater and wiith'all possible progessionson it.There' from geologital sources, it always ends aree progresips For fungi, protista, insect up with moire enrichment of nutrients in larvae, worms, fish, algae, etc. Each the water - more enrichment means more _Sp roh and rowingtorganisms eventually It,it,cannot compete-successfully, it will clump.ansi deposit.The nature of growth Be replaced by.those that can compete. liftsom free growth to rooted forths, under prevailing conditiOA at the tit'ne: starting in the shallow,s. Another ConditionS shift rapidly with rapid growth. progression occurs (Figures 9 and 10).

4 It is this relationship that favors profuse IV :ilhe inter ctiopf bacteria or fungi and nuisance growth of algae below significant algtOrre 8) are particulgrly , waste discharges. There is a tremendous sigpffic .to eu.trophicAtionj pool of carbon dioxide available in geological formations and in the airs A The b cteri,a orFrthe saprophytic group" TranSfer to the water is signiant and amorehem tend to work on preformed encourages algal productivity ar4 eventual brga.materials -. pre-existing organics eutrophication of any body of water, but, frodead or less favored organisms. this does not occur as rapidly as when the . Alg 1 dells produce theyorganics from water body is super saturated with CO2 lig t ...eArgy chlortppyll and mineralized from bacterial decay of wastewater

. ntlt netts.This is a happy combination dischtrges or benthie deposits from them. foboth: The algae release the oxygen ,4use by the bacteria while the bacteria .. .elease. the cOlvneeded by thealgae.

A.,

5 _r

4 , 183 . , r (*.

Algae and Cultural Eutrophication LAKEZONATI I' AN AQUATIC PLANTS

II LITTCM,ZIAL--

/

LIMNETIC

rzo FU NDAL

DO

3

DEAD 0 ALGAE.. I ik OXIDATION )°SEWAGE DEAD . BY. BACTERIA ° BACTERIA . 0 LIGHT

/"---- MINERALS ;),;:""\ M `g0 a 4 MIRA 'MUG.

6 189 Algae and Cultural Eutrophication

PITIORAL ZONE, AND, APUIVITIC-PLAHT DEPTH

SEED PLANTS

10 METERS FEROS.

20 me

,40 m' CHARAPHYTES 70m LAD5PHORA

*

o B Nitrogen arid phosphorus ate essential A We produce more nutrients per capitaper"' for growth. ,They also are prominently. day in the United States than in other considered ineutrophication control. nations and much more today than 100 Algal cell.ma't4s is about 50% carbon, 4 years ago. More people in population 15% nitrogehind appoximately 1% centers accentuate the problem. phosphorus not considering luxury uptake in. excess of immediate use. Phosphorus B Technology is available to remoZ most ks considered as the most controllable of the nutrients from the water carriage *niti.rig nutrient.It's control is corn- system. , plicated bythe feedback of P frotn.tenthic sediments and surface wash. Phosphorus 1 This technology will not be used untess removal means solids removal. Good water is recognized to be in sho'rt clarification is essential to obtain good supply. removal of P.- This also means improved -. . removal of other nutrients- a major 2 It will not be Used villep.we'PfaCe advantage of the P removal route.Both , realistic dommodity value on the water N & P are easily converted from one form and are willing to pay for, cleanup for to another; most form are water rense,putposes. 4....

CRemoval musebe followed by isolation of V SUMMARY acceptable gases to the atmosphere acceptable solids into the spa.....for iense Control dfteutrophication is npt entirely or storage. Water contact cannot be possible. takes must eventually fill with prevented, but it must be limiie4 olthe * benthic sediments, silt-Lace wast and enrichment" of the water body is hastened. 'vegetation.Natural processes, eventually 6 .causefilling. Increased nutrient discharges frorR added activities grossly increase filling rqte. A -0 4 a 190, Al ae and Cultural Eutro shicatio4

REFERENCES ThiS outlin was prepared by F. J. Ludzack, 1" A collection of articles one,Biosphere Chemist, National Training Center, Sci Am. 223'':(No.,'3) ,jP. 44-208. VD, OWPO, USEPA, Cincinnati, Ohio -- . ember 1970. 0-, i'l04 45- , (c 2 Bartch, A,. E. and Ingram,',. M. criptors: Algae, Eutrophication Stream life and the P-Plluff6n, Environ- ment. Public Works 90:(NO7,) 104-- -July 1959. , . '',4-'

. 4 o. ,,z

tr A

1r et. ?...k V. Tt, j. . V # 0 4 'A 'S , ..Is 1 - R . " 9 , # '03'; 044 . ° .0 # , Nc(. , 44 , .9% - IA s UV, 41.4 -4 4

, eis. igra . . JL, 4. I . ! s °-1.; . It* :4. 5. 192 t r . . .4p

6 6 CALIBRATION AND USE OF,.PLANKTON COUNTING EQUIPMENT

. I Another pracedureis to select,a value IINTRODUCTION o _ . '. '' Sor each of the variables involved, (inter- -A With the exception of factory-set .. pupillary distance,-t oOm, etc.) and, calibrate the scope at that combintion: instruments,' no two microscopes can . , be counted, upon to provide exactly.the Then each time the scope is to be used for' . same magAificat4n witfiay given com- 9. quantitative work, re -set each,variableitd - 4 the Value selected. A separ.ate multipli- bination-of ocuaarl and objectives. For a .0 .accurate quantitative studies, it is.there- . cation factor must be-calcalated for each , fore necessary to standardize,or "valibrate" adjustment which changes ,the magnification each.instrument against a. known standard: of'the insfronent. is. scale. One scale frequently used is a .. . .. Microscope slide on which two millimeters Since the Whipple Square can be used to measure both linear dimensions and ; are subdivided into tenths, and two addi-,- square areas, both shtild berecorded on tional tenths'are subdiviaed into hIndredths. an appropriate form.A. suggested format "Figlure 3. `,. is ,shown in Figure 6. : " , , B In orcier to provide an accurate measuring./. . .. device in thwmic'roscope, aWhipple (Data written. fn are used asan illustration 'Plankton Counting Square or reticule and are not intehded to apply to any (Figure 2a) is installed in one ocular Pareicular.microicope. An, unused form . (there are many gifferent types of reticules), -0- is includeckas Figure 6-A: ),.. . A ''',1 S N. This square i8 theoretically of such p. sire It .i i 4. e . t , . t that with a 10X objective, a 10x ocular, andla tube length of 160 mm, -the image 4 v.- UTHE CALIBRATION 'PROOEDUR:E ...4 . ... 1,6 , t, of thesquare covers a square area on the-- . . , slide one-mm on a slide: Since this A.Installing theWhipple `Square or'Reticuie, .; . o 41... .44 4 objectiirg is raceItattained, however, Irnost ' To install the-Fetionleip the'dcuti,r. AwtcPosco, apes must b4 standardized.or 41' do., (Usually-the right one on a binodnWr --:. 4141e at ea". as described bei,low in orzter, , microscope)4 carefully unscreVhe'upper, . to ascertain the actual size,orthe,Whipple ,.; rid e re tleg 1e on ,Square, aseen} throw litte microscopes ,.*'4.1e rutmounti ng a.place th th$ dircular diaphragm or shelevihichwill , (herein tet'iefeilreq as'the'tWhipple r 4eld"r. prTkreseis's,Chematically,- be foul* af roxiinatery half way loy.tinside' If the (f'igare`*4): eplace the mo,untl.ng &Fick c. 'reprzlte'd Figt2rfts-5 and'7. observe thoCha k* gs on'the reticule.If " Whipple eyepiece is to be used atmore 4P they are not ih sha p foc s, remove and". than (Inb. magnification, it 'must be rgcali; turn the reticule or ',orated for each. A basic tYrit of monocular . .- microbcope.is shown in Figure1. , . Onreticules with the.markings etched-on . CMicroscopes with two-eyepieces (binocular) one side of a glass disc, the etched sur- are a convenience but not essential.' Like face tan tisually,be recognized by shitliqg modtrn car3-Ithey are not only great f the disc at the proper angle in a light. "performers," but also complicated' to The markings will usually be in the beat t service or, in this instance, calibrate. , ,focus' with the etched surface awn:. If 'the On some instruments, changing the inter- markings are sandwiched between tvp,g14.as. ---pupalary distance also changes the tube discs cemented togefher, both aides ate length, on others it does not.The "zoom" aiSe, and the foCils may not be quite as featureon certain scopes is also essentially sham d a syste'm.for.changiug the tube length. . B Obdervation of the Stage Microingir The resultant is that inddition to calibra.- V. tion at each combination of eyepiece and Replace the ocular in the- Microscope and objective, any otherf actor which may.i. tobser4e the stage mitrometer as 's illus- ' ' affect magnification must also betconsidered ' trated schematically in Figure 51* Calibration -In some instances this may mean setting up of the Whipple Squar.e. On a suitably ruled a table of calibrations at- a series of micro- drin`stith as the one illustrated' Figure 6, Calibration Data,' record the°aCtual distance,_ :,scope settings. in millimeters subtended by the image of

, sr. 192, tqvt61'.miC. id:-6.76 201. c. .; alibration and Use of Plankton Cbunting Equipment

AI

the entire WhiPpletfield and also by each rim of an_empty cell fOr the above depth of its subdivisions.This should be measurements, as the coverglass is supported determined for each significant settling of, by the highest points of the rim only, which the interpupillary distance for a bin,ocular ate very difficult to identify.Use the average microscope, and also for' each combination of all depth measurements as the "true" depth of lensesipmp/oyed.Since oculars and of the cell. ,To simplify. calculations below, "it objectives marked with identical magnifi- will be assumed that we are dealing with a _ cation, and since microscope frames too cell with an average depth of,exactly 1.0 mm. may differ, the serial or other identifying number of those actually calibrated should be recorded.It is thus apparent that the IVPROCEDURE FOR STRIP COUNTS USING determinations recorded will only be valid 'THE SEDGEWICK-RAFTER CELL when used With the lenses Vted an,on that pdrticular microscope. APrinciples C Use of the 20X Objective. Since the total area of the ce'l is- 1000 mm2, . the total volume is 1000 mm or 1 ml. A Due to the short working distance beneath "strip" the length of the cell thus constitutes q a 46X (4mm) objective, it is impossible a volume (Vi) 50 mm long, 1 mm deep, Arid to focus ,to the bottom. of the,Sedgewick- the width of -the Whipple field. Rafter plan)cton,counting cell with this'lens. A 10X (16mm) lens on the other hand The volume of such a strip in riffn2 is: "wastes" space.between,the front of the leris ..nd the coverglass,, even when focused 50 X" width of field X depth, on the bettom o` Thee In order to take Ire m-oseteffickenruse possible of this cell 50 X w.X then, an objective of intermediate focal0 length is'desirable: ,A lens with a Local 56 w length of approximatelr8 mm, having a I maerlification of 20 or 21X will meet these In theigexample.given below on the plate reqtairements.Subh lenses are available. entitlffd Calibration Data, at a magnification from American mantfacturers and are of approximately 200X 'with an ,interpupillary recommended for this type of-work, setting of "60", the width of the Whipple field is recorded as 'approximately 0.55 mm (or 550 microns).In this case, the volume . IIICHECKING THE CELL of the strip is: The internal dimensions of a Sedgewick-Rafter%* V1= 50 w =° 50 >n.55 27.5 (mm3) plankton counting celL8liciuld be 50 mm'long by 20 mm wide by 1 rnm7cleep (Figure 8). . , r BCalculation of Multiplier F, actor The actualhorizontardimensions of each new In Drder to convert plankton counts per cell should be checked With calipers, and the strip to counts per ml, it is simply,. depth of the ceA checked at several points ,necessary to multiply the count obtained around the edge using the vertical focuSing by, a factor (F1) which represents the scale engraved on the fine adjustment knob of number of times the volume of the strip .most microscopes.OrPe complete rotation of examined (V1) would be contained ink 1 ml or the knob usually raises or lowers the ohjeqtive 1000 mm3. Thus in the example given 1 mm or I00 microns (andspach single ina.At above: equals 1 micron). Thus, approximately ten volume of cell inmm3 turns of the fine4adjustment knob shotild raise F,1 = the focus from the bottom of the cell to, the vo ume e75.1717-01.rimml- underside 'of a coverglass testing on the rim. 1000 1000 Make thesegmeaswicements-on an empty cell. 27.5 36.36 The use of a No. I or-l-r/2,i24 X 60 mm 1 coverglass is recommended rather than the = approx. 36 'heavy coverglass that comes with the S-R cell, as the thinner glass will somewhat con-, If more than one strip is to be counted, form to any irregularities of the cell rim the factor for twd, three, etc. , /strips (hence, also making a tighter seal and reduc- could be calculated separately using the ing evaporation when in actualse).Do not same relationship'outlined above, changing attempt to focus on the upper s'face of the only the measurement-for-the length of

