PB-242 008 PLANKTON ANALYSIS TRAINING MANUAL ENVIRONMENTAL PROTECTION AGENCY MARCH 1975 DISTRIBUTED BY: National Technical Information Service U. S. DEPARTMENT OF COMMERCE ------- EPA-430/1-75-004 PB242008 PLANKTON ANALYSIS TRAINING MANUAL U.S. ENVIRONMENTAL PROTECTION AGENCY OFFICE OF WATER PROGRAMS ------- BIBLIOGRAPHIC DATA SHEET 1 Kcport No EPA-430/1-75-004 PB 242 008 4 1 itlr and Subtitle Plankton Analysis (141) training manual 5. Report Date March 1975 6. 7 A in hurts) t. M. Sinr.lmr, IVlnnij.il Coordinator 8. Performing Organisation Kept No. 9 !'• ifornuiif <>rg mi/moil N.imt mil Aildr U. S. Environmental Protection Agency, OWPO MPOD, National Training Center Cincinnati, OH 45268 10. Project/Task/Work Unit No 11. Contract/Grant No. 12 Sponsoring Org.ini/at ion Name and Address Same as #9 above. 13. Type of Report & Period Covered final 14. 15. Supplementary Notts 16 Abstracts A manual which covers the broad field of plankton analysis, including reference outlines on classification and identification of algae and zooplankton, limnology of plankton, techniques of collection, and laboratory methods of analysis. 17 Key Words and Document Analysis 17o. Descriptors Biomass; Ecology; Plankton; Zooplankton 7b. Identif icrs/Upen-l ndcd I crms Plankton Analysis 7c. < O^AI I I icld/droup 06 F 8 Availiibilily Sum mi nt Release to the public 19. Security Class (This Report) UNCLASSIFIED |21. No. of Pages 20. Security Class (This Page UNCLASSIFIED tev 107H I NIXWSI I) HY ANSI AND UNI SU> THIS FOKM MAY BE RFPRODUCED USCOMM-DC 6295-P74 ------- EPA-430/ 1-75-004 March 1975 PLANKTON ANALYSIS This course is offered for professional personnel in the fielda of water pollution control, hmnolo ’, and water supply Primary empiasis is given to practice in the identification and enumeration of organisms v hich may be observed in the microscopic examination of wat r Methods for Ihe chemical and instrumental evalua’ ion of plankton are compared with the reaults of microscopic examination in an extensive practical exercise Problems of significance and control are also considered ENVIRONMENTAL PROTECTION AGENCY Office of Water Program Operations TRAINING PROGRAM ------- CONTENTS Title or Description Outline Number Limnology and Ecology of Plankton 1 Biology of Zooplankton Communities 2 Optics and the Microscope 3 Structure and Function of Cells 4 Types of Algae 5 Blue-green Algae 6 Green and Other Pigmented Flagellates 7 Filamentous Green Algae 1 8 Coccoid Green Algae 1 9 Diatoms 10 Filamentous Bacteria 11 Fungi and the “Sewage Fungus” Community 12 Protozoa, Nematodes, and Botifers 13 Free-living Amoebae and Nematodes 14 Animal Plankton 15 Laboratory: Identification of Diatoms 16 Preparation of Permanent Diatom Mounts 17 Laboratory: Identification of Animal Plankton 18 Techniques of Plankton Sampling Programs 19 Preparation and Enumeration of Plankton in the Laboratory 20 Calibration and Use of Plankton Counting Equipment 21 Determination of Odors 22 Determination of Plankton Productivity 23 141. 3.75 ------- 2 Contents ‘ Iitle or Description Outline Number Laboratory Proportional Counting of Plankton 24 Laboratory Calibration of Plankton Counting Equipment 25 Laboratory. Fundamentals of Quantitative Counting 26 Algal Growth Potential Test 27 Algae and Actinomycetes in Water Supplies 28 Algae as Indicators of Pollution 29 Odor Production by Algae and Other OrganIsms 30 Plankton in Oligotrophic 31 The Effects of Pollution on Lakes 32 Application of Biological Data 33 The Problem of Synthetic Organic Wastes 34 Significance of “Limiting Factors’ to Population Variation 35 Nutrients: The Basis of Productivity 36 Algae and Cultural Eutrophication 37 Control of Plankton in Surface Waters 38 Control of Interference Organisms in Water Supplies 39 Case Preparation and Courtroom Procedure 40 Key to Selected Groups of Freshwater Animals 41 Key to Algae of Importance In Water Pollution 42 Foreword Classification-Finder (Part I and Part II) 43 ------- LIMNOLOGY AND ECOLOGY OF PLANKTON INTRODUCTION A Most Interference Organisms are Sm all. B Small Organisms generally have Short Life Histories. C Populations of Organisms with Short Life Histories may Fluctuate Rapidly in Response to Key Environ- mental Changes. D Small Organisms are Relatively at the Mercy of the Elements E The Following Discussion will naiyze the Nature of These Ele- ments with Reference to the Res- ponse of Important Organisms. PHYSICAL FACTORS OF THE ENVIRON- MENT A Light is a Fundamental Source of Energy for Life and Heat. 1 Insolation is affected by geo- graphical location and mete- orological factors. 2 Light penetration in water is affected by angle of incidence (geographical), turbidity, and colør. The proportion of light reflected depends on the angle of incidence, the temperature, color, and other qualibes of the water. In general, as the depth increases arithmetically, the light tends to decrease geo- metrically. Blues, greens, and yellows tend to penetrate most deeply while ultra violet, vio- lets, and orange-reds are most quickly absorbed. On the order of 90% of the total illumination which penetrates the surface film is absorbed in the first 10 meters of even the clearest water. ‘ Turbidity may originate within or outside of a lake. a That which comes in from outside (aflochthonous) is predominately thert solids (tripton). b That of internal origin (auto- chthonous) tends to be bio- logical in nature. B Heat and Temperature Phenomena are Important th Aquatic Ecology. The total quantity of heat avail- able to a body of water per year can be calculated and is known as the heat budget. 2 Heat is derived directly from in- solation; also by transfer from air, internal friction, and other sources. C Density Phenomena Density and viscosity affect the floatation and locomotion of microorganisms. a Pure fresh water achieveg its maximum density at 4 C and its maximum viscosity at 0 C. b The rate of change of density increases with the temperature. 2 Density stratification affects aquatic life and water uses. a In summer, a mass of warm surface water, the epilimnion , is usually present and separated from a cool deeper mass, the hypolimnion , by a relatively thin layer known as the the rmoclme . b Ice cover and annual spring and fall overturns are due to successive seasonal changes in the relative densities of the epthmmon and the hypo- RI. MIC. ecO. 4c 8.72 1—1 ------- LImn9 and I cology of Plankton limnion, profoundly influ- enced by prevailing meteoro- logical conditions. c The sudden exchange of water masses having differ- ent chemical characteris- tics may have catastrophic effects on certain biota, may cause others to bloom. d Silt laden waters may seek certain levels 1 depending on their own specific gravity in relation to existing layers already present. e Saline waters will also stratify according to the relative densities of the various layers. 3 The viscosity of water Is greater at lower temperatures. a This is important not only In situations involving the control of flowing water as in a sand filter, but also since overcoming resistance to flow generates heat, it is significant In the heating of water by internal friction from wave 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). D Shore development, depth, Inflow - outflow pattern, and topographic features affect the behavior of the water. E Water movements that may affect organ- isms include such phenomena as waves, currents, tides, seiches, floods, and others. Waves or rhythmic movement a The best known are traveling waves . These are effective only against objects near the surface. They have little effect on the movement of large masses of water. b Standing waves or seiches occur in all lakes but are seldom large enough to be observed. An “internal seich’ is an osciflation in a density mass within a lake with no surface manifestation may cause considerable water movement. 2 Langmuire spirals (or Langmu.tre circulation are a relatively mass- ive cylindrical motion Imparted to surface waters under the influence of wind. The axes of the cylinders are parallel to the direction of the wind, and their depth and velocity depend on the depth of the water, the velocity and duration of the wind, and other factors. The net result is that adjacent cylinders tend to rotate In opposite directions like meshing cog wheels. Thus the water betwedn two given spirals may be meeting and sinking, while that between spirals on either side will be meet- ing and rising. Water over the sinking areas tends to accumulate flotsam and jetsam on the surface in long conspicuous lines. Masses of microcrustacea attemping to stay near the surface may impart a reddish color to this water, and i is thus often referred to as the “red dance.” The rising water on the other hand, having recently come from some depth, may (at least in the oceans or large lakes) have a bluish appearance, and is Imown as the “bl dance.” a This phenomenon is of consider- able importance to those sampling for plankton (or even chemicals) near the surface when the wind is blowing. Grab samples from 1-2 ------- Limnolozy and Ecology of Plankton either dance might obviously differ consideral.ily. and 1.1 a plankton tow is contemplat- ed, it should be made across the wind In order that the net may pass through a succession of both dances. b Langmuire spirals are not usually established until the wind has either been blowing