234 a

Ca,libration and Use of Plankton Counting Equipment

G

Fig req.. THE COMPUU MICROSCOPE . A) coarse adjustment; 33) finadjustment; tuKned toward the object being studied.In C) arm or pillar; D) mecha cal stage which thiscase J is.a 40X, K is a 010X, L is a 20X, holds slides and is movable n two directioris and M is a lOX objective.The productof by means of the two knobs;) -pivot or joint. . the magnification power of the objective being. This should not be used or'' rokenll while used times the magnification power of the counting plairkton; F) eyepie e (or ocular cf: eyepiece gives the total magnification of .he fr figure 4); G) draw tube. T k will be found microscope.Different makes of microscopes on monocular microscopes nly (those having employ objectives of slightly differentpowers, only one eyepiece). AdjOsent of this tube but all are approximately equivalent.N) stage is very helpful in calibradn the microscope of the microscope; 0) Sedgwick-Rafter cell in for quantitative cotutt.ing (Se 5. 5. 2. 2. ). place for observatiori; P) substage condenser; H) bddy tube.In some mak s of microscopes Q) mirror; R) base or staid; note: for this can be replaced with a body tube having information on the optibal system, consult two eyepieces, thus maldrig the 'scope into reference 31(Photo by Dbn*Moran. ). a."binocular. "I) revolving nosepiece on which the objectives are m unted; J) through M are objectives,, wily one if which can be . Calibration and Use of Plankton Counting Equipment I 1

Ar a

a b

o 0

. t. )( (

m11111101 I

d

Figure 2 Types ,of -eyepiece micrometev,discs or the margin of the large square to facilitate' reticules (reticules, graticules, 'etc.). the estimation of sizes of organisms in When dimensions -are mentioned in the different parts of the field.(b) Quadrant folloWing description, they refer to thp ruling with 8. 0 mm circle, for counting.' bacteria in milk smears for example.(c) markings on the reticule disci and not to Linear scale 5. 0 mm divided into tenths,. .the inpapurements subtended on the micro- ent of linear diniehsions. scppe Slide. ,The latter must be.determined (d) Portohr icule for estimating the size by calibration procedures such as those of pdrticlese.- The sizes of the series of discs described elsewhere.(a) Whipple plankton is based onthesquare root of two so that the- counting eyepiece. The fine rulings in the areas of successive discs double as:they subdivided square are sometimes. extended to . progress in size.

23-4 , Jil Calibration and Use y Plankton Counting Equipment

strip counted, Thus or. . rtwostrips in the Thus in the example cited aboire where at example cited above: an approximate magnificiation of 200X and 0 with an interpupillary setting of 60, the V2= .100W = 100 X 0.55 = 55 mm3 width of the Whipple field is .55 mm. Then: 1000 1000 .0 18. 2 F = 36.36 or approx. 36 55 1 -5 F2= V1 .1? ., above)' It will however be noted that F = 2 If two strips are counted: - I Likewise a factor F3 for three strips Fl ..55 would equal 7 or approxima ely 12, etc. and F2 = ITT20 ,= 18. 2 = approx. 18, etc. 10 C\' An-Empirical "Step-Off" Method his same value could be obtained,withou without e use of a stage micrometer by refully A simpler but more empirical procedure oving the cell sidewise across the field for determining the.factor is to consider f vision by the use of a mechanical stage., that if'a. strip 20 mm wide were to be Count the number of Whipple fields ih the counted the length of the cell, t13,9.t the width of the cell.There should be ap)roxi- entire 1000 mm3 would lie included 'since ately 36 in the instance cited above. the cell is 20 mm wide and 1"mm.deep. This 20 mm strip width can be equated to EPARATE COUNT USING THE 1000 mm3.If a strip (or the total of 2 or SEDGEWICK-RAFTER L more strips) is less than 20 mm in width, the quotient/of 20 divided by this width will A Circumstances of Use be a multiplier factor for converting from count per strip(s) to count per ml. The use of concentrated samples, local' established programs, or other circumstantes

Figure 3. STAGE MICROMETER The type illustrated has two millimeters divided into tenths, plus two additional tenths subdivided into hundredths.

LI] 1mm Enlargement of Micrometer Scale

. '23-5

r.,

Calibration and Use of Plankton Counting Equipment ,

ti

CALI limn ON OF WHIPPLE SQUARE as seen with 10X Ocular and 43XObjective (approximately 430X total magnificatiOn)

4' L .

f' "Small squares" subtend one Ufth of large squares. .005r trim or 5 2p

0 Wh;PpleSquare as seen through ocular:" "Large square" subtends r ("Whipple field") one Tenth of entire Whipple I\ Square: :026 nun or 26p

Apparent lines of sight subtend .26 mm or 260p on stage micrometer scale

a Min "IP\ (1000 .01,mm (10p) PORTIOltripf MAGNIFIEDIMAGE OF STAGEMICROMETER SCALE --10.

11

*, , Figure 5

CALIBRATION OF THE WHIPPLE SQUARE .

, The apparent relationship of the Whipple micron ethr with g magnification of 'Square is shown as it is viewed through a approximately "430X (10X ocular .and 43X microscope,while looking at -a stage. objective).

19.0r, 23-7 Calibration and Use of4PlanktOn Counting Equipment

MICROSCOPE CALIBRATION DATA

Mictoscope No.6,q3 79

%.

Tube Apr "oximate Length, or Linear dimensions of Whipple Factor for .11k41.gnification Interpupillatiir squares in millimeters*. Conversion Setting Whole Large Small to count/ ml

100X, obtained with (2 S-8 Stripe) Objective r , Serial No.

5,710 9,Vaol) Sp I. /30 O. 11.3 0, oc226 8.9 andOcular N Serial No ,60 /, / / 5- 0, /I Ij5 o.ta,2, 9,o

A:194.74'AI) 70 I. /DO 0.1/0'0. 0;.2,2 9./

. , 200X, obtained with 6 (2 8-8 Strips) Objective Serial No. 'ilgfV.PI..nd 6-0 0.5000 05:40,0//a /7.9 and Ocular Serial No. (,0 0.55000550.0//0 /.19. 2 (2967gLGox) 70 0.5Y50 nsg"00/09 /1.3 , .

(Nannoplanlcton). 400X, obtained with (cell-20 fields ) Objective , Serial No. ..

2.19/134/49X) i i D ; .005 . and Ocular . Serial No 0 , . 0.. 005 .,lo.. , o 05 AT . ',1967.9Z00,0 . 4 . . *1 mm z' 1000 microns

. 11V1icroscope calibration data.The form situations.' For example, "Interpupillary shown is suggested for the recording of Setting" could beyeplaced by "Tube Length" data pertaining to a particular microscope. or the "2.S-11 Stripscould be replaced by Headings could be modified to suit local "per field" or "per 10 fields." , Figure

I 4

Calibration and Use of Plankton Counting (Equipment .

MICROSCOPE CALIBRATION.DATA

Tube Linear dimensions of Whipple Factof for . Approximate l_iigth, or Ir squares in millimeters*. Conversion 4:- _MagnificationInterpupillaity to count/ ml Sett ing whole if Large I Small

100X, obtained With (2 S-R Strips) Objective ' . Serial No; . , . . . . , and Ocular . Serial No . . . . .

. . . .

200X, obtained with (2 S-R Strips) Objective Serial No.

Wn7ii Serial No.

(Nannoplankton) 1 400X, .obtained with (cell- 20' fields )s _ . Objective . . . Serial No. - . . . , , . . .. . , . .:

F , and Ocular . , Serial No. ice. . .: . . . . d - . lrnm = 1000 microns AQ, pl. 8. 10. 60,

FigUre MICROSCOPE CALIBRATION DATA Suggetted work sheet for the calibration'of a microscope,Details will need to be adapted to the particular instrument and situation. (.. . . . Calibratfon and Use of Plankton Counting Equipment yr .

a

r

01.

a

S-A COVER GLASS .14Bilir WHIPPLE SQUARE

PLANKTERS WATER IN 1MM A S-R CEJL '/InAnn I - *,\,,.\s\% A

\;4-\\ 11 I-THICKNESSS-R SL1DE

.Figure 7 A cube of water as seen through a Whipple square at 100X magnifitation in a Sedgewick-Rafter.cell The figure is draWb as if the micro cope were focused on the bottom of the tell, making.yisible only th'ose organisms lying on the bottom of the cell.The little =bug (copepod) halfway up, and the algae filament at the top wataldbe out of focus. The focus must be rhoved up and down in order to.study,lor count) the entire cube.

0 2 0°1 n O Ca14 ration and,Use of Plankton Coun nig..Equipment'

1 may make it necessary to employ'the mare NANNOPLANKTON COUNTING conventional technique of counting one or more separate Whipple fields inste of . For counting nannoplankton using, the high the strip count method. The basic retion- dry power (10X ocular and 43X objective) ships outliqed above still hold, namel and the "nannoplankton counting cell" (Figure 9) which is 0.4 mm deep, a minimum 'volume cell in mm3 of 20 separate Whipple fields is suggested. F /-\ volume examined inmn--73 The same general relationships presented above (Section IV)-can be used to obtain a. B Princ1 ies Involved multiplier or factor (F5) to convert counts per 20 fields to counts per ml. The volue examined in this case will consist of one or more squares the dimen.?,_ To take another example from Figure 4, at sionsof the Whipple field in area and 1 mm an approximate.magnification of 400X and an in depth (Figure 7). Common practice inthrpuPillary setting of 70 (see also Figure .3) for routine work is to examine 10 fields, we observe that one side of the Whipple field but exceptionally high or low counts or measures 0. 260 mm. The volume of the other circumstances may indicate that fields examined is thus obtained as follows: some other number of fields should be employed.In this case a "pr field" = side2 X depth X no. bf fields' 4 5 factor may be determined to be subsequently 36 divided by the number of fields examined = 0.26 X 0.26 X 0.4 X 20 = .54 mm as with the strip count. The following 1 000 description however is based on an assumed and F5 = (approx. )1 .850 ' count of 10 fields. ,C Calculation of Multiplidr Factor It Should be nbted that the volume of the nannoplankton cell, .1 ml, is of-no significance As stated above, the total volume in this particular calculation. represented in.the fields examined con- Asts of the total area of the Whipple fields multiplied by the depth. REFERENCESi 2 V4=(side of Whipple field)X depth- 1 American Public Health Association, et. al. Standard Methods for the Examination (1 mm) X no. of fields counted) / of Water, Sewage, and Industrial Wastes. 14th Edition, 'Am. Public Health Asgoc. For example, let us assume an approxi-* ., New York. 1976. mate magnification of 100X (see Figures - 6 and 7 and.an interpupillary setting of 2 Jackson, H. W. and Williss,L. G. "50".The observedleneth of one side The Calibration and Use of Certain of the Whipple field in this case is 1:13 Plankton, Counting Equip. enta Trans. fmm. -The calculation of V Am. Mic, Soc. LXXXI(1tk:96-103. 1962. 4is thus: 2 V4 =sideX depth X no, of fieldS 3 Ingram; W. M. and Palmer; C. M. , . Simplified Procedures for Colle4ing, 1.13 X 1.13 X 1.5< 10 = 12.8mm3 Examining, and Recording Plankton in :Water.Jour. Am. Water Works. The multiplier faCtor is obtained as Assoc. 44(7): 617-724.1952. above (Section IV ,A): 3. 4 Palmer, C. M. Algae in Water Supplies. F volume cell in mna . U. S. D. H. E. W. Public Health Service ..C` 4 vol xaminecl in mm3 Pub. No. 657.1959. .i0 (approx.) 78 5Palmer, C. PI,. and Maloney; T. E. A 8 New Counting Slide for Nannoplankton. American Soc. Limnol and Oceanog. ( one field were counted, the factor Special Publications No; 21.pp. 1-6. wo d be IL, for 100 fields it would 1954. b e ? . ) . ) -

.` A - 0 20° 23-11 *,* rs -,

Calibration and Use of.--- Plankton Counting Equipment

O

Area I litrips,, Uncounted Counted

1,--,\ Figure 8 . , . . \ Sedgewick-Rafter, counting cell showing bottom scored across fdr ease in counting strips.The "strips" as shown in the illustration simply represent the area counted, and are not marked On the slide.The conventignaPdi,mensions are 50 X 20 )< 1 mm, buf _ these should b'e checked for accurate work. ,

/

:Jr, .trrrrrr-7.1en r. Ii f:( Figure 9 t at / Nannoplanktpn cell.* Dimensions of the circular part of the cellare 17.9 mm diameter X 0.4 mm depth. When covered with a coverglass, the volume contained is 0. I ml. The channels for the introduction of sample,and the release of air are 2-mm wide and approximately 5 vim long.. This slide is designed to be used with the 4 mm or 43X Thigh dry) objective. 4

'6Welch, PaulS.Limnological Methods, Dyinking Water. John Wiley and Sons. BlakistonCompany. Phila. T,oronto.. New York.1948. 1948. 49±7 7' Whipple, G.C., Fair, G. M., and ,,This Aline was prepared by HI W. Jackson, Whipple,M, C. The Micrqscopy of forrrier Chief Biologist, National Training 9 Center, and revised by R. M. *Sinclair, 4quatic tiologist, National Training Center OTD, OWP01.USEPA, Cincinnati, Ohio 45268 . Descriptors: Plankton,Microscopy : .4 .203 , .$

23..12.0. .s`` a A

LABORATORY: PROPORTIONAL COUNTING OF MIRED LIQUOR

. . . , I OBJECTIVE ,C Proportional Counting

To learn the techniques of propoftional 1 Fifty- count 8ounting of mixed liquor, samples and 0 become familiar with cornmon types aMoving the slide at random count of microorganisms. each type, until a total of 50 organisms have been "jo.9kted:

II MATERIALS q b Tally the results and compute the percentage Of each type. A Mixed liquor samples, each con- . taining a number of microscopic forms. IV RESULTS

'B Glass slides, cover slips, and dropping A Record your results on the board. 4- pipets. , B Discuss the methods and the use of the proportional count results. III PROCEDURES A Make a wet mount of the sample(s) proviaed./Do not alloy,' the sliltle This outline ,wfts prepared by Ralph. Slnclair, mountto evaporate. Add a drop ,National Training and Operational Technology to the Side as necessary. Cbnter, OWPO, 14SEPA, Cincinnati, 'Ohio 4526 . 13 First, scan the slide. On the sheet \, marked, "Conimon types of Protozoa 'Descriptors: Analytical TechniqUes, .4 . and I\ietazoa", make a check next to Activated Sludge each type you find.If you find,,a S type not illustrated'ipake a simple -sketch on the .reverse of the above , sheet. The objective here is to becoine familiar with allaof the. 0 Common types of, microorganisms o .4 4,) found in the sample.. 44

7.. X laboratory: propor,i6nal CoUnting ofMixed Liquor

o .

ESTIMATING THE SIZE OF 'A PROTOZOAN

o

10x OBJECTIVE 10x EYEPIECE = 100

TRACHELOPHYLLUM PUSILLUM

4

40x OBJECTIVE :10x EYEPIECE 5,--400. TOTAL.

O v .

e.

.

24-2 Laboratory: Proportional Counting of Mixed Liquor

COMMON TYPESOF,PROTOZOA AND METAZOA

14Ki5 ROTIFER - For size comparison with othermicroorganisMe

Aspidisca STALKED CILIATES 111 I.'. EpistylisI .1 9 0 17,1 C ,E5 c.) 1 8 Crawl' ng 't telotroch rvr 0 stage Lateral Posterior PER ITRICHS 2 ta 11 12 z NEMATODE

NAKED 0CC. cc 13 14 Lu AMOEBAE FLAGELLATES Chironomus 4:10-t OTHER PROTOZOA WORM-LIKE MULTICELLULAR ANIMALS.

I

4-3 Laboratory: Proportional Counting pf Mixed Liquor

PROPORTIONAL COUNTS-- 7 PROTOZOA AND METAZOANS Date Analyst random swoop across slide Count Organisms in Plant Do not search for #articular typos Ignore rare organisms ``" .Stop attotal count of oither 50 or 100 organisms. 1 TOTAL 50-0. TOTAL 100 Tally each organism twice, once in a total taffy box, nd once in the appropriate tally box below r. 5

Paramecium Vaginicola 10 15

r?- Imp Aspidisca tel otrochs Pleuromonas Trachelophyllum--ry6 -96. .11 ."T

Amoebae 7-% 7Aelosoin a 19

4 WI*

1. 6 Epistylis ROTIFER

207 ; Laboratory: Proportional Counting of Mixed Liquor

PROPORTIONAL COUNTS- PROTOZOA AND METAZOANS° Date Analyst Count organisms in a random sweep across slide Plant Do not'search for particular types Ignore rare organisms Stop at a total count of either 50 or 100 organisms

TOTAL 50 TOTAL 100 Law- Tallt each organdun twice, once in r. a total tally box,and once in the appropriate tallybox below 9 1 1 M.m.=

IN= =ION Im= Vaginicola -14 -36 -34 15 =IP 2 6 N110, 1 imi

Aspidisca telotrochs Pleuromonas Trachelophyllum a. 1s

Ionotus- -34 19.

MMIMMINO

Im10..0

1=IMMMI

IMMIAMM

IMMIMMIM

/ Stentor Epistylis ROTIFER

2033 it ).

Laboratory: Proportional Counting of Mixed Li . , .

A

C

PRO,PORTIONAL COUNTS- PROTOZOA AND METAZOANS % t Data ..t Analyst Count organisms in a random sliver" across slide Plant DO not search for particular types Ignore tars organismr=- Stop at a total count of either 50 or 100 organisms

TOTAL SO TOTAL 100 --aw Tally each grganIsm twice. once in a tole, tally box, and once in the A appropriate tally box below 1 1 I

Paramecium ti 2 l6 15-

n*.

Aspidisca telotrochs' Pleuromonas Trachelophyllum 11 0.

Litonotus

,11 11011.111110

Stentor Epistylis =44 ROTIFER

) .209 . ,I 0 1

. , Laboratory: PropOrtional Counting of Mixed Liquor:

<55

PliaiPORTIONALCOUNTS- PROTOZOA AND METAZOANS Date Anal* Count organisms in a random sweep across slide Plant Do not search for particular types Ignore rare organisms Stop at a total count of either 50 or 100 organisms

TOTAL 50 TOTAL 100 --ow Tally each organism twice, once 7'in total tally box, and once in the appropriate tally box below !") "Iv 1 / Paramecium -16 6

;NA -

telotrochs Pleuromonas Trachelophyllum 11 16

Amoebae Aelosoma 4

Stentor,"_ Epistylis

210 Laboratory: Proportional Counting of Mixed Liquor

PROPORTIONAL COUNTS.- PROTOZOA AND METAZOANS Date Analyst Count organisms in a random swoop across slide Plant Do not search for particular types Ignore-ram organisms Stop at a total count of *idler 60 or 100 organisms

TOTAL 60 Tally each organism twice. once in a total tally box, and once in thi appropriate tally box below

Peranema

1.1

5. Pleuromonas trachelophyllum 3. 3

.4.1

Amoebae °soma Litonotus % . 4 4

o: Epistylia' ReITIFER. r A *:*

Laboratory: Proportional.Counting of Mixed Liquor /

PROPORTIONAL COUNTS7..

r PROTOZOA AND METAZOANS

Count organisms in a random sweep across slide Plant Do not.march for particular types Ignore' ram organisms Stop at a total count of either 50 or 100 organisms

7'4 TOTAL 50 TOTAL 100 --am- Tally each organism twice, once in a total tally box,and once in the appropriate tallybox below .) 1 1

Paramecium Th 2 6

Aspidisca telotrochs '*Pleuromonas TrachelophyIlum% 3 11

Litonotus Amoebae 4 1 19 .,1..

1001114

Stentor F.pistylis ROTIFER

" 212 .11

s, KEY TO SELECTED GROUPS OF FRESHWATER ANIMALS

The following key- is intended to prOvide animal as a mernbe-r sof the group Phylum an introduction to same of the more PROTOZOA.If you selected "lb' s, proceed common freshwater animalsTechnical to the couplet indicatedContinue-this language is kept to a minimum. process,until the selected statement is terminated with the name.).4 a group,. In using this key, start with the first couplet (la, lb), and select the alternative If you wish more information About the that seems most reasonable.If you - group, consult references.(See reference selected,"la" you have identified the list. )

.S

el

N1. O

251-1 BI.4 21b. 6. 76 213 Key to Selected Grou s of Freshwaternimals

:"

la The body of the organ' n comprising 5aSkeleton or shell present.Skel- 15 a single microscopic iependent eton may be external or internal. cell, or many similar indepen- , . dently functioning cells associated 15bBody soft and/or wormlike, in a colony with little or no differ- .'Nr 'Skin may range from soft to ence between the cellsi. e., with- parchment-like. out forming tissues; or body co.-n- prised of rnaSses of nultinucleate 6aThree or'more pairs oell 19 protoplasm.Mostly microscopic, formed jointed legs prent. single celled animals.- Phylum ./0Tf113.0PODA (Fig. 4) O t- Phylum PROTOZOA 6bLegs or appendages, if present, 7 limited to pairs of bumps or hpoks. lb The body of the organism com- 2 ,-"lobesor tenacles; if present, prised of many cells of different soft and fleshy, not jointed. kinds, i.e..vforming tissues. . May be microscopic or macro- Body strongly depresited or 8 scopic. flattened in cross section.