for an extended period, or else is blowing rather hard. Their presence can be detect- ed by the lines of foam and other floating material which coincide with the direction of the wind. 3 Currents a Currents are arhythrnic water movements which have had major study only in ocean- ography. They primarily are concerned with the trans- location of water masses. Th ’ may be generated inter- nal]y by virtue of density changes, or externally by wind or runoff. b Turbulence phenomena or eddy currents are largely re- sponsible for lateral mixing in a current. These are of far more importance in the economy of a body of water than mere laminar flow. c Tides 1 or rather tidal currents, are reversible (or oscillatory) on a relative- ly long and predictable period. They are closely allied to seiches. For all practical purposes, they are restricted to oceanic (especially coastal) waters. If there is no freshwater inflow involved, tidal currents are basically “In and out.” $1 a significant amount of freshwater is added to the system at a constant rate, the outflowing current will in general exceed the inflow by the amount of freshwater input. There are typically two tidal cycles per lunar day (approx- irnately 25 hours), but there is continuous gradation from this to only one cycle per (lunar) day in some places. Estuarine plankton populations are extremely influenced by local tidal patterns. d Flood waters range from torren- tial velocities which tear away and transport vast masses of substrate to quiet backwaters which may inundate normally dry land areas for extended periods of time, in the former case, planktonic life is flushed away completely; in the latter, a local plankton bloom may develop which may be of immediate significance, or which may serve as an inoculum for receding waters. F Surface Tension and the Surface Film 1 The surface film is the habitat of the “neuston”, a group of particular importance in water supplies. 2 Surface tension lowered by surfactants may eliminate the neuston. This can be a significant biological observation. III DISSOLVED SUBSTANCES A Carbon dioxide is released by plants and animals in respiration, but taken in by plants in photosynthesis. B Oxygen is the biological complement of carbon dioxide, and necessary for all animal life. C Nitrogen and phosphorus are fundamental nutrients for plant life. 1 Occurm great dilution, concentrated by plants. 1—3 ------- Umnology and Ecol 4 gyof Plankton z The distribution of nitrogen compounds is generally corre]at- ed with the oxygen curve, espe- cially In oceans. D Iron, manganese, sulphur, and silicon are other minerals important to aquatic life which exhibit biological stratification. E Many other minerals are present but their biological distribution in waters is less well known, fluorine, tin, and vanadium have recently been added to the “essential” list, and more may well follow. F Dissolved organic matter Is present in even the purest of lakes. V BIOLOGICAL FACTORS A Nutritional Classification of Organisms 1 Ho].ophyt ic or independent or- ganisms, like green plants, pro- duce their own basic food elements from the physical environment. 2 Holozoic or dependent organisms, like animals, ingest and digest solid food particles of organtc origin. 3 Saprophytic or carrion eating organisms, like many fungi and bacteria, digest and assimilate the dead bodies of other organ- l ms or their products. B The Prey-Predator Relationship is Simply one Organism Eating Another. C Toxic and Hormonic Relationships 1 Some organisms such as certain blue green algae and some ar- mored f]agellages produce sub- stances poisonous to others. 2 AntibIotic action in nature Is not well understood but has been shown to play a very influential role In the economy of nature. V BIOTIC COMMUNITIES (OR ECOSYSTEMS) A A biotic community will be defined here as an assemblage of organisms living in a given ecological niche (as defined below). Producer (plant-like), consumer (animal-like) and reducer (bacteria and fungi) organisms are usually Included. A source of energy (nutrient, food) must also be present. The essential concept In that each so-cafled community is a relatively independent entity. Actually this position is only tenable at any given instant, as individuals are constantly shifting from one community to another in response to stages in their life cycles, physical conditions, etc. The only one to be considered in detail here is the plankton. B Plankton are the macroscopic and microscopic animals, plants, bacteria, etc. floating free in the open water. Many clog filters, cause tastes, odors, and other troubles In water supplies. 1 Those that pass through a plankton net (No. 25 sIlk bolting cloth or equivalent) or sand filter are often known as nannoplankton (they usually greatly exceed the “net” plankton in actual quantity). 2 Those less than four microns in length are sometimes called ultraplankton . 3 There are many ways in which plankton may be classified: taxo- nomic, ecological, industrial. 4 The concentration of plankton varies markedly In space and time. a Depth, light, currents, and water quality profoundly affect plankton distribution. b The relative abundance of plankton in the various sea- sons is generally 1 spring, 2 fail, 3 summer, 4 winter 1—4 ------- L1xnnolo v and Ecology of Plnnkton 5 Marine plankton include many larger animal forms than are found in fresh waters. C The benthic community is generally considered to be the macroscopic life living In or on the bottom. D The perip yton community might be defined as the microscopic benthos, except that they are by no means confined to the bottom. Any surface, floating, or not, is usuaUy covered by film of living organisms. There is frequent exchange between the periphyton and plankton communities. E The nekton is the community of larger, free- swimming animals (fishes, shrimps, etc.), and so is dependent on the other communities for basic plant foods. VI THE EVOLUTION OF WATERS A The history of a body of water determines its present condition. Natural waters have evolved in the course of geologic time to what we Imow today. B In the course of their evolution, streams in general pass through four general stages of de relopment which may be called: birth, youth, maturity, and old age. 1 Establishment of birth. In an extant stream, this might be a ‘dry run” or headwater streambed, before it had eroded down to the level of ground water. 2 Youthful streerns; when the strear.i bed is eroded below the ground water level, spring water enters and the stream becomes permanent. 3 Mature streams; havc wIde valleys, a developed flood plain, deeper, more turbid, and usua]i r warmer water, sand, mud, silt, or clay bottom materials which shift with increase In flow. 4 In old age, streams have approa- ched base level. During flo3d stage they scour their bed and de- posit materials on the flood plain which may be very broad and fiat. During normal flow the channel is refilled and many shifting bars are developed. (Under the influence of man this pattern may be broken up. or tem- porarily interrupted. Thus as essen- tially “youthful” stream might take on some of the characteristics of a “mature” stream following soil erosion, organic enrichment, and increased surface runoff. Correction of these conditions might likewise be followed by at least a partial rever- sion to the “original” condition.) C Lakes have a developmental history which somewhat parallels that of streams. The method of formation greatly influences the character and sub- sequent history of lakes. 2 Maturing or natural eutrophication of lakes a If not already preseot, shoal areas are developed through erosion of the shore by wave action and undertow. b Currents produce bars across bays and thus cut off irregulars areas. c Silt brought in by tributary streams settles out In the quiet lake water. d Rooted aquatics grow on shoals and bars, and In doing so cut off bays and contribute to the filling of the lake. e Dissolved carbonates and other materials are precipitated In the deeper portions of the lake in part throdgh the action of plants. 1—5 ------- Umno1o y and Ecolouv of Plankton I When filling is well advanced sphagnum mats extend out- ward from the shore. These mats are followed by sedges and grasses which finally Convert the lake into a marsh. 3 Extinction of lakes. After lakes reach maturity their progress toward filling up is accelerated. They become extinct through: a The downcutting of the out- let. b Filling with detritus eroded from the shores or brought in by tributary streams. c Filling by the accumulation of the remains of vegetable materials growing in the lake itself. (Often two or three pro- cesses may act concurrently) When man hastens the above process, it is often called “cultural eutrophication.” V I PRODUCTIVITY A The biological resultant of all physical and chemical factors is the quantity of life that may actually be present. The ability to produce this “biomass” is often referred to as the “productivity” of a body of water. This is neither good nor bad per se. A water of low producti- vity is a “poor” water biologically, and also a relatively “pure” or “clean” water; hence desirable as a water supply. A productive water on the other hand may be a nuisance to man or highly desirable. Some of the factors which influence the productivity of waters are as follows: B Factors affecting stream productivity. To be productive of plankton, a stream must provide adequate nutrients, light, a suitabic temperature, and time for growth to take place. Youthful streams, especially on rock or sand substrates are low in essential nutrients. Tempera- tures in mountainous regions are usually low, and due to the steep gradient, time for growth is short. Although ample light is available, growth of true plankton is thus greatly limited. 