2a _Body or colony usually for'm'ing 3 7bBody oval, round, or shaped like -10 irregular masses or layers some anfinverted."U" in Cross section. times 'cylindrical, goblet shaped, vase shaped, or tree like.Size 8aParasitic inside bodice of higher range from barely visible to animals, Extremely long and flat, -divided into _Sections like a-Roman . , girdle.' Life historymay involve: 2b Body or colony shows some type 4 an intermediate host.Tape worm of definite symmetry. Class' CESTODA 51. 4 0 '3a Colony surface rough or bristly 8b "Body a single unit.Mouth and in appearante under microscope digestive systempresent, but no ,or hand lens.Grey, green, or amts. } brown. Sponges. Phylum PORIFERA (Fig. 1) 9aExternal or internM parasite of higher animals.Sucking discs 3b CoyiriSr surface relatively smooth. present for attachment. -Life his- 'General texture of mass gelatinous, tory may involve two or more in- it transparent. Clumps of minute termediate hosts or stages.Flukes. individual organisms variously Clpss TREMATODA drstributed. Moss animals, , bryozoans. 9bFr e living.. Entire body covered I' Phylum BRYOZOA (Fig. 2) withlocomotive cilia.Eye areas in head often appear "crossed". 4a Microscopic: Action of two Free living fllitworms.' ciliated (fringed) lobes at an- ClassjURBELLARIA (Fig. 6) terior (front) end in life often gives appearance of wheels., 10a Long, slender; with snake -like Body often segmented, accordian- - motion in life.Covered with glis- like.Free swimming or attached. tening'cutiele.Par-asitic or free- Rotifers or wheel 4nimalcaes. living. Microscopic tosix feet in Phylum TROCHELMIIITHES length. Round worms. (Rotifers) (Fig. 3)" Phylum NEIViAttIELMINTHES (Fig. 7) 4b Larger, wormlike, or having 5 strong skeleton b shell. 10b. Divided into ,sections pr seginents, 11

. . 2 1 .4 25 -2 O , 4' Key to Selected Groups of Frethwater Animals

10c Vnsegmented, head bluntone 18, sucking parasites on higher animals, or two retractile 'tentacles. often found unattached to host. Flat pointed, tail. . Leaches. Ma's HIRUDINEA (Fig. 98) IlaHead a more or less well-fornied, hard, capsule with jaws, eyes, 15a Skeleton internal, of true bone. 40 and antennae.. (Vertebrates) Class INSECTA order DIPTERA (Figs. 8A, 8C) 15b Body covered with an external 16 skeleton or shell. 1lbHead structure pOft: except 12 (Figs. 10, 13 17, 18,- 24, jaws (if present). 8E.). 25, 28) 0

12a Head conical gr rounded, lateral 13 16a External skeleton jointed, shell 19 appendages not conspicuous or covers legs. and other appendageS, numerous. often leathery.in nature.. r phylum ARTHROPODA 12b Head somewhat broad and blunt, 14 Retractile jaws usually present. 1-6b External shell entire, not jointed, 17 unless composed of two ri- ..00*L Soft fleshy lobes or tentacles, -7- often somewhat flattened, may be like halves. present in the-head region. Tail (Figs. 10, 11, 12) usually narrow.Lateral lobes or fleshy appendages on each '17a. Half inch or less in length. Two segment unless there is a large. leathery, clam-like shells.Soft, sucker disci at .rear end. parts inVittle include delicate,. Phylum ANNELIDAN ig.9)/ jointed appendages.Phyllopods or branchiopods. .13a Minute dark colored retractile? Class CRUSTACEA, Sub,classes jaws present, body tapering BRANCHIOPODA (Fig.. 12) somewhat at both ends, pairs or and OSTRACODA (Fig. 11) rings of bumps or "legs" often present, even, near tail. 17b /Soft parts covered with thin 18 Class INSECTA Order DIPTERA skin, mulcous:produced; no: jointed legs. (Fig. 8)' phylum.MOLLUSCA

13b No jaws, sides of body generally 14 18a Shell single, may be a spiral cone. parallel except at ends, Thicken- Snails. ed area or ring usually present ClasS.GASTROPODA (Fig. 13) if not all the way back on body. 4 Clumps of minute bristles on most 18b Shell double, two halves, hinged segments. Earthwo-rms, sludge- at one point.Mussels, clams. worms. Class BIVALVIA (Pig. 10) 0 Order OLIGOCHAETA 194 Three pairs of regular walking 29 14a Segments with brittles and/or fleshy'' legs, ortheir rudiments. Wings lobes-orlother extensions. Tube present in all ad'Ults and rudiments builders, borers, or burrow6rs.. iz*some larvae, :Often reddish or greenish in Class INSECTA (Figs. 22;. 2 color. Brackish or fresh water. 25, 26, 2ft:'29) Nereid, worms. Order POLYCHAETA (Fig. DA) 19b More than three pairs'of legs 20 apparently` present. 14b Sucker disc at each end, the'large e one posteriOr. xrnal.blood- 20a Body elongated,. head broad and flat ._ 25-3 r Key to Selected'Groups Frvshwater Animals a

J with strong jaws.Appendages follow- 25aAppendages leaflike, flattened, ing first, three pairs Of legs are rclund- more than' ten pairs. tppering filaments. Up to 3 Subclass BRANCHIOPODA inches long. 5obson fly and fish fly (See 22 a) larvae. 1 Class liNISCTA. Order' 25bAiiiimal less than 3 mm, in length. MEGALOPTERN (Fig. 14) Append4ges more or less slender and jointed, often used for walking. 20b FOU1' or more pairs of legs. 21 S'hells opaque.Ostracods,. (Fig. 11) Subclass OSTRACODA 21a Four pairs of legs.Body rounded, bufbous, head minute.Often 2C) Body q series of six or More brown or red. Water mitOs. similar segments, differing. t mainly imsize. Phylum ARTIIROPODA, Class ARACIINII)A, Order ACARI 261) Front part of body enlarged into (Fig. 15) a somewhat separate body unit cqcophalothora0 often covered 21b Five or more pairs of walliing 22 with a single-piece of shell (cara- or swimming legs: gills, two pace).Back part (abdomen) may be pairs of antennae.Crustaceans. relatively small, even folded Phylum ARTIIROI'ODA, underneath front part. (Fig.19b) Crass CRHSTACE 27a Body coropressdgaterally, 22a Ten ormore pairs of f d, organism is tall and thin.Seuds. leaflike \swimming and atory atnphipods. appendages. Mhny sped wim Subclass AMPIIIPODA (Fig. 17) constantly in /life; some swim upside doWn,Fairy shrimps 27b Body' compressed dorsoventrally, pityllopods. o'rbranchipods. i.e., organism low and britad. 4 Subclass BRANCBIOPODA A Flat gills contained in chainber (Fig. 16) irteneath tail,Sowbugs. Subclass ISOPODA (I+ ig. 18) 22b.1,ess than ten pairs of swimming 23 or respiratory appendages. 28a Abdomen extending straight out hohind, Gilding in two small pro- 3. 23a Body arid legs inclosed 24 jections. One or two lai-ge masses' of valved (2 halves) shell wliich Nay eggs are often attached to female. or may not completely hidethem. Locomotion by means of two enlarged, unbranched antennae, the only large 3b Body'and legs mht enclosed in 26 appendages on the body. 'Copepods. bivalve shell. May he large 9r Subclass COPEPODA (Fig: 19) minute. (Figs. 17, 18, 19) 28b Abdomen extending rut behind ending in,an expanded "flipper" or swim- 24aOne pair.of branched antennae ming paddle.Crayfish or craw fish. enlarged for locomotion, extend Eyes on movable stalks.Size range outside of shell (carapace). usually from one to six inches. Single eye usually visible. 4 Subdlass DECAPODA "Water: fleas" Subclass CLADQCERA (Fig. 12) 29a Two pairs of functional wings, 39 . N one pair may be more or less har- 24b Locomotion abcornplished by 25 dened as protection for the other body legs, not by antennae. pair.Adult insects which normally live on or in the water. 25, 28) 25-4 21G 0 "b . . Key to'_Selected Groups of FreshwaterAnimals

. es Mb No functional wings, though 30 is wide.Alderflies, .fishflies, sand pads in which wings are develop- dobsonflies. ing may be visible. Some may Order MEGALOPTERA (Fig. 22, 14) . resembleadult insects very 5' closely, others pay differ ex- (34b Gene ally rounded in cross section.. tremely from adults. Late al filaments if present tend to b long and thin. A few forms 30aExternal pads or cases in which 35 . extremely flattened, like a suction wings develop clearly v cup.Beetle larvae. 24,26,27) Order C014tOPTERA (Fig. 23) $0b More or less wormlike, or at 31 lir least no external evidence of - 35a Two or three filaments or other 37 wing development. .structures extending out from end Of abdomen. 31aNo jointed legs present.. Other. ,structures such as hooks, sucker 35b Abdomen ending abruptly, unless discs, breathing tubes may be terminal sment itself is extended present.Larvae of flies, as singlerticture.(kgs. 24A, 24C) midges, etc. 4. Order DIPTERA ('Fig. 8) 36a Mo th parts adopted for chewing. *# FrOnt o cc civered by extensible 31bThree pair,s of jointed thoracic 32 folded mouthparts often called a legs, head capsule well formed. .; "mask". Head broad, eyes widely . spaced. Nymphs of dragonflies 32aMinute (2-4mm) living on the or dar'n,ing needles. water surface film.Tail a . Order ODONATA (Figs.24A, 24C, 24E) strong organ that can be hooked into a "catch" beneath the 361) Mouthparts for piercing and sucking. thorax. 'When rele-ased animal Legs often adapted for water lo- jumps into the air.No wings comotion. Body forms various. are ever grown. Adult Water bugs, water scorpions, water tails. boatmen, backswimmers electric Order COLLEMBOLA lightbugs., water stridems water measurers, etc. 32b Larger (usually over 5 mm) 33 Order IIF-MIPTERA (Fig. 25) wormlike, living beneath the surface. 37a extensions (caudal' filaments) two. --Stonefly larvae. 33aLive in cases or webs in water. Order pLEcoRTErifi (Fig. 26) Cases or webs have a silk foundation to which tiny sticks. 371) Tail extensions three, at times 38 stones, and/or bibof 46bris greatly reduced in size. d. Abdominalsegments are attached. 1 often with tiiinute gill filaments. .. 38a Tail extensions long and Blender. Gen,erally cylindric in shape. ;Rows of hairs may give. extensions Caddisy larVae: a feather-like appearance.

be Order TRICHOPTERA (Fig. 21) Mayfly larvae. Order EPtAMEROPTRA 33b' Free living, build-no cases. (Fig. 27) 34a Somewhat flattened in cross 38b Tail extensions flat, elongated section apd massive in appear- plates. Head broad With widely ances Each abdominal segment spaced eyes, abdomen relatively with rrather stout, tapering, lateral long and slender. Damselfly filaments about as long as body nymths: Order ODONATA.(1`if. 24D'

25.-5 Ku to Selected Groups. of 'Freshwater Animals

515

39aExternal wings or wing covers""" 42a Paired rippendages arelegs 43 form a hard protective dome over theinner wings folded 42b Paired appendages are fins. beneath, and over the abdomen. gills covered by a flap a(las. (operculum).----T rue'Tishes -Order COLEOPTERA Class PISCES (1}g.28) 39b E'xterinal wings leathery at base. 43a'Digits with claws, nails, or hoofs 44 -Membranaceous at tip.Wings sbmetime;;°yer6r short.Mouth- 43b Skin naked. No claws or digits. parts fciVpiering and sucking Frogs, toads.\ and salamanders. Body form Various.True bugs. ClaSS A MPHIBIA Order FIEMIPTEFIA (Fig. 25)

40a Appendage present in,pais. 42 44a, Warip blboded; :\ 4'5 (fins, legsv-wings) ',N "f''\ 40b..N,o paired appendages.Mouth 41 .44h Cold bloodedBo y covered a round suction disc. with horny scalesr plates 'Class REPTIL A --"" 41a Body- long and slenderSeveral 45aBody Covered with fathers typles along side of head Birds Lampreys."' Class A VES Sub Phylum VERTETillATA Class 'N'('7.0STOMATA 45bBody entered with hair "Mammals 41h 13odv plump, ovalTail extending Class MAMMALIA out abruptlyLarvae of frogs and toads,Legs appear one 'at a,til-ne during metamorphosis to adult form.Tadpoles. Class 41MPHIBIA

A

a

a

5 5

4 25 -6 4 Key to Selected Groups of.Ereshwater Animals, c

II REFERENCES - Invertebrates REFERENCES - Fishes

1 Eddyi,,Samuel and Hodson, A. C. 1 American Fisheries Society. A List of I , litionornic Keys to the Common Common and Scientific Names of Fishes Animals of the North Central States. J from the United States and Canada.- Burgess Pub. Company, Minneapolis. Special Publication No. 2, Am. Fish 162 p.1961. soc.Executive Secretary AFS., Washington Bld. Suite 1040, 15th & 2 Edmondson, W. T. (ed.) and Ward and New York Aventie, N.W. Washington, Whipple's Freshwater Biology.John DC 20005.(Price $4.00 paper, Wiley & Soni, New-york. pp. 1-1248. $7. 00 cloth).1970 1959. 2 Bailey, Reeve M. A ReVised List of 3 Jahn, T. L.nd Jahn, F. F. How to Know the Fishes of Iowa with Keys for the Pr to Wm. C. Brown Company, Identification, IN: Iowa Fish and aubuque, Iowa. pp. 1-234. 1949. Fishing.State of Iowa, Super. of Printing.1956 (Excellent ,color 4Klotsr-Elei-e"B.The New Field Book'of pictures). Freshwater Life.G.P. Putnam's ns. 398 pp..1966 3 Eddy, Samuel. How to Know the Freshwater Fishes.Wm. C. Brown 5 Kudo, R. Protozoology.Charles C. Company, Dubuque, Iowa. 1957. Thomas, Publisher, Springfield, Illinois. pp. 1-778. 1950. or. .,.4Hubbs, C. L. and Laglesi, K. F.Fishes of,the Great Lakes Region.Bull. 6Palmer, E. Lawrence.Fieldbook of Cranbrook Inst. Science, Bloomfield N,atural History.Whittlesey House, Hills, Michigan. 1949. McGraw-Hill Book Company, Inc. New York.1949. 5- Lagler, K. F. Freshwater Fishery Biology.Wm. C. Brown 'Company, 7Pennak, R. W.. Freshwater Invertebrates Dubuque, Iowa. 1952. of the United States.The Ronald Press ,Company, New York.pp. 1-769,1953 6Trautman, M. B.'The- Fishes of Ohio. Ohio State University Press; Columbus,. 8Piinentel, Richard A.Invertebrate 1957. (An outstanding example of a Identification Manual. Reinhold State st zcly)% Publishing Corp.151 pp. 1967. Pratt, HW.- A Manual of the Common Descriptors: Aquatic Life, Systematics. - I Invertebrate Animals Exclusive of Insects.The Blaikston Company, Philadelphia, Pa. pp. 1-854.1951 ...wlA,.

Key to Selectsid Groups of Freshwatei. Ahimap

tiy .\ t cei

1. SeaTilla spieules Up to .2 mm.lpag.

'IA. Rotifer, Folyarthra 3C. Rotifer, Philodina Up to .3 mm.' Up to .4 mm. 3B. Rotifer, Keratella 2B. Bryozoal mass. Up to Up to .3 mm. several feet diam.

214. Bryozoa, Plumatella. Individuals- up to 2 mm. Intertwined masses may he very extensive. 1-

4A. Jointed leg 4B. Jointed leg 4C. Jointed leg Caddisfly Ctayfish Ostracbd

5. Tapeworm head, Taenia .Up to 25 yds. long -

6B. Turbellaria, Dugesia Nematodes. Free living Up to 1:6 cm. aria, Mesostdma forms commonly up to J IA? a %ATI. --I mm.. occasionally 25-8 more. x: 2210 .-

, . Key to Selected Groups of Fregliwater Animals A

c I

813. Diptera, Mosquito pupa. Up to 5 mm.

(

I 8E. Diptera, cranefly 13A. Dipt, rd, :Mosquito larvae 8C. Diptera, chironomid .pupa. I:p to 2. 5 cm. Up to 15 mm. long. , I larvae.' Up to 2cm.

I

9D. Diptera,. Rattailed maggot 9A. Annelid, Up to 25 mm. without tube. I segmented worm, up to 1/2 meter I

I

I 1013. AlasiniOnta, endview. - i. r I

10A, Pelecyopod, Alasmidonta 0 I - Side view,. up' to 18 Cm.long. 9B. Annelid, leech up to 20 cm.

I 1

:12B.Branchiopod, 12A. BratichioPod,, Bosmina. Up - I Daphnia. Up ____.., to 2mm. to 4mm.

411A. Ostracod, Cypericus Side view,: up to 7 mm.- 4

. 1113. Cyperus, end view. 25 -9 $

ii.ey togrjeted-Orotts-of Festmate Animals

a 13. Gastropod,CaMpeloma Up to 3 inches. 15. Wkter mite, up to 3 mm.

14. Megaloptera,Sialis ALderfly larvae Up to 25 min,

9. 16. Fairy Shrimp,Eubranchipus Up to 5 cm.

.11

I. J10111111Ilk, 18. Isopod,Asellus 4Wert Nt, Up-to 25 mm. /1.7C\ iffillite. 41Io I 17. Amphipod,pcmaiaporefa Up to'25 mm.

O

19A. Calanoid copepod, 20. Collembola, Podura yclopoid copepod Up to 2 mm. , Female 19B. Up to 3 am. Female 25,10 Up to 25 mm. 0 2°4#0*.e 9 Key to Selected Groups of Freshwater Animals

3 21A. 21B. 21C.

21D. 21E. Trichoptvra, 1 aryalcases, 22.. Megaloptera,--alderfly Up to 2 cm. mostly 1-2 cm. e

23A. Beetle larvae, 23B.Beetleklarvae,24A. Odonata, dragonfly Dytisidael Hydrophilidae nymph up to 3,or Usually about 2 cm. Usually about 4cm cm. 4

Odonata, tail of damselfly A nymph 24E, Odonata, front view (side view) of dragonfly-nymph Suborder showing "mask" Zygoptera partially extended (24B, D) Suborder Anisoptera

. Odonata, damselfly (24A, E, C) nymph (top view) 24C. Odonata, tail of dragonfly nymph (top view) 25-11 223 Key to Selected Groups of Freshwater Animals

25A. Hemiptera, Water Boatman About 1 cm. 258. Hemiptera, Water Scorpion 26. Plecopte ra, About 4 cm, Stonefly nymph Up to 5cm.

1

27 .Epheme ropte ra. 28A. Coleoptera, Mayfly nymph Water Up to 3cm. beetle. Up to 4 cm. , 28Bs. Coleoptera. Dytiacid beetle Usually up to 4 CM. 1

29B. 4Diptera. Mosquito Up to 20 mm. 29A. Diptera, Crane fly. Up to 23 cm. 25 -12

, A.

A .Kay FOR THE INITIALSEPARATION OF SOME COMMON PLANKTON ORGANISMS

1. No chlorophyll present, 'unless 'through ingestion 8 1. At least some chlorophyll present 0 2. Pigments not in plastids Oyanophyta 2. Pigments in one or more plasts 3

3. Cell wall .of over-lapping halves and distinctly sculptured Bacillariophyta '3.Cell wall not of over-lappingchalves, , or if so, then not sculptured 4

4. Pyrenoids present; color usually. bright green Chlorophyta 4. Pyrenoids absent; color green, yellow-green yellow-brown 5

5. Bright green, motile, usually with- one 'anterior flagellum Euglenophyta Yellowish to brownish, motile or not 6 J .st.1.5.! 6,,With a ,distinct lateral groove, motile Pyrrophyta 6. '"Without a lateral. groove 7 I ,- '7.Seldom' motile; unicellular, colonial or filamentous , . . .Xanthophyta 7. Motile, unicellular or colonial r . . .Chrysophyta

8. Unicellular; naked or enclosed in a smooth or sculptured shell 9 8. Multicellular; body usually with a distinct exoskeleton...... 11 9. Amoeboid; sometimes with shell, no cilia or flagella Ameoboid Protozoa 9. Actively motile; never with shell; cilia or flagella obvious 10

10. Body more or less covered by short cilia; movement "darting" . Ciliate Protozoa 10.. Body with one or more flexible whip-like falgella; movement "continous" .;Flagellate Protozoa

11.Shell bivalved (clam-like) r .12 11.Shell not compoOd of two halves 13

12. With distinct head anterior to valves Cladocera 12. No head anterior to valves Ostracoda .13.Usually microscopic; body extended -.into a tail or foot' with one or more toes Rotifera t . 14 \ 13.Usually macroscopic if rilatur ; ' A

14. Appendages bilateral; heed not prominent ...... ,- Copepoda 14. Appendages unilateral; head prominent Phyllopoda. o o

.. CHLOROPHYTA A Key,,, to' Sorrre of the Comnion Filamehtous Genera

Alf , 1. Filaments unbranched _ 2 1. Filaments branched (sometimes parenchymatous) 12 ..

2.Chloroplast single, parietal band 3 2.Chloroplast one or more, If parietal not a band' 5 3. Chloroplast encircling more than half the cell (Nap-ring like)- Ulothrix 3.Chloroplast .encircling less than hdlf the cell. ... 4 4.Filaments of indefinite length; cells with square ends Hormidivm 4.Filaments usually short,of 3.8 cells, with ends round Stichococcus

5.Cell wall of Ilk pieces; pyienoids Iaaing < Microspora 5.Cell wall not of H pieces

6.Some cells with apical caps Oedogonium 1 6.Cells without apical taps 4. 7

7. Chloroplast (s) parietal 8 Chloroplast (s) axial.. ti t 9 8;Chloroplast one or more, spiral bands.. Spirogyra 8.Chloroplast several, longitudinal bands Sirogonium

9.Cell walls- without a median constriction, 10 9.Cell walls with a median constriction . 11

I. 10. Chlor plast stellate ; Zema 10.Chlor Oast an axial band Mouygngeotia

11..Filamentscylindrical Hyalothaca 11. Filaments triangular, twisted Desmidium

12.Coenocytic dichotomously, branched, with constrictions Dichotomosiphon 12.Filaments with regular cross walls 13 I o # 4* 13.Parenchymattius, discoid,epiphytic . a.. Coleochaete 13.- Not parenchymatous 01°4 -- ° 14.Main axis cells much broader than branch cells..... _._. .-Draparnaltia- 14.Main axis and branch _cells 'approximately- thesanie breadth 16' .. 15, Maitraxi arilateral branches attenuated into long multicellualr hairs ,.16 15.Axis and branches not attenuated into long multicellular hairs d'.. 17 16.Sparsely or loosely branched Stigeoclonium. 16. ;1Densely and compactlybrarich d Chaetophora ..t e .0. 0.

17; Cells bearing swollen or bulbous-bd.sed setae 18 17. Cells without setae 19 1 . 18, Swollen-based ,setae on dorsal surface of cells;rostrate \ epiphytes; little or not al all branched Apbanochaete` 18. Bulbous-based setae terminal..onbranches;,itot S . . prostrate epiphytes . , Bitlbochaete _ - a , -, 19. With .terminal and/or intercalary akinites ° Pithophora 19.. Without ,distinctive akinites 20 ;r1 20. Cells of erect filaments becoming shbrter and broader toward filament apex;-UsuallY growing on back of turtles; branching only . from base ' . , Ba.sicladia . .20;Thallus not as above . 21

21. Filaments irregularly branched; branches short 1- or few celled. . zoclonium 21. Filaments repeatedly°branched; branches narrowed toward tips C dophora

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CHLOROPHYTA A Key to Some Common Non Filamentous Genera 4

I. Unicellular 2 1. . . . Colonial - 1 27 .. , - I 2.Motilein vegetative -state,, flagella 2 -4 3. 2.Non- motile in *vegetative state . 5 ..: ... 4.. . 3.'Cells-with 2 flagella 4 47' .3.Cellswith .4 flagella Carteria

4.'Cellenclosed by bicovex shell.... .r Phacotus 4.Cell.not enclosed by a'Shell . . ",' Chlamydomonas : . : 5. Cellswith median constriction (often slight), or of chloroplast only.. . 6 5. Cellswithout a median constriction 15 . .. 6. .. Cellslunate : Closteripal o 6". Cells not lunate in any degree i # 7

7. Cellscylindrical, noticeably 'longer thanbroad.ti''' 8 7.cellsalmost .never cylindrical; flattened or triangular in apical view 9 8. \ength2-3 times the breadth, contriction nearly lacking. . ...Cylindrocystis 8, ength much greater than breadth, nodulose at constriction Pleurotaenium , . ___. 9. Cellstriangular in end view 1 Staurastrum 9. Cellsnot triangular in end vie)w - z : '10.. 10. Semicells with lateral incisions, appearing lobed / 11 10.Semicells without lgeral incisions 12

11. Lateral incisions few, shallow, lobes rounded...... Euastrum 11.-Lateral incisions many, deep, lobes angular Micrasterias

12.Semicells with radiating arms ,.. Staurastrum Semicells without arms; spines, granules,or teeth may be present 13 13.Semicells without spines Cosmarium Ot404. 13. Semicells with spines 14 14.Spines few, usually at the apicalcorners 'Arihrodesmus 14,Spines numerous, scattered Xanthidium

15. Cells elongate, sometimes needle-like. . .. 16 ,, 15.Cells 'spherical,- ovid, angular; not needle-like 18 --., 46.Ceiis with terminal setae Schroederia 16.Cells without terminal setae . . .17: Cells acicular, withOut a row of pyrenoids. ..Ankistrodesmus 17. Cells acicular, very long, with a row, of 10-12 pyrenoids Closteropsis ..e 18. Cells wittioitt spines br processes 19 18. Cells with spines or processes 23 . 19. Cells embedded- in a gelatinous matrix 19.1 Cells without a. gelatinous matrix. 2201.