2 As the stream flows toward a more Imatur condition nutrients tend to accumulate, and gradient diminishes and so time of flow increases, temperature tends to increase, and plankton flourish. Should a heavy load of Inert silt develop on the other hand, the turbidity would reduce the light penetration and consequently the general plankton production would diminish. 3 As the stream approaches base level (old age) and the time avail- able for plankton growth increases, the balance between turbidity, nutrient levels, and temperature and other seasonal conditions, determines the overall produc- tivity. C Factors Affecting the Productivity of Lakes The size, shape, and depth of the lake basin. Shallow water is more productive than deeper water since more light will reach the bottom to stimulate rooted plant growth. As a corolla ry, lakes with more shoreline, having more shallow water, are in general more productive. Broad shallow lakes and reservoirs have the greatest production potential (and hence should be avoided for water supplies). 2 Hard waters are generally more productive than soft waters as there are more plant nutrient minerals available. This is often 1—6 ------- FACTORS AFFECTING PRODUCTIVITY Geographic Location 2V Human Geological Latitude Influence Formation Longitude Altitude Sewage Climate A griculture Mining Primary Nutritive Materials Nature o LIUIUW ui Trans- -Light Bottom A llochthonous parency Penetration Depos2t$ Materials Ba sin I - . Trophic Nature of a ration O2 Penetra Seasonal Cycle and Stratification and Littoral Circulat. Stagnation Utilization Region Growing Season r ------- L1mnolo y and Ecology of Plankton greatly influenced by the character of the Boll and rocks in the watershed, and the quality and quantity of ground water entering the lake. In general, pH ranges of 6.8 to 8.2 appear to be most productive. 3 Turbidity reduces productivity as light penetration is reduced. 4 The presence or absence of thermal stratification with its semi-annual turnovers affect productivity by distributing nutrients throughout the water mas 9. 5 Climate, temperature, pre- valance of ice and snow, are also important. D Factors Affecting the Productivity of Reservoirs The productivity of reservoirs is governed by much the same principles as that of lakes, with the difference that the water level is much more under the control of man. Fluctuations in water level can be used to deliberately increase of decrease productivity. This can be dem- onstrated by a comparison of the TVA reservoirs which practice a summer drawdown with some of those in the west where a winter drawdown Is the rule. 2 The level at which water is re- moved from the reservoir is also important. The upper epilimnion may have a high plankton turbi- dity while lower down the plankton count may be less, but a taste and odor causer (such as Maiio- monas)may be present. There may be two thermoclines, with a mass of muddy water flowing between a clear upper epilimnion and a clear hypolimnion. Other combinations ad infinitum may occur. 3 Reservoir discharges also pro- foundly affect the DO, temperature, and turbidity In the stream below a dam. Too much fluctuation in flow may permit sections of the stream to dry periodically. yin CLASSIFICATION OF LAKES AND RESERVOIRS A The productivity of lakes and impound- ments is such a conspicuous feature that it is often used as a means of classification. 1 Ol1gotro c lakes are the geologically younger, less produc- tive lakes, which are deep, have clear water, and usually support Salmonoid fishes. 2 Mesotropic lakes are generally intermediate between oligotrophic and eutrophic lakes. They are moderately productive, yet pleasant to be around. 3 Eutrophic lakes are more mature, more turbid, and richer. They are usually shallower. They are richer In dissolved solids; N, P. and Ca are ab nidant. Plankton is abundant and there is often a rich bottom fauna. Nuisance conditions often appear. 4 Dystrophic lakes - bog lakes - low in pH, water yellow to brown, dissolved solids; N, P. and Ca scanty but humic materials abun- dant; bottom fauna and plankton poor, and fish species are limited. B Reservoirs may be classified as storage, or run of the river. 1 Storage reservoirs have a large volume in relation to their Inflow. 2 Run of the river reservoirs have a large flow through In relation to their storage value. 1—8 ------- Limnology and Ecology of Plankton C According to lot .ation, lakes and regervolrq may be classified as polar, temperate, or tropical. Differences in climatic and geographic conditions result in differences in their biology. D’ THE MANAGEMENT OR CONTROL OF ENVIRONMENTAL FACTORS A Liebig’s Law of the Minimum states that productivity is limited by the nutrient present in the least amoung at any given time relative to the assimilative capacity of the organism. B SheLford’s Law of Toleration: C The artificial introduction of nutrients (sewage pollution or fertilizer) thus tends to eliminate existing limiting minimums for some species and create intolerable maximums for other species. 1 Kn.Dwn limiting minimums may sometimes be deliberately maintained. 2 As the total available energy supply is Increased, productivity tends to increase. 3 As productivity increases, the whole character of the water may be changed from a meagerly productive clear water lake (oligotrophic) to a highly pro- ductive and usually turbid lake (eutrophic). 4 I.utrophication leads to treatment troubles. D Control of eutrophication may be accom- plished by carious means 1 Watershed management, ade- quate preparation of reservoir sites, and pollution control tend to maintain minimum limiting nu- tritional factors. 2 Shading out the energy of insola- tion by roofing or inert turbidity; Suppresses photosynthesis. 3 Introduction of substances toxic to some fundamental part of the food chain (such as copper sul- phate) tends to temporarily inhibit productivity. X SUMMARY A A body of water such as a lake rep- resents an intricately balanced system in a state of dynamic equilibrium. Modification imposed at one point in the system automatically results m compensatory adjustments at associated points. B The more thorough our knowledge of the entire system, the better we can judge where to impose control measures to achieve a desired result. REFERENCES 1 Chamberlin, Thomas C., and Salisburg, RoUin P., Geology Vol. 1, “Geolo- gical Processes and Their Results”, pp i-xix, and 1-654, Henry Holt and Company, New York 1 1904. 2 Dorsey, N. Ernest. Properties of Ordinary Water - Substance. Reinhold Publ. Corp., New York. pp. 1-673. 1940. 3 Hut cheson, George E. A Treatise on Limnology. Jonn Wiley Company. 1957. 4 Ruttner, Franz. Funlamentals of Umnzlogy. University o Toronto Press. pp. 1-242. 1953. 5 Tarzwell, Clarence M. xperimental Evidence on the Value of Trout 1937 Stream Improvement in Michigan. American Fisheries Society Trans. 66.177-187. 1936. MInIm ,m I I oII of IoI.r.tIOn Ab.,n( •. —— — D.n...Ing Abm.d.n.. ..ogo of Op%Imum M.,In om IIn,lt of of f.. br _________________ (fr.aI.,.t .buod.nc. Abjent Oerret. Iflg AbwidsnCl 1—9 ------- Limnology and Ecology of Plankton 6 Us DHEW, PHS. Algae and Metropolitan Wastes, transactions of a sefnlnar held April 27-29, 1960 at the Robert A. Taft Sanitary Engineer- ing Center, Cincinnati, Ohio. No. SEC TRW61-3. 7 Ward and Whipple. Freshwater Biology (Introduction). John Wiley Company. 1918 8 Whittaker, R. H. Communities and Ecosystems. Macmillan, New York. 162 pp. 1970. 9 Zakdln, V. I. and Gerd, Sr. Fauna and Flora of the Lakcs and Reservoirs of the USSR. Avail- able from the Office of Technical Services, U. S. Dept. Commerce, Washington. DC. This outline was prepared by H. W.Jackson, chief Biologist. National Training Center, DTTB, MDS, OWP, EPA. Cincinnati, OH 45268. 