20; Gelatin obvious, lamellate; dhloroplast Cup-shaped ...... -Gloeocystis 20. Gelatin sometimes obvious, chloroplast, star-like Asterococcus 4

21. Cells spherical , -.±. ..,22 21..:Cells angular Tetraedron . . ., . . 22. Cell wall smooth Chlorella . 22.-Cell wall sculptured' . . Trochiscia . ' 7 24 23. Cells angular . s ... .. 23. ps spherical or. oval,with spines .-. .. . 23 -'.,.: . . . - , , . 24. Angles with furcated _processes .. ., -, Tetraedron .-...-2-4. Angles with spines ... IN Polredriopsis . .. 12

. .. . ._ ..,G lenkinia 25. Cells spherical, spines delicate c, ...... "...... 26 ... 25: Cells oval, spines evident .. . 14 .,1/4 26. Spines localized at ends of cell ... La.geheimii- -

26. Spines distribbted over cell . . Franceia

27..Motile,. each cell with 2 equal-length flagella .t? 28 27. Non-motile invegetative state 33

28. Colony. a "flat" plate 29 28. Colony spherical or ovid 30

29. Colony "horse-shoe" shaped Platydorina 29. Colony quadriangular or circular ,Gonium

30. Cells 8-16, crowded-pyrifOrm 1 . .Pandorina 30. Cells more than 16, not crowded, spherical .or nearly so 31 . 1. 31. Celld more than 300 in number. Volvox 31. Cells less than 300 in nitalier 3?

32. Cells (16) - 32k nur per Eudorina- 32..Cells 64-128 -(256), in number . Pleodcirina . _

33. ,Cells of colony lying in one plane 34 33. Cells not in a conspicuous single plane ,37

. . . I . 34'. Colony, ciroillar (rarely cnuciate) ...'Pediastrum 34. Colony not circular I 38 . .4 40,

. 1 2 2 26.5 ,

35.Colony, a flat strip; cells side by side ,Scenedesmus 35,Colony quadriangular , 36 Colony usually large, cells in 4ts, no *pines Crucigenia 36.Colony of 4 cells, leach with 1 or 2 marginal spines...... Tetrastrum 37. Cells acicular (needle-like) Ankistrodesmus 37.Cells not acicular 38

38.Colony without a gelatinous envelope 39 38.Colony with a more or less conspicuous gelatinous envelope 47

39. 2-8 oval enclosed by a distinct sheath .Oocystis 39.ICells hot enclosed by a sheath 40

40. Cells with long Spities or setae 41 40.Cells without spines or setae 42 41.Colony pyramidate, cell spherical,long spines Errerella . . 41.Colony quadrate, - or a tetrahedron, . . . long setae i .. Micractinium go - . \ 42.Cells linear, radiating from acommon center A ctinastrum. 42.Cells no linear 43, ...... ,. 43.Cells .strongly lunate' often "back-to-back" Selenastrum 43.Cells not. lunate - 44

-44..` Cells arranged in a hallow sphere 45 44.Cells not arranged in a hallow sphere 46

... 45.cellsspherics1,sometimes joined by processes '.1,..- . . . Coelastraim .45:' e.Cells not spherical, oute* angles extended into stout, blunt ,teeth or spines .7.-;.-.:...... ,

46.Cells uniform, spherical,in groups of 4:- 8 rlo 46. -Cells notluniformi ellipsoid, (oblong) or reniform Dim . ., 4 7 , Cells curved tlo strongly lunate 1. . 48 ., Cells, not curved or innate"- ...... && , ..&& ..49 A ..d. . 8.Cells innate., loosely arranged in colony Kirchneriella . 48.Cells curved or reniform, colony compact, distinct Neptirocytium .. orr . i ' 49 Cells connected by' branching central strands. Dictyosphaerium '49.Cells not connected by stands 50

\ 50.Cells cyli -drical or fusiform \ 51 or 50.Cellssprical or s ghtly ovoid 52 ..'.v-, .- .. 4,,,...**N,... . - . 41 , ..4,.. 51:Celli in parallel "buns f 2 - 8 ' . r .Quadrigula. .51.Cells longitudinally arranged, not ,grouped laterally Ela\katothrix . 4 77 .

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52. Glosocystis - Cells ellipsoid or ovoid, envelope lamellated ft. 52. Cells spherical, or nearly so, . . . 53

53. Chloroplast axial,star - shaped Asterococcua` 53. Chloroplast parietal, not star- shaped 54

54, Cells enclosed by lamellated sheaths. . Gldeocystis 54..Cells in homogenous envelopes Sphaerocystis

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III I ETIGLENOP HYTA A 4. I 4111 i. ..) ie.r 1. Vegetative cells sessile Colacitnn 1 1. Vegetatiye cells- motile 2

1 .. '2.Cells with _chlorophyll 3 , -a 2.Cells without chlorophyll .. . . 8 3..Protoplast° withina lorica or teat Trachelomonas I 3. Protoplast naked, no lorica 4 ,.. 4.Body strongly metabolic ... .Euglena 1 4.Body -rigid . , 5

5.With two laminate, longitudinal chloroplasts Chryptoglena I 5.With numerous chloroplasts . 6 .- _, 6.Body conspicuously flattened, sometimes twisted , PhacuS I , i 6.Body not compressed, radially syMmetrical 6 .3,% .. 7.Body broadly ellipsoid to ovoid Lepocinclis I 7. Body elongate, narrow Euglena e.Cells with one flagellum .. iistisia 1 8. Cells with two flagella Peranema

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i KEY TO SOME COMMON DIATOM GENERA (BACILLARIOPHYTA) OF MICHIGAN

.Diatains in Valve View

1.4Valves without a dividing line or cleft; markings of' valve radiate about a central 4point.(.centric diatom genera) 2

2. Frustules usually in filaments or zig-zag chains; rectangular in girdle viewi, valve round, oblong, triangular or elongate 3

a.Frustules cylindi:ical; inarkings4rominent on girdle;sucus preseht; seldom seen in valve view Melosira

3. Frustule not cylindrical 4 4.Valve ovate to oblong; two horns or processes present on valve face, scatter small spines often present Biddulphia

. 4.Valve not ovate to oblong, horns or processes absent ,5

5.,Valve face appearing as two; three sided pieCes giving the appearance of six processes Hydrosira

..5. Valve face three to several times longei° than broads; margins undulate.: "costae" evident Terpsinoe

2.Frustules usually solitary (sometimes forsning short chains) 6 6.. Frustules usually elongated; many intercalary bands; frustules cylindrical 7

7.Each valve with a single long spine Rhizosolenia

7. Each valve with two long,spines.

6. Valves discoid; cylindrical; or spherical; somtimeswith spines or a. 8 processes ...... 9, 8.Ornamentation of valves in two concentric parts of unlike pattei71 CyclotelIa

8. 'Ornamentation of valves radiate; continuous from. center to margi9 of valves 9

9.. Ornamentation of valves distinctly radiate; rows of punditae single at center becoming multtheriate at the margin; margin of valve with recurved spines Stephanodiscus

9. Ornamentation of .valves not distinctly radiate or,becoming multiseriafe at margin 10

233 10.Isolated large punctum evident at the margin *pines not evident ....r Thalassiosira

10. Isolated punctum not evident at the margin,' short spines present, Cascinodiscus 1."Valve_ with a- dividing line ,ocleft; marking of wall bilaterally disposed to an axial or exce tric line (pennate diatom genera) 11

11. Both valves with a'pseudoraphe 12

12. Valves asymetrical to['one axis 13

13.Valve asmetrical to longitudinal axis, striae present . .Ceratoneis

. 13.Valve apymetrical to transverse axis 14

14.Valvep clavate 15 14;:Valves not claate; striae present; valves wittr-unequal capitate ends; often forming star like colonies. .

15. ."Costae" present, frustules may be joined face to face to form fan like filaments. Meridion

15."Costae: present, frustules single (appeawri as asymetrical ) Opephora

12.Valves symetricalto both axes 16 16. Frustules septate or "costate"; often appearing in zig- zag chains 17

17. Septae present; usually many partially septate intercalary bands; valves triundulate Tabellaria

17.4. Septae absent; prominant "costae" on valve. . .Diatoma

16. Frustules occuring Ire or attached in filaments; sometimes formingscicles ...... 18 ' 18.Frustulea typically forming long filaments; usually not more than 5 or 6 times longer than broads; often appearing costate . . . Fragilaria

18.Frustules usually solitary or forming. fascicles; usually many times longer than broad Synedra (note: the above two genera are actually seperated only of the basis of growth habit)

11.At least one valve with a pseudoraphe 19 19. One valve with a true raphe the other valve with a pseudoraphe '20

20.Valve asymetrical to the 'transverse axis; partial terminal septae; bent about the transverse axis Rhoicosphenia .

20.Valves symetrical to both axes. . 21.

21. Valve elliptical; valves with- a marginal and/or submarginal hyaline ring; often loctiliferous; bent about the longitudinal

axis .. . Cocconeis

21.Valves usually lanceolate or linera lanceolate"; bent - 'around the transverse- axis Acfmanthes (Those with a sigmoid raphe` are sometimes put in Ac4nanthidium or Eucocconeis)-

19.Both valves with a 'raphe 22

.22.Raphe median or nearly so, never completely marginal; not in a canal 23

23. Valkres sigmoid'in outline ft 24. 24. Raphe sigmoid; punctae in two series; one tran verse and one longitudinal row forming a 900 angle. .. Gyrosigma

24. Raphe sigmoid; punctae in three series forming, a angles of other than 90° Pleurosigma

23.Valve not sigmoid in outline 25

25.Valves symetrieal to-,both axes s .. .26'

26.Frustules with sept'ate intercalary bands 27

27.Intercalary bands with marginal loculi, piInctae distinct Mastagloia . 27. Intercalary _bands with two large faramen along apical axis, punctae indistinct..,...... Diatomella . 2,6. Frustules without septate intercalary bands 28

28.Valve face with a sigmoid saggital keel; , "hourglass" shape outline in girdle. view. . .Amphiprora . . 28.Valve face without a sigmoid saggital keel . . .- .. . 29 M1' ' 1 . ,, .., 29.Valve with undulate or zig-zag irregular logitudinel lines or blank spaces . . . . Anomoeneis

29.Valve without undulate or zig-zag irregular logitudinal' lines or ,blank spaces 30 . T r t

26-11

2 3 5 1N. 30.Valve with thickened, non-punctate central' area; (stauros) pseudoseptae sometimes present, longitudinal lines and blank spaces lacking ...... - . Stauronels

30.Valve with or without stauros, septal absent 31.1/4

31. Valve with longitudinal lines or blank spaces . .. 32 32.Proximal ends of raphe usually curved in opposite directions; "defaut regulariei." toward valve apkces;. longitudinal blank spaces. Neidium 7 j. 32. Proximal ends of raphestraight; valves with fine striae that appear as costae; longitudinal 'line near margin. Caloneis

31..Valves without longitudinal lines or blank spaces 33

-33.Valves with siliceous ribs along each side of the raphe 34 34. Raphe bisects siliceous ribs on valve; central t area small and orbicular; striae and punctae very distinct Diploneis, r, 34. Raphe short; less than 1/2 length of valve; central area long and narrow; terminal nodules evident, elongate 3 5 F- 35.Raphe short; 1/4 or less the length of the valve; .striae not evident Amphipleura 35. Raphe longr; usually about 1/3 the length of. the valve; striae. usually fine, but evident. .

33. Valve without siliceous ribs 36 36.Valves with smooth transverse costae; raphe often ribbon like.

36.Valves with transverse-striae 37 37.Raphe sigrnoid Scoliopleura 37.Raphe straight `38 '38.Raphe in straight and raised keel Tropodoneis

38.Raphe 'straight and not in a keel 39

39.Striae daubly punctate, central area long and narrow..Brebessonia

39.Striae single to lineate, central area variable. . . .Navicula .., 1 Valves symetical to one axis only I 40 25; ...... 4 40. Valves symetrical to the longitudinal axis .. ..., 41 41. Punctae in one series; longitudinal line absent Gomyhbnema

41. Punctae in two series; longitudinal line present' Gomphoneis . .. \ . t

40.Valves symetrical to transverse axis. . .. t 42 .42.Raphe short; vestigial terminal; with evident terminal nodules, central nodule lacking 43 A 43.Colonial; forming tree like coldriies; valves usually with evident spines Desinogonium

43. Usually nbt in colonies or if colonial, forming only short chains or stellate clumps 44

44. Cells shaped like. the femur of a chicken. . ..Actinella

44.Valves various shaped; lunate to nearly straight; valve often with undulate dorsal and/or ventral margin; raphe prominant in gir le view 45

45. Dorsal margin convex,ve4tralmargin slightly cOncave, both margins sinvate-dentste. . . Amphicampa

45_. Dorsal margin convex, Ventrd(l margin straight to concave, one, both or neither margin wavy, pseudoraphe often present on ventral margin . . .Eunotib.

42.Raphe not vestigial; usually as nearly as 'long as the valve;. valves usually cymbiform 46

- . '46.Valves convex; central nodule usually lies very close to the ventral margin; both. raphe visible In girdle view .. . .Amphort.

46. Valves flat or nearly so; raphe a smooth curve with the`.same curvature, asthe axial field; raphe not visible in girdle s view Cymbella

22.Raphe marginal and in a canal .. . . . 47.Valves 'with a.single canal that is usually marginal but may appear to be somewhat central 48

c: 48.Valves symetrical to, both axes . . . . 49'

49. Tranaver,se internal septae that appear as "costae"; canal nearly Median .Denticula:

49. Transverse "costae" lacking; carnial dots present. . . Nitzschia 0'

48. Valves asymetrical to longitudinal axis r-50

50."Costae" quite evident 51

51. Axial field forming an acute angle at the central nodule; raphe along the ventral margin of valve Ephithemia

51. Axial field forming a less acute angle at the central nodule; raphe along the dorsal margin, of the valve . .Rhopalodia

50. "Costae" not evident; carnal dots along the raphe 52

52. Raphe of, One valvediagonally opposite the raphe on the othet valve Nitzschia

52. Raphe.of one valvedirectly Opposite the raphe on the other valve Hantzschia

47. Valves with a canal next to both lateral margins 53

53. Valve face transversely undulate; band of short costae aloqg each . r lateral margin' appearing' like a row of beads Cymatopleura

53. VOve faceortot transversely undtilate 54

54. Valve shaped like a Sadd Camyylodiscus

54. Valve face flat; either isopolar or heteropolar; sometimes the frustule -is slightly spiral in shape ... ., . . .Surirella

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t CHRYSOPHYTA A Key to Some More or Less Common Genera

1. Filamentous, branched Phaeothamnion Not filamentous 2

2.(Unicellular 3 2.Colonial ., 3.Protoplast enclosed by a lorica 4 3. Protoplast not enclosed by a lorica . 6

4.Epiphytic or epizooic \ 5 ..., 4.Motile; cells with siliceous scales many of vh ich have long, siliceous spines Mallomonas

5. Epiphytic; lorica flask-shaped Lagynion 5..Epiphytic or epizooic; lorica cylindrical Epipyxis(= Hyalobryon)

., 6. Motile; protoplast naked Ochromonas (art associ) 6.Non-motile; protoplast with long delicate, pseudopodia . - Rhizochrysis

7.Sessile; each cell In a long, cylindrical lorica Epipyxis (Hyalobryon) 7.Motile 8

8. Each cell within a companulate;basally pointed lorica Dinobryon 8. Lorica absent 9

9.Colony bracelet-shaped Cyclonexis 9.Colony spherical - 10

10. Colony bristling with long siliceous rods Chrysosphaerella 10.Colony without siliceous rods from each cell .: 11 . 11. Colonies not enclosed by a gelatinous sheath / Synura . 11. Colonies enclosed by a distinct gelatinous sheath 12

12. Shorter flagellum more than 1/2 length of longer flagellum Uroglenopsis 12.Shorter flagellum less than 1/2 length. of longer. flagellwn Uroglena

239 26-15 . .

.r"

CYANOPHYTA A Key to Some of the Common Genera

colonial . 2 1. Cells not in trichomes; unicellular or 10 1. Cells in trihomei 3 2.Colony with some regular arrangementof cells Colony amorphilius, no definite form 6 / 2. . 4 .3.C-olony a flat 'plate 5' 3. Colony spherical, cells peripheral .. Cells regularly arranged Merismopedfa 4. Holop,edium, 4. Cells irregularly arranged Colony with a centralbranching system Gomphosphaeria 5. Coelosphaerium 5. Colony withOut a central branching system

. many celled 'Colony mostly few celled 9 6. it4 Cells elongate Aphanothece Cells spherical 8 Microczstis 8.Cells close together 8. Cells more than 2-'3 diameters apart Aphanoc4iisa Cells usually hemispherical,with of without, definite gelatinoussheaths. .Chroococcus 9. Gloe ocapsa 9. Cells sphNgal or olod, 'gelatinoyssheaths very distinct

1 0. Trichomes without sheath (notfilamentous) 10. Trichomes in a sheath (filamentous) _ , 12 . ,. . 11. "Heterocysts absent / 14 11.-Heterocysts present' ,

c,12.Trichomes straight..... Osciliatoria 13 12. Trichomes' regula y spiralled Arthrospira 13. Cross walls dist Spirulina 13. Cross walls lacking .

, 15 14,Heterocysts terminal eterocysts intercala'ry .1 s- g, -18., Cylindrospremurb 15. -Trichrnes cylindrical ... .16 Trichomes ,attenuate. Trichomes\golitary Calottirix . .17 14. Trichomes in masses ..

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26.'46

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17.Trichomes without akinetes Rivularia 17.Trichome's with akinetes 1 Gloeotrichia

18.Trichomes straight, parallel in bundles Apha.nizomenon Trichomes solitary, or :if in masses, not parallel 19

11. 19.Trichomes solitary, or if numerous, then not in a firm gelatinous es, matrix . .._. , . .- .Anabaena 19.'Trichomes entangled in a firm gelatinous matrix Nostoc -;.' ', . 20. Many parallel trichemes in a sheath...... ,t' Microcoleus 20.A single trichome or row of irichomet in a sheath 21

21.Filaments not branched .. . . 22 21.Filaments. branched Cl 23

22.Sheath obvious firm . .4 Lyngbya 22..Sheath indistinct; delicate Phormidium

23.Filaments with false branching 24 23./Filaments with true branching 26 24. , Without heterocysts Plectonema 24,With heterocysts. 25

25.False branches arising single Tolypothrix 25.F*alse- branches in 'pairs- .. --- . Scytonema . . --, 26. Trichomes always uniseriate Haplosiphon 26.'Trichomes wholly or in part nultiseriate Stigonema

; XANTHOPHYCEAE A Key to' Som Common Genera

Filamentous. . 2 1. Not filamentous 4

N Z.Filaments branched, siphopceaous -Vaucheria 2.Filaments not branched 3 3.Cell wall of stout H pieces,cells spmetimes barrel-shaped Tribonenv. 3.Cell wall of delicate H'pieces, cells short and cylinaileal*. . , .

4.Cells embedded in a gelatirious matrix .. ... - ..,,. 4 5 4.Cells not embedded in a gelatinous matrix .. Ifs 7

5.Colonial gelatinous envelope dichotomously branched . . °Mischococcus 5.Colonial gelatinous enveloi4 not, branched . .. 1, . ..6 . , . te, 6.Colonial envelope, 'tough Hheav-P cartilaginous Botryococcus 6.Colciniatl envelope' iAratery;:coloniis-small, 'free floating Chlorobotrys

8. 7. Cells epiphytiak. 0 , , 7.Cells not epiphytic,. . .'.,. .° .. ' ...... -.; 11., . , ...... t 8.Stipe long,seta-like, :, -- Stipitococcus 8. Stipe short; dr cell-sessile ...... °: .. ,,,' Peroniella

9.Cell vase-like, apex flattened °. . . .. ,.', .i..l',.-, Stipitococcus . ° .4.4'. . .:' .., .... Peroniella 9.Cell oval or spherical ...... ' ... ../: .. . Ct ,- . , ,:, . 10.CelTs ' cylindricai, ends broadly rounded . . ..°'.. ' . , .. .; .. . .,. Ophiocytium , 10.Cells not ,cylindrical 4 . Characiopsis . - t . , .. . t . 11.Cells cylin ricalstraight or contorted . .-.:...... - . OPhiocytium 11.'Cells glob ,with rhyzoids, on soil :-. ... - Botrydium t

,

4

242 4,ii4,

Acicular:needle-like in shape.*.(Ankistrodesmus) . A*. Aerial:algal habitat on moist soil, rocks, trees, e ; involving a, thin film of water; sometimes only somewhat aerial. Akinete; A type of spore formed by the transformation of a vegetative cell into a thick- wallet resting cell,containing a concentration of food material.(Pithophora)

Amoeboid:like an amoeba; creeping by extensions of highly plastic proto lasm (pseudopodia).(Chrysamoeba)

Amorphous:without definite shape; without regular form. Anastomose:to sepa and come together again; a meshwork. Antapical:the posterior orear pole or -region of,.an orgaririm, or of a colony or cells. Anterior:.., the forward end; toward the top. Antheridium:a single cell or a series of cells in which male gametes areproduced; a multicellular, globular male organ in the Characeae; more properly called a globule (a complicated and specialized branch inwhich antheridial cells are produced. cr. a f Antherozoid:male sex cell; sperm.

Apex:the summit, the terminus, end of a projection, of an incision, or of a filament , ,zt of cells. ,Apical:. Forward tip. APlanospore4 non- motile, thick-welled spore formed_ many within an tinspecialized vegetative cell; a small resting ftp or e.

A ibuscular:branched or growing like a tree or bush. Armored: See theca. s- . AtAktuate:narrowing to a point or becoming reduced in diame er.(Gloeotrichia) ik . Autospores:spore-like bodies cut out- of the contents ore cell which arefirsmall replicai of the parent cell and which only enlarge to become mature plants.(Coelastrum) . - Axial:along a median line bisecting an object either transversely or longitttilinally (especially the later,. e.g.. an axial chloroplast). 7 Bacilliformirod- shaped. Bilobed;with two lobes or extensions.

243 Bipapillate:- with. two`` small protrusion's; nipples. Biscuit- shaped:a thickened pad; Pillow-shaped. ,Bivalve (wall):wall of cell which is in two sections, one usually slightly larger than the others. .- Blepharoplast:a/granular body in a swimming organism from which a. flagellum arises. ., Bristle:a stiff hair; a nee-like spine.(Mallomonas) r

CapPlate:with an enlargement or a head at one end.(as in some species of Oscillatoria) Qarotene:Orange-yellow plant pigment of which there are four kinds in algae; a hydrocarbon, C, H.

Chlorophyll;.- a green pigment of which there 11 are five kinds in the algae chlorophyll-a , occurring in all of the algal divisions. Chloroplast:a body of various shapes within the cell containing the pigments of which chlorophyll is the predominating one.

Chromatophore:body 'withina cell contining the pigments of which some other tan chlorophyll is the predo inating one;" may be red, yellow, yellow-green or brown. Go, t Chrysolaminarin:See leucosin. Coenobium:a colony with 2N number of cells.(Scenedesmus)' Coe ocytia!with thultinucleate cells, or -cell-like units; a multinucleatp-,...non-cellulat plant.(Vaucheria) Collar:A thickened ring or neck surrounding the opening in a shell or lorica through which a flagellum projects from the 4nclosed organism.(Trachelomonas)

Colony:a group or closely associated cluster of. cells, adjoined or merely inclosed py a -a common investing mucilage or sheath; cells not arranged in a linear series to form a filament;,4either aggregate or coenobium. Constricted:cut in or ipOseds usually form two opposite points on a cell so that an isthmus is formed between two parts or cell halves; indented as at the joints between cells of a filament.(Cosmarium) 9 Contractile vacuole:a small vacuole (cavity) which, is bounded by a membran that pillsates, expanding and contracting. Crenulate; wavy with small scallops; with small crenations, , 4 Crescent: an arc of a circle; a curved figure tapering to horn-like points from a wider, cylindrical midiegion, - - .Cross wall:a cyoss petition.

L 24

1, Cup- shaped:a mere or less complete plate (as a chloroplast) which lies just within - the cell w41, open at one side to forlif a cup. Cylindrical:a figure, round in cross section). elongate. with parallel lateral margins when seen 'from the side, the ends square or truncate.(H7alotheca)

Daughtercells:cells produced directly from the division of 'a primary or parent cell; cells produced from the same mother cell. I.

Dichotomous:dividing or branched by repeated forkings, usually into two equal portions OD segments. Pisc; Disc-shaped:a flat (usually circular) figure: a circular plate. Distal:the forwar or anterior end or region as op osed to the basal end. Ellipsoid:an ellipse, a plane figure with curved margins, the poles more sharply rounded than the lateral margins of an elongate figure.

Epiphyte:living upon a plant,' sometimes living 'internally' also. Euplankton:true or openwater plankton(floating) organisms. Eye spot:a granular or complex of granules (red orbrowil) sensitive t6 ligh) and related to responses to light by swimming organisms of spores.(Pandorina) False Branch:a, branch formed by lateral growth of oneor'both ends of a broken filament a branch not formed by lateral division of cells in an unbroken filament.(Tolypothrix) Filament:a thread of cells; one or more rows of cells; in theikuegreen ,algae the thread of cells together with a sheath that inay.be present, the thread of . cells alone referred `to as a trichome. * Flagellum (flagglla):a relatively coarse, whip-like organ of locomotion, arising from a special, granule, the *blephIroplast, within a cell.(Eugl4na)

Fucoxanthin:a broWn pigment predominant in the Phaeophyta and Chrysophyta. (Synura) Fusiform:a figure broadest in the iijuidegion and gradually tapering to both polesWhich may be acute or bluntly rounded; shaped like a spindle.(Closferium)

Gamete:a sex cell; cells which unite to produce a fertilized egg orzygospdre.

C Gas vacuole:See pseudovacuole. /Gelatin (gelatinous)a mucilage-like substance. Glycogen:a starch -like Storageproduct questionably identified in food granules of the Cyanopjyta.(Chroodocpcus)" j. Gregarious:an association; groupings of individuals not necessarily joinedtogether but closely associated.

.245 26-21 ."

, .

Gullet:a canal leading frdrn the opening offlagellated cells into the reservoir in the anterior end.(Euglena)

Gypsum:granules of calcium sulphate which occur in the vacuoles of some desmids.(Closterium)

Haernatoohrome:a red orbrange pigment, . especially in someChlorophyta rand, Euglenophyta, which masks the green chlorophyll,

Heterocyst:an enlarged cell in some of thefilamentous" blue- green algae, usually empty and different in shape from the vegetative cells,(Anabeana) .e, Hold-fast cell:a basal cell'of afilament or thallus, differentiated to form an an attaching organ:(Oectgonium)

H- pieces:wall of overlapping H- shaped structures. T rib onema) Intercalary:arranged in the same series, as spores' or heterocysts occur in series with the vegetative cells rather than 'beingterminal or lateraliN., (heterocyst of Anabeana) . 4 Laminate:plate-like; layered... Literal groove:a groove in encircling thecell.(CeratiumY Leucosin:a whitish food reserve characteristic of many'of,the Chrysophytam especially the Heterokontae; gives S. metallic lustre to cell'CoMents. (Difibbryor,1), Ili Lorica:a shell-like structure of varying shapes whichhouses ai4 organism, .has an opening through which organs of locomotionare.' extended. (11.nobryon)

. Lunate:crescent-shaped; as of the new moon shape.(Selenastrum) Median construction:See constricted. S. Metabolic: changing shape in tion-es in many Euglena. ' Micron:a unit of microscopical measurement:._ 1 if a millimeter by using a micrometer' in the eyepiece of themicrosco . as been calibrated with a standard stage micrometer; expressed the symbol..

I et Moniliform:arranged like a string of beads; beadlike; lemon-shaped.'( Anabeana) " Mother. the -cell which by mitosis or by internal cleavage gives rise to ogler' cells (usually spores). o MUltinucleate:with many nuclei. . Multiteriate:cells arranged in more tligr one row; a filament two or morecells in deter.(Stigeonema) 6' - motion cau sed by cilia or flagella. (Volvox) . Oblong:a curved figtxice, elongateWith the ends broadly rounded but moresharply curved. "than the lateral margins. . . .., , ,. . %., 2.46 Obovate:an ovate figure, broader at the anterior end than at the posterior..

Oogonium:a female .sex organ, usually an enlarged cell; an egg se.

Oval:an elongate, curved figure with convex margins and with ends broadly and symmetrically curved but more sharply. so than the, lateral margin. Ovoid:shaped like an egg; a carved figure broader at one end than at the other. Paramylum:a solid, starch-like storage product in the Euglenophyta.(Euglena) Parietal:along the wall; arre.neekl at the circumference; marginal as opposed to central or azial in location.' .

Pellicle:a thin membrane.( Euglena) ti Peridinin:a brown pigment characteristic of the Dinoflagellatg.(Ceratium) Perip1ast:bounding membrane of cells in Euglenoids and Chrysophytes. P,hycocyanin: 'blue pigment lound in the Cyanophyta, and in some Rhodophyta. t I Phycoerythrin:a, red pigment found in the Rhodophta, and in some Cyanophyta. Plankton:organisms0%, srffting in the ivater, or if swimming, not able to move against currents. 4 Plastid:a body or organelle of the cell,either containing pigments or in some instances colorless. Plate:sections, p6lygonal in shape, composing the cell wall of some Dinoflagellata (the thecate or armored dinoflagellates). Poster-ior:toward the rear; the end oppoSite the forward (anterior) end of a cell or of an organism.' ° . - Protoplast:the living part of the cell; the cell membrane and its contents usually enclosed by a'ce1.1 wall of dead material..1 Pseudocilia:meaning false cilia; flagella-like structures not 'used for locomotion as in Apiobystis and Tetraspora. Psgrdopa'renchymatious:a false cushion; ,a pillow like mound of cells (usually attached) which. actually is a` compact series of short, often branched filaments.(Coledchaete)

Pseudovacuole: .Meaning a fse vacuole; a pocket in the cytoplasm of many blue-green algae Which co ains gas ors mucilage; is light refractive.(Microcystis)

, 1 Pyrenoid; .a4otein,body around which starch or 'paramylum collects in a cell, usually _buried in a chloroplast but sometimes free within the cytoplasm. (Oedogonium) Pyiform:pear- shaped.(Pandorina)

t 26-,23 ,

Reniform:kidney- shaped; bean- shaped.(Dimorphocacus)

Replicate::infolded; folded back as in the cross walls of sonic specks of Spirogyra; not a plane or straight wall. Reticulate:ntt4ii, arranged fo form a network; with ormings.

Scale:siliceous or inorganic material covering the cell. (Mallomonas) -c* Semicell:a ,eell-half, as in the Plaeoderm desmids in which the cell has two parts that Pare mirror images of one another, the two parts often connected by a*. narrow isthmus.' (Stain estrum) Septum:a cross-partition, cross wall or a membrane complete or incomplete through the short diameter of, a cell, sometimes parallel with the long axis. Setae:a hair,usually arising from within a cell wall; or a hair-like extension ibrined,py tapering of a filament of cells to a fine point. Sheath:a covering, usually of mucilage,: soft or firm; the covering of a colony of cells, or an_envelopd about one or more filaments of cells. ,Siphonous:a tube; a thallus without cross partitiOns (Vaucheria) Solitary:unicellular; solitary.(Chlamydomonas)., Spine:a sharply-pointed projection from the eel], wall.(Mallomonas) Sproangium:a cell4(-sometimes an unspecialized vegetative cell) which gives rise to . spores; the case which forms aboi t the zygospores in the Zygnemalales. Star- shape d:See stellate ,Stellate:with radiating projections from a common center; star-like.(Zygnema) 'Stigma:see eyespot.

-Sutiire:,a groove between plates, as in some Dinalagellata; a cleft-like crack or line in some zygospores of the Zygnematacete:; (Ceratium) .- ' ,Thallus:a plant bodywhich' is not differentiated into root, stem and lealorgans; a ; , frodd; the algal plant. , '1 Theca; Thecate:a firm outer Agfall;'a shell, sometimes..with plgtes, as in the

. .Dinof ia gellata,(Peridinium) .'

. . iTest:a shell Or covering external to the0 yell itlf.See Lorica. J '' Transverse furrow (groove):a-groove extending ground the cell as in the Dinoflagellata. ( Ceratium) Trichrome:in blue-greent% a series of cells joined end to end.(Oadilltoria)

I

. 26.44 24 True Branch:a branch formed by means of lateral division of cells in a main filament. Includes all branched algae except those blue -green algae with false branching.

Tychoplankton:the plankton of waters near shore; organisms floating andentangled among weeds and in algal mats, not in the open water ofa lake or stream. Undulate:regularly wavy. Unicellular:Seeviolitary.. Vegetative:referring to a non-reproductive stage, activity,or.cell as opposed to activities and stages involved in reproduction, especially sexual reproduction. Xanthophyll:a yellow pigment of several kinds associated, With chlorphyll, C46H5602 Zoospore:an nimal -like spore equipped with flagella and usually .with an eye-spot.

This key was prepared by Dr. Matthew H. Hohn, Professor of Biology, Central Miohigan University, Mt. Pleasant, Michigan.

Descriptors: Plankton, Identification Keys

26-25 24 9 s PAGES 1-198"FIELD KEY TO SOME GENERA OF ALGAE" AND "CILIATED PROTOZOA" REMOVED DUE TO COPYRIGHT RESTRICTIONS.