1-10 ------- BIOLOGY OF zooPLANKroN COMMUNIT S I CLASSI}’ICATION A The planktonic community is composed of organisms that are relatively independent of the bottom to complete their life history. They inhabit the open water of lakes (pelagic zone). Some species have inactive or resting stages that lie on the bottom and carry the species through periods of stress, e. g. • winter. A few burrow in the mud and enter the pelagic zone at night. but most live in the open water all the time that the species is present in an active form. 13 Compared to the bottom fauna and flora, the plankton consists of relatively few kinds of organisms that are consistently and abundantly present. Two major cat- egories arc often culled phytoplankton (plants) and zooplankton (animals), but this is based on an outmoded classification of living things. The modern tendency is to identify groupings according to their function In the ecosystem: Primary pro- ducers (photosynthetic organisms), consumers (zooplankton). and decomposers (hetero- trophic bacteria and fungi). C The primary difference then is nutritional, phytoplankton use inorganic nutrient elements and solar radiation. Zooplankton feed on particles, much of which can be phytoplankton cells, but can be bacteria or particles of dead organisms (detritus) originating in the plankton, the shore region, or the land surrounding the lake. 0 The swimming powers of planktonlc organisms is 80 LimIted that their hori- zontal distribution is determined mostly by movements of water. Some of the animals are able to swim fast enough that they can migrate vertically tens of meters each day, but they are capable of little horizontal navigation. At most, some species of crustaceans show a general avoidance of the shore areas during caln weather when the water is moving more slowly than the animals can swim, By definition, animals that are able to control their hori7ontal location are nekton, not plankton. E In this presentation,a.minimum of clas- sification and taxonomy is used, but it should be realized that each group is typified by adaptations of structure on physiology that are related to the plank- tonic mode of existence. These adapta- tions are reflected in the classification. II FRESHWATER ZOOPLANKTON A The freshwater zooplankton is dominated by representatives of three groups of animals, two of them crustaceans’ Copepoda, Cladocera, Rotifera. All have feeding mechanisms that permit a high degree of selectivity of food, and two can produce resting eggs that can withstand severe environmental conditions. In general the food of usual zooplankton pop- ulations ranges from bacteria and small algae to small animals. B The Copepoda reproduce by a normal biparental process, and the females lay fertilized eggs in groups which are carried around in sacs until they hatch. The immature animals go through an elaborate development with many stages. The later stages have mouthparts that permit them to collect particles. In many cases, these are in the form of combs which remove small particles by a sort of filtration process. In others, they are modified to form grasping organs by which small animals or large algae are captured individually. C The Cladocera (represented by Daphnia ) reproduce much of the time by partheno- genesis. so that only females are present. Eggs are held by the mother in a brood chamber until the young are developed far enough to fend for themselves. The newborn animals look like miniature adults, and do not go through an elaborate series of developmental stages in the water as do the copepods. Daphnia has comb-formed filtering structures on some of its legs that act as filters. HI. AQ. 29.5.71 2-1 ------- Biology of Zooplankton Communities 1) Under some environmental conditions the development of eggs is affected and males are produced. l ertilized eggs are produced that can resist freezing and drying, and these carry the population through unsatisfactory conditions. E The Rotifera are small animals with a ciliated area on the head which creates currents used both for locomotion and for bringing food particles to the mouth. They too reproduce by parthenogenesis during much of the year. but production of males results in fertilized, resistant resting eggs. Most rotifers lay eggs one at a time and carry them until they hatch. III ZOOPLAN1cTON POPULATION DYNAMICS A In general, zooplankton populations are at a minimum Lfl the cold seasons, although some species flourish in cold water. Species with similar food requirements seem to reproduce at different times of the year or are segregated in different layers of lakes. B There is no single, simple measurement of activity for the zooplankton as a whole that can be used as an index of production as can the uptake of radioactive carbon for the phytoplankton. However, it is possible to find the rate of reproduction of the species that carry their eggs. The basis of the method is that the number of eggs in a sample taken at a given time represents the number of animals that will be added to the population during an Interval that is equal to the length of time it takes the eggs to develop. Thus the potential growth rate of the populations can be determined. The actual growth rate, determined by successive samplings and counting, is less than the potential, and the difference is a measure of the death rate. C Such measurements of birth and death rates permits a more penetrating analysis to be made of the causes of population change than tf data were available for population sue alone. I) Following is an indication of the major environmental factors in the control of I ooplankton. I Temperature has an obvious effect Ln its general control of rates. In addition, the production and hatching of resting eggs may be affected. 2 Inorganic materials Freshwater lakes vary in the content of dissolved solids according to the geological situation. The totaL salinity and proportion of different dissolved materials in water can affect the pop- ulation. Some species are limited to soft water, others to saline waters, as the brine shrimp. The maximum pop- ulation size developed may be related to salinity, but this is probably an indirect effect working through the abundance of nutrients and production of food. 3 Food supply Very strong correlations have been found between reproduction and food supply as measured by abundance of phytoplankton. The rate of food supply can affect almost all aspects of pop- ulation biology including rate of mdi- vidual growth, time of maturity, rate of reproduction and length of life. 4 Apparently in freshwater, dissolved organic materials are of little nutri- tional significance, although some species can be kept if the concentration of dissolved material is high enough. Some species require definite vitamins in the food. 5 Effect of predation on populations The kind, quantity and relative pro- portions of species strongly affected l y grazing by vertebrate and inverte- brate predators. The death rate of Daphnia is correlated with the abun- dance of a predator. Planktivorous fish (alewives) selectively feed on larger species, so a lake with alewives is dominated by the smaller species of crustaceans and large ones are scarce or absent. 6 Other aspects of zooplankton Many species migrate vertically con- siderable distances each day. Typically. migrating species spend the daylight hours deep in the lake and rise toward the surface in late afternoon and early evening. Some species go through a seasonal change of form (cyclomorphosis) which is not fully understood. It may have an effect in reducing predation. 2 -.2 ------- Biology of Zooplarikton Communities REFERENCES 1 Baker, A. 1. An Inexpensive Micro- sampler. Limnol. and Oceanogr. 15(5): 158—160. 1970. 2 Brooks. .J. L. and Dodson, S. I. Predation. Body. Size, and Corn- posItion of Plankton. Science 150: 28-35. 1965. 3 Dodson, Stanley I. Complementary Feeding Niches Sustained by Size- Selective Predation, Lirrinology and Oceanography 15(1): 131-137. 4 Hutchlnson, G. E. 1967. A Treatise on lininology. Vol. II. Introduction to Lake Biology arid the Llmnoplanktou. xl+ 1115. JohnWtley& Sons, Inc., New York. 5 Jossl, Jack W. Annotated Bibliography of Zooplankton Sampling Devices. USFWS. Spec. Sd.: Rep. -Fisheries. 609. 90 pp. 1970. 7 Lund, J. W. G. 1965. The Ecology of the Freshwater Plankton. Biological Reviews, 40:231-293. 8 UNESCO. Zoolplankton Sampling. UNESCO Monogr. Oceanogr. Methodol. 2. 174 pp. 1968. (UNESCO. Place de Fortenoy, 75, Paris 7e France). 6 LIkens, Gene E. and Gilbert, John J. Notes on Quantitative Sampling of Natural Populations of P lanktonic Rotifers. Limnol. arid Oceanogr. 15(5): 816—820. ThiS outline was prepared Dy W. ‘:. c1mondson, Professor of Zoology, University of Washington, Seattle, Waehington. 2—3 ------- Biology of Zooplankton (omm initics VIG(JltF. I SEASflNA CIIANGESOI ’ ZOOPIJANKTON IN LAKE ERKEN, SWEDEN 1957 Each panifl shows the abundance of a species of animal. Each mark en die vertical axis represents 10 tndividuala/liter. r4auwerck, A. 1963. Die Beziehungen zwischen Zooplankton end Phytoplankton im See Erken. Symbolae Botanicae Upsaliensis, 17:1—163. . 1 P K A K J J A S 0 N U 2-4 ------- FIGURE 2 REPRODUCTIVE RATE OF ZOOPLAN1------- Biology of Zooplankton Communities PROTOZOA Difflu gia Amoebae CUiates Epistylis Codonella Stentor 2—6 ------- 1U U Y 01 L.OOPJ.aniaofl ‘.ommunitie i Cladocera ROTIFERA ARTHROPODA Crust cea Nauplius ]arva c( copepod I neecta - Cheoborus y chaeta Polygarthra Brachionu.e Copepoda 2—7 ------- [ 3io1o r of Zooplankton Communities PLANKTON IC BIVALVE LARVAE Ii spined (fin attached) staple (gill attached) Glochidia (Unionidae) Fish Parasites (1-3) Veliger Larvae (Corbiculidae) Free Living Planktonic (4-5) Pediveliger atlachcs byssus lines) 38O j 3771L 2 3 veliger 5 pediveliger 2—B ------- OPTICS AND THE MICROSCOPE I OPTICS An understanding of elementary optics is essential to the proper use of the microscope. The microacopist will find that unusual pro- bleme in Illumination and photomicrography can be handled much more effectively once the underlying ideas In physical optics are understood. A Reflection A good place to begin is with reflection at a surface or interface. Specular (or regular) reflection results when a beam of light leaves a surface at the same angle at which it reached it. This type of reflection occurs with hi ily polished smooth surfaces. It is stated more pre- cisely as Sriell’s Law, i.e., the angle of incidence, 1. is equal i IFie angle of reflection, r (Figure 1). Diffuse (or scattered) reflection results when a beam of light strikes a rough or Irregular sur- face and different portions of the incident light are reflected from the surface at different angles. The light reflected from a piece of white paper or a ground glass is an example of diffuse reflection. Figure 1 SPECULAR REFLECTION - SMELL’S LAW BI. MIC. 18.6.68 Strictly speaking, of course, all reflected light, even diffuse, obeys Snell’s Law. Diffuse reflected light is-made up of many specularly reflected rays, each from a a tiny element of surface, and appears diffuse when the reflecting elements are very numerous and very small. The terms diffuse and specular , referring to reflection, describe n ot so much a difference in the nature of the reflection but rather a differ- ence In the type of surface. A polished sur- face gives specular reflection, a rough surface gives diffuse reflection. It Is also important to note and remember that specularly reflected light tends to be strongly polarized In the plane of the reflect- ing surface. This is due to the fact that those ra whose vibration directions lie closest to the plane of the reflection surface are most strongly reflected. This effect is strongest when the angle of incidence is such that the tangent of the angle is equal to the refractive index of the reflecting sur- face. This particular angle of Incidence is called the Brewster angle . B Image Formation on Reflection Considering reflection by mirrors, we find (FIgure 2) that a plane mirror forms a virtual image behind the mirror, reversed right to left but of the same size as the object. The word virtual means that the image appears to be In a given plane but that a ground glass screen or a photographic film placed in that plane would show no image. The converse of a virtual image is a real image. Spherical mirrors are either convex or con- cave with the surface of the mirror repre- senting a portion of the surface of a sphere. The center of curvature is the center of the sphere, part of whose surface forms the mirror. The focus lies halfway between the center of curvature and the mirror surface. 3—1 ------- Ontics and the Microacooe Q Ic1 Figure 2 IMAGE FORMATION BY PLANE MIRROR Construction of an Image by a concave mirror follows from the two premises given below (Figure 3) IMAGE FORMATION BY CONCAVE MIRROR I A ray of light parallel to the axis of the mirror must pass through the focus after reflection. 2 A ray of light which passes through the center of curvature m t return along the same path. A coronary of the first premise Is; 1 A ray of light which passes through the focus is reflected paraUel to the axis of the mirror. The image from an object can be located using tJ familiar lens formula 1 1 — + — p q f where p distance from the object to the mirror q distance from the image to the mirror f focal length C Spherical Aberration No spherical surface can be perfect in its image-forming ability. The moat serious of the imperfections, spherical aberrations occurs In spherical mirrors of large aperture (Figure 4). The rays of light making up an image point from the outer zone of a spherical mirror do not pass through the same point as the more central rays. This type of aberration is reduced by blocking tl outer zone rays from the Image area or by using aspheric surfaces. Figure 4 Sk’HERICAL ABERRATION BY SPHERICAL MIRROR Figure 3 3-2 ------- Opti s and the Microscone D Refra tion of light Turning now to li ’i . . , ‘ i .itls ’ I thait iiiiu’roi we find that thc nio ’ t i iiipoi Lint Ii.i ii li i — istic is refi a turn. R’fi .ii I ion i ’. ’Ii ’ i Lu the change ol cIire tioji .iiidlui v.’k.i ity iii light as it pasece Ii urn iiiii’ iii ‘diii iii Lu another. The ratio of tlit’ v&’lot lt HI . 1 1 1 (or more corre tl in .i v.i tin nil to Lii . velocity in the In ed in in i .tl led Lii. refractive ind ’ . Sons’ t)pI . .l v.ilu, ’ ol refractive inde Inca ‘,ui’t’d with .floiio— chrornatii light (sodiu i ii I) lint’) ,i r.’ I ii,tt’d in Table I. Refrac tion c’auei’s un oh n’s t mum r ed in a n ccliim flu of lnghc’r refra tive indi’x than air to appear loser to the surface than it actually Is (l ’igure 5). This eth’. t may Actual depth Figure 5 REFRACTION OF LIGHT AT INTERFACE be used to determine the refractive index of a liquid with the microscope. A flat vial with a scratch on the bottom (inside) 18 placed on the stage of the microscope. The microscope is focused on the scratch and the fine adjustment micrometer reading is noted. A small amount of the unknown liquid is added, the scratch is again brought into In. u’ and the ne micrometer reading i ’s talut ii. l”inally , thi mu rosr.ope is re- fui usi’d until th . s .urfai e of the liquid appears i ii ‘..h.u r p fo. us, The micrometer reading is t.ilo’n agdin and, with this information, the rifra tivi index m y be calculated from the ‘ iniplifi .d t’qua Lion actual depth ii has tive m.ndx apparent depth I Rl”l”ltA(’T1VI ’ INDICES OF COMMON MATF ’RIAI.S MI”ASURFD WITH SODIUM LIGHT Vzn uum 1.0000000 Crown glass 1.48 to 1. 61 Air 1.0002918 Rock salt 1.5443 CO 2 1.0004498 Diamond 2.417 Watir’ I. 3.340 Lead sulfide 3.912 When the situation is reversed, and a ray of light from a medium of high refractive index passes through the interface of a medium of lower index, the ray is refracted until a critical angle is reached beyond which all of the light is reflected from the interface (Figure 6). This critical angle, C, has the following relationship to the refractive i dices of the two media sin c .! 2 where ni W”ien the second medium is air, the formula be Omes REFLECTION AT CRITICAL ANGLE sin C nI // Figure 6 Air MQdium Image Object 3—3 ------- Optics and the Microscope E Di8pcrsion Dispersion is another important property of transparent materials. This is the variation of refractive index with color (or wavelength) of light. When white light passes through a glass prism, the light rays are retracted by different amounts and separated into the colors of the spectrum. This spreading of light into its component colors is due to di8pcrs ion 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 wavelengths. Because of dispersion, determination of the refractive index of a substance re- quires designation of the particular wave- 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 substances. Table 2. DISZ’ERSION OF REFRACTIVE INDICES OF SEVERAL COMMON MATERIALS Refractive index D line C line (yellow) (red) 5893A 6563k F line blue 4861A Carbon disulfide 1.6523 1,6276 1.6182 Crownglass 1.5240 1.5172 1.5145 Flint glass 1.6391 1. 6270 1. 6221 Water 1.3372 1.3330 1.3312 The dispersion of a material can be defined quantitatively as n (yellow) - dispersion n (blue) - n (red) - n (593m i) - - n (486m i) - n(656m 1 ) where n is the refractive index of the materiai at the particular wavelength noted In the parentheses. There are two classes of lenses, con- verging and diverging, called also convex and concave , respectively. The focal point of a converging lens is defined as the point at which a bundle of light rays parallel to the axis of the lens appears to onverge after passing through the lens. The fo al length of the lens is the distance from the lens to the focal point (Figure 7). Figure 7 CONVERGENCE OF LIGHT AT FOCAL POINT G Image Formation by Refraction Image formation by lenses (Figure 8) follows rules analogous to those already given above for mirrors: I Light traveling parallel to the axis of the itlns will be refracted so as to pass through the focus of the lens. 2 Light traveling through the geometrical center of the lens will be unrefracted. The position of the image can be determined by remembering that a light ray passing through the focus. F, will be parallel to the axis of the lens on the opposite siae of the lens and that a ray passing through the geometrical center of the lens will be unrefracted, F Lenses 3—4 ------- Optics and the Microscope l .’iguri’ 8 IMAGE FORMATION HY A CONVEX LENS The magnification. M, of an image of an object produced by a leno is given by the relationship M image size image distance q object size object distance p where q distance from image to lens and p distance from Dbject to lens. H Aberrations of Lenses Lenses have aberrations of several types which, unless corrected, cause loss of detail in the image. Spherical aberration appears in lenses with spherical surfaces. Reduction of spherical aberration can be accomplished by diaphragmlng the outer zones of the lens or by designing special asphern al surfa es in the lens system. Chromatic dberratlOn is a phenomenon caused by the variation of refractive index with wavelength (dispersion). Thus a lens receiving white light from an obje4 t will form a violet Image closer to the lens and a red one farther away. Achromatic lenses are employed to minimize this effect. The lenses are combinations of two or more lens elements made up of materials hay uig different dispersive powers The use of monochromatic light is another obvious way of eliminating chromati aberration. Astigmatism Is a third aberration of spherical lens systems It occurs when obje t point ’ are not locatcd on the optical ixJs of the lens and results in the formation of an indistuu t image. The simplest remedy for astigmatism is to place the objet t lose to the axis of the lens system. Interferern e Phenomena Interferen e and diffraction arc two phe- nomena whit h are due to the wave haracter- istit s of light, The superposition of two light rays arriving simultaneously at a given point will give rise to interference effects, whereby the intensity at that point will vary from dark to bright depending on the phase diffcren es between the two light rzi The first requirement for interference is that the light must come from a single source. The light may be split into any number of paths but must originate from the same point (or coherent source). Two light waves from a coherent source arriv- ing at a point in phase agreement will reinforce each other (Figure 9a). Two light waves from a coherent source arriv- ing at a point in opposite phase will cancel each other (Figure 9b). U Figure 9a. Two light rays, 1 arxl 2, of the same frequency but dif- ferent amplitudes, are in phase in the upper diagram. In the lower diagram, rays 1 and 2 interfere constructively to give a single wave of the same fre- quency and with an amplitude equal to the summation of the two former waves. r i p q 3—5 ------- Qp_tiçs and the Microscope ------- Optics and the Microscone Each dark band represents an equivalent air thickness of an odd number of half wavelengths. Conversely, each bright band is the result of an even number of half wavelengths. With interference illumination, the effect of a transparent object of different re- fractive index than the medium in the microscope field is I a change of light intensity of the object if the bat kground is uniformly illumi- nated (parallel cover slip), or 2 a shift of the interference bands within the object if the background consists of bands (tilted cover slip) The relationship of refractive indices of the surrounding medium and the object Is as follows ns= n ( 1 + 360t where n 8 refractive index of the specimen nm refractive index of the surrounding medium 0 phase shift of the two beams, degrees K wavelength of the light thickness of the specimen. J Diffraction In geometrical optics, it is assumed that light travels in straight lines. This is not always true We note that a beam passing through a slit toward a screen creates a bright band wider than the slit with alter- nate bright and dark bands appearing on either side of the central bright band, decreasing in intensity as a function of the distarxe from the center. Diffraction describes this phenomenon and, as one of its practical consequences, limits the lens in its ability to reproduce an image. For example, the image of a pm point of light produced by a lens is not a pm point but is revealed to be a somewhat larger patch of light surrounded by dark and bright rings. The diameter, d, of this diffraction disc (to the first dark ring) is given as - 2.44fx d D where f is the focal length of the lens, K the wavelength, and D the diameter of the lens. It is seen that in order to maintain a small diffraction disc at a given wave- length, the diameter of the lens should be as large as possible with respect to the focal length. It should be noted, also, that a shorter wavelength produces a smaller disc. If two pm points of light are to be distin- guished in an image, their diffraction discs must not overlap more than one half their diameters. The ability to distinguish such image points is called resolving power and is expressed as one half of the preceding expression resolving power 1. f K II THE COMPOUND MICROSCOPE The compound microscope is an extension in principle of the simple magnifying glass. hence it is essential to understand fully the properties of this simple lens system. A Image Formation by the Simple Magnifier The apparent size of an object is determined by the angle that is formed at the eye by the extreme rays of the object. By bringing the object closer to the eye, that angle (called the visual angle ) is increased. This also increases the apparent size. However a limit of accommodation of the eye is reached, at which distance the eye can no longer focus. This limiting distance is about 10 inches or 25 centimeters. It is at this distance that the magnification of an object observed by the unaided eye is said to be unity. The eye can, of course, be focused at shorter distances but not usually in a relaxed condition. A positive, or converging, lens can be used to permit placing an object closer than 10 inches to the eye (Figure 12). By this means the visual angle of the object is increased (as is its apparent size) while the image of 3—7 ------- Optics and the Microscope Figure 12 VIRTUAL IMAGE FORMATION BY CONVEX LENS the object appears to be 10 inches from the eye, where It 18 be8t accommodated. B Magnification by a Single Lens System The magnification, M, of a simple magni- tying glass is given by M + 1 where f focal length of the lens in centimeters. Theoretically the magnification can be increased with shorter focal length lenses. However such lenses require placing the eye very close to the lens surface and have much image distorUon and other optical aberrations. The practical limit for a simole magnifying glass is about 20X. In order to go to magnifications higher than 20X, the compound microscope is required. Two lens systems are used to form an enlarged image of an object (Figure 13). This is accomplished in two steps, the first by a lens called the obiective and the second by a lens known as the eyepiece (or ocular) . C The Objective The objective is the lens (or lens system) closest to the object. Its function is to reproduce an enlarged image of the object in the body tube of the microscope. Objectives are available in various focal VirtuS ituagi Figure 13 IMAGE FORMATION IN COMPOUND MICROSCOPE lengths to give different magnifications (Table 3). The magnification is calculated from the focal length by dividing the latter into the tube length, usually 160 mm. The numerical aperture (N. A.) is a measure of the ability of an objective to resolve detail. This is more fully discussed in the next section. The working distance is in the free space between the objective and the cover slip and varies slightly for objectives of the same focal length depending upon the degree of correction and the manufacturer. There are three basic classifications of objectives achromats, fluorites and apochromats. listed in the order of their complexity. The achromats are good for routine work while the fluorites and apo- chromats offer additional optical corrections to compensate for spherical, chromatic and other aberrations. Eye Ey 1p4t 1 Image Obh,c tiuS 3—8 ------- Op tics and the Mi ’ roscopi Table 3. NOMINAL CHARACTFRISTICS 01” USUAl MJCROSCOi ’E OBJECTIVES Nominal focal length mm 56 32 16 8 4 4 18 2.5X 0.08 40 SO 5 0.10 25 16 5 10 0.25 20 0.50 1.3 43 0.66 45 0.85 90 1.30 80X 30X .3.9 90X 250X Another system of objectives employs reflecting surfaces in the shape of concave and convex mirrors. Reflection optics, because they have no refractmg elements. do not suffer from chromatic aberrations as ordinary refraction objectives do. Based entirely on reflection, reflecting objectives are extremely useful in the inlrared and ultraviolet regions of the spectrum. They also have a much longer working distance than the retracting objectives. The body tube of the microscope supports the objective at the bottom (over the object) and the eyepiece at the top. The tube length is maintained at 160 mm except for Leita instruments, which have a 170-mm tube length. The objective support may be of two kinds, an objective clutch changer or a rotating nosepiece I The objective clutch changer (“quick- change” holder) permits the mbunting of only one objective at a time on the microscope. It has a centering arrange- ment. so that each objective need be centered only once with rcspcct to the stage rotation The changing of objec- lives with this system is somewhat awkward compared with the rotating nog up ieee 2 The revolving nosepice allows mounting three or four objectives on the microscope at one time (there are some nosepieces that accept five and even six objectives). In this system, the objectives are usually noncenterable and the stage is centerable. Several manufacturers pro- vide centerable objective mounts so that each objective on the noseplece need be centered only once to the fixed rotating stage. The insides of objectives are better protected from dust by the rotating nosepiece. This, as well as the incon- venience of the so-called “quick-change” objective holder, makes it worthwhile to have one’s microscope fitted with rotating nosepiece. D The Ocular The eyepiece, or ocular, is necessary in the second step of the magnification process. The eyepiece functions as a simple magni- fier viewing the image formed by the objective. There are three classes of eyepieces in common use huyghenian. compensating and flat-field. The huyghenian (or huyghens) eyepiece is designed to be used with achromats while the compensating type is used with fluorite and apochromatic objectives. Flat-field eyepieces, as the name implies, are employed in photo. micrography or projection and can be used with most objectives. It is best to follow the recommendations of the manufacturer as to the proper combination of objective and eyepiece. Nominal magnif. Working Depth Diam. of Resolving Maximum Eyepiece N. A distanec focus field power, white useful for useful max mm _ mm. light, magnif. magnif. 8. 5 4. 4 7 8 2 1.4 25X 2 1 0.7 500X 25X 0. 7 1 0. 5 0. 4 660X 15X 0. 5 1 0. 4 0. 35 850X 20X 0.2 0.4 0.2 0.21 1250X l2X 3—9 ------- Optics and the Microscope The usual magnifications available in oculara run from about 6X up to 25 or 30X. The 6X is generally too low to be of any real value while the 25 and 30X oculars have slightly poorer imagery than medium powers and have a very low eyepoint. The most useful eyepieces lie in the 10 to 20X magnification range. E Magnification of the Microscope The total magnification of the objective- eyepiece combination is simply the product of the two individual magnifications. A convenient working rule to assist in the proper choice of eyepieces states that the maximum useful magnification (MUM) for Uae miLroscopt? in 1 • 000 times the numeri- cal aperture (N.A.) of the objective. The MUM is related to resolving power in that magnification In excess of MUM gives little or no additional resolving power and results in what is termed empty magnification . Table 4 shows the results of such combinations and a comparison with the 1000X N.A. rale. The under- lined figure shows the magnification near- est to the MUM and the eyepiece required with each objective to achieve the MUM. From this table it is apparent that only higher power eyepieces can give full use of the resolving power of the objectives. It is obvious that a lOX, or even a 15X, eyepiece gives insufficient magnification for the eye to see detail actually resolved by the objective. F Focusing the Microscope The coarse adjustment is used to roughly position the body tube (in some newer microscopes, the stage) to bring the image into focus. The fine adjustment is used after the coarse adjustment to bring the image into perfect focus and to maintain the focus as the slide is moved across the stage. Most microscope objectives are parfocal so that once they are focused any other objective can be swung into position without the necessity of refocusing except with the fine adjustment. The student of the microscope 8hould first learn to focus In the following fashion, to prevent damage to a specimen or objective: 1 Raise the body tube and place the speci men on the stage. 2 Never focus the body tube down (or the s.tage up) while observing the field through the eyepiece. 3 Lower the body tube (or raise the stage) with the coarse adjustment while care- fully observing the space between the Table 4. MICROSCOPE MAGNIFICATI ON CA LCU LATED FOR VARIOUS OBJECTIVE - EYEPIECE COMBINATIONS Objective Focal Magni- length fication Eyepiece MUMa (1000 NA) 5X lox l5X 20X 25X 56mm 3X l5X 30X 45X 60X 75X 80X 32 5 25X 50X 75X 100X l25X bOX 16 10 50X 100X 150X 200X 250X 8 20 100X 200X 300X 400X 500X 4 40 200X 400X 600X 800X 1000X 660X 1.8 90 450X 900X 1350X 1800X 2250X 1250X aMUM maximum useful magnification 3—10 ------- Optics and the Microscope objec’tivc’ and slid, and permitting the two to come close together without touching. 4 Looking through the microscope and turning the fanc adjustment in such a way as to move the objective away from the specimen, bring the image into sharp focus. The fine adjustment is usually calibrated in one- or two-mit ron steps to indicate the vertical movement of the body tube. This feature is useful in making depth measurements but should not be relied upon for accuracy G The Substage Condenser The substage holds the condenser and polarizer. It (an usually be focused in a vertical direction so that the condenser can be brought into the correct position with respect to the specimen for proper illumination. In some models, the conden- ser is centerable so that it may be set exactly in the axis of rotation of the stage, otherwise it will have been precentered at the factory and should be permanent. H The Microscope Stage The stage of the microscope supports the specimen between the condenser and objective, and may offer a mechanical stage as an attachment to provide a means of moving the slide methodically during obser- vation. The polai izing microscope is fittcd with a circular rotating stage to which a mechanical stage may be added. The rotating stage, which is used for object oi ientation to observe optical effects, will have centering screws if the objectives are not centerable, or vice versa It Is tin- desirable to have both objectives and stage centerable as this does not provide a fixed reference axis. The Polarizing Elements A polarizer is fitted to the condenser of all polarizing microscopes. In routine instru- ments, the polarizer is fixed with its vibration direction oriented north-south (east-west for most European Instruments) while in research microscopes, the polarizer can be rotated. Modern instru- ments have polarizing filters (such as Polaroid) replacing the older calcite prisms. Polarizing filters are preferred beause they I are low-cost, 2 require no maintenance, .1 permit use of the full condenser aperture An analyzer, of the same construction as the polarizer, is fitted in the body tube of the microscope on a slider so that it may be easily removed from the optical path. It is oriented with its plane of vibration perpendicular to the corresponding direction of the polarizer. J The Bertrand Lene The Bertrand lens is usually found only on the polarizing microscope although some manufacturers are beginning to include it on phase microscopes. It is located In the body tube above the analyzer on a slider (or pivot) to permit quick removal from the optical path. The Bertrand lens is used to observe the back focal plane of the objective. It i convenient for checking quickly the type and quality of illumination, for observing Interference figures of crystals, for adjust- ing the phase annull In phase microscopy and for adjusting the annular and central stops in dispersion staining. K The Compensator Slot The compensator slot receives cornpensators (quarter-wave, first-order red and quartz- wedge) for observation of the optical prop- erties of crystalline materials. It is usually placed at the lower end of the body tube just above the objective mount, and is oriented 450 from the vibration directions of the polarizer and analyzer. L The Stereoscopic Microscope The stereoscopic microscope, also called the binocular, wide-field, dissecting or 3—11 ------- Optics ana the Microscope Greenough binocular rnkroscope , is in reality a combination of two separate compound microscopes. The two micro- scopes, usually mounted in one body, have their optical axes inclined from the vertical by about 70 and from each other by twice this angle. When an obje t is placed on the stage of a stereos opac microsope, the optical systems view it from slightly different angles, presenting a stereoscopic pair of images to the eyes, which fuse the two Into a single three-dimensional image. The objectives are supplied in pairs, either as separate units to he mounted on the microscope or, as in the new instruments, built into a rotating drum. Bausch and Lomb was the first manufacturer to have a zoom lens system which gives a continuous change in magnification over the lull range. Objectives for the stereomicroscope run from about 0. 4X to 12X, well below the magnification range of objectives available for single - objective microscopes. The eyepieces supplied with stereoscopic microscopes run from 10 to 25X and have wider fields than their counterparts in the single-objective microscopes. Because of mechanical limitations, the stereomicroscope is limited to about 200X magnification and usually does not permit more than about 120X. It is most useful at relatively low powers in observing shape and surface texture, relegating the study of greater detnU to the monocular microscope. The stereomicroscope is also helpful in manipulating small samples, separating ingredients of mixtures, pre- paring specimens for detailed study at higher magnifications a rxl performing various mechanical operations under micro- scopical observation, e. g. mic rornanipulation. III ILLUMINATION AND RESOLVING POWER Good resolving power and optimum specimen contrast are prerequisites for good microscopy. Assuming the availability of suitable optics (ocular, objectives and substage condenser) it is still of paramount importance to use proper Illumination. The requirement for a good illumination system for the microscope is to have uniform Intensity of illumination over the entire field of view with independent control of intensity and of the angular aperture of the illuminating cone. A Basic Types of Illumination There are three types of illumination (Table 5) used generally I Critical. This is used when high levels of illumination intensity are necessary for oil immersion, darkfjeld, fluores- cence, low birefrmgence or photo- micrographic studies. Since the lamp filament is imaged in the plane of the spec imen, a ribbon filament or arc lamp is required. The lamp must be focusable and have an iris diaphragm, the position of the filament must also be adjustable in all directions. 2 K hler. Also useful for intense illumi- nation, K hler illumination may be obtained with any lamp not fitted with a ground glass. The illuminator must, however, be focusable, it must have an adjustable field diaphragm (iris) and the lamp filament position must be adjust- able in all directions. 3 “Poor man’s”. So-called because a low- priced illuminator may be used, this method gives illumination of high quality although of lower intensity because of the presence of a ground glass In the system. No adjustments are necessary on the illuminator or lamp filament although an adjustable diaphragm on the illuminator is helpful. All three types of illumination require that the microscope substage condenser focus the image of the illuminator aperture in the plane of the specimen. In each case, then, the lamp iris acts as a field diaphragm and should be closed to just illuminate the field of view. The differences in these three types of illumination lie in the adjustment of the lamp condensing lens. With poor man’s illumination there is no lamp conden- ser, hence no adjustment. The lamp should be placed close to the microscope so that 3—12 ------- Optics and the Micros Table 5 COMPARISON OF CRITICAL, KOHLER AND POOR MAN’S ILLUMINATION Critical Kohier Poor mants Lamp fijament ribbon filament any type any type Lamp condensing lens required required none Lamp iris required required useful Ground glass at lamp none none present Image of light source in object plane at substage iris none Image of field iris near object plane in object plane near object plane Image of substage iris back focal plane of objective back focal plane of objective back focal plane of objective the entire field of view is always illuminated. If the surface structure of the ground glass becomes apparent in the field of view the substage condenser is slightly defocused. Critical Illumination With critical iflumuiation the lamp conden- ser is focused to give parallel rays, focus- ing the lamp filament on a far wall is sufficient. Aimed, then, at the substage mirror, the substage condenser will focus the lamp filament in the object plane. The substage condenser iris will now be found imaged in the back focal plane of the ob- jective, it serves as a control over con- vergence of the illumination. Although the substage iris also affects the light intensity over the field of view it should most decid- edly not be used for this purpose The intensity of illumination may be varied by the use of neutral density filters and, unless color photomicrography is anticipated, by the use of variable voltage on the lamp filament. Ko’ilcr illumination (Figure 14) differs fiom ritical illumination in the use of the lamp ondenqer. With critical illumination the lamp condenser focuse the lamp filament at infinity, with Kohier illumination the lamp filament is focused in the plane of the substage condenser iris (also coincident with the anterior focal plane of the substage condenser). The functions of the lamp conderts er iris and the substage condenser iris in controlling, respectively, the area of the illuminated field of view and the angular aperture of the illuminating cone are precisely alike for all three types of illumination. Critical illumination is seldom used because it requires a special lamp filament and be- cause, when u8ed, it hows no advantage over well-adjusted Kohler illumination. Kohier Illumination To ar ange the microscope and illuminator for Kohler illumination it is well to proceed through the following steps a Remove the diffusers and filters from the lamp b Turn the lamp on and aim at a con- venient wall or vertical screen about 19 inches away. Open the lamp diaphragm. c By moving the lamp condenser, focus a sharp image of the filament. It ahould be of such a size as to fill, not necessarily evenly, the microscope 3—13 ------- OptIc. and the M crosLope Critical KObler Eye E yepoint Ocular mans 0 Focal plane ‘ Focal plane Objective 0 Preparation 0 Substage condenser © Lump condenser Light source / 3—14 ------- - — - Opti and the’ Microscope 8Ul tJ g i 011 (1 ’ iist’I Opeiiiiig. I i it dot’ . i t , 1110¼’ i tile i.i nip W I \ Ii ‘iii tht v.’ i i i It) eiila I I hit I iI.i l iii ill I 1 1 1. 1 141, rt’fui ii d l’ui n the lamp .iiicl . 1 liii it IL Lii , liii it)— St ‘ i ’’ Ill I ci (ii t I .1 It) lilt II it.., ill iii ‘,j Ills III LIlt II I ’ S (Ill .itt ti t.s ,I Ii dist.in ’ r) t l’Izi . .i ‘ , )I 1111111 UIi ttit ill Is I I).,s lI Iq stage U i I fos ti ha rpi v. ith (I PX) objt ’¼ Live. Open I u II) tii i’ apel Liii e diaphragm in (1w sub 1.igi condi’nsc’ If the light too bright, tcnporarli place a neutral density filter or a diffuser in the lamp. f (‘lose the lamp diaphragm, or field diaphragm, to about a I-cm opening. Rack the mit roscope substage con- denser up and down to focus the field diaphragm sharply in the same plane as the specimen. g Adjust the mirror to center the field diaphragm in the field of view. h Remove the 16-mm objective and replace with a 4-mm objective. Move the specimen so that a clear area is under observation. Place the Bertrarxl lens in the optical path, or remove the eyepiece and insert an auxiliary telescope (sold with phase contrast accessories) in its place, or remove the eyepiece and observe the back aperture of the objective directly. Remove any ground glass diffusers from the lamp. Now OI)Serve the lamp filament through the necroscope. Jf the filament does not appear to be centered, swing the lamp housing in a horiiuntal arc whose center is at the field diaphragm. The purpose is to niaintaLn the field diaphragm on the lamp in its centered position. If a vertical movement of the filament is required, loosen the bulb base and slide it up or down. If the base is fixed, tilt the lamp housing in a vertit al arc with the field diaphragm is the inter of n i3vein nt (again rntle.ivt ,, ing to keep tlit ’ lamp din— phi., got in tht iitcrt ’d position). II you h vc in i’,tet i’d this step. you 11.1 VI ui t onip! i .Iii d th iii .s t d iffit ult put lion (Ilettil uiiu i o i ope lamps Ii ,IVI iii IU’,tfll(flt ’, to niave the bulb iiiilept i l, ntiy ol Pit Ia ma housing to ‘.iiiiplify this ‘,ti p. p Put th ‘ ,pit men in phi’ i, I iplac i ’ tI i y ,pit , t’ and (hi di’ ’, ir , ’d ob pet — tivi .intl I (‘lOt US k Open or lose th field diaphragm until it just disappears from the field. 1 Ob’.erve the back aperture of the objctive, preferably with the Bertrand lens or the auxiliary telescope, and close the aperture diaphragm on the substage condenser until it is about four-fifths the diameter of the back aperture. This is the best position for the aperture diaphragm, a posi- tion which minimizes glare and maxi- mizes the resolving power. It is instructive to vary the aperture dia- phragm and observe the image criti- cally during the manipulation. m If the illumination is too great, insert an appropriate neutral density filter between the illuminator and the condenser. Donotuse the con- denser aperture diaphragm or the lamp field diaphragm to control the intensity of illumination Poor Man’s Illumination Both critical and K hler illumination re- quire expensive illuminators with adjust- able focus, lamp iris and adjustable lamp mounts. Poor man’s illumination requires a cheap illuminator although an expensive illuminator may be used if its expensive features are negated by inserting a ground glass diffuser or by using a frosted bulb. Admittedly an iris diaphragm on the lamp would be a help though it is not necessary. a The illuminator must havp frosted bulb or a ground glass difiuser. 3—15 ------- Optics and the Microsi ope It should lii pos . ibli to (lint t it iii the genera I ii iret tion of tlii 1il14tJ gi mirror, vi r y • loi..e Liii ’ ii’ to 01 iii plaL e the I (Of. b [ ‘ocus on .iiiy prupix .1 LiOn .iIL i r tilting th( iiiirror to illu otitia Li the field. Remove the top li ’ii of the on il i’ii i,i ’r and, by r It king Liii t ondeiisi r up or, moi often, down, hi ing into lot us (in the same plum’ js the s;wt iin.n) a finger, i’m ii or othe i Olijet t pLo id in the same general region is the ground glass diffuser on the lamp. The glass surfa itself an then hi focused in the plane of the spe im ‘n. d Ideally the ground glass surfa e will Just fill the field of view when centered by the substage mirror, adjustment may be made by moving the lamp closer to or farther from the mi ro- scope (the position might be marked for each objective used) or by cutting paper diaphragms of fixed aperture (one for each objective used). In this Instance a lamp iris would be useful. e Lower the condenser Just sufficiently to defocus the ground glass surface and render the field of illumination even. f Observe the back aperture of the objective and open the substage con- denser iris about 75 percent of the way. The final adjustment of the 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 only with neutral density filters or by changing the lamp voltage. Proper iUummation is one of the most im- portant operations in microscopy. It is easy to judge a microscopist’s ability by a glance at his field of view and the olijec- tive back lens. U i eso1viiig Power Tlii riMilving power of the mitroscope is tt.. ihility Lu distinguish separate details of ii i ly .p i. ‘d mo roscopic structures. i’hi Lliior i Lit al limit of resolving two ili’.i riLi pointi., a distan t’ X apart, is I. 22 ) 2N.A. whi ii wavi I ngth of light used to iltuminjte the specimen N. A. - numer icil aperture of the objc c ti v (‘ Substituting a wavelength of 4, 500 Angi.troms and a numerical aperture of I. 3, ibout the best that can be done with visible light, we find that two points about 2, 000A (or 0. 2 micron) apart can be seen as two separate points. Further increase in resolving power can be achieved for the light ma roscope by using light or shorter wavelength. Ultraviolet light near 2, 000 Angstroms lowers the limit to about 0. 1 micron, the lower limit for the light microscope. The numerical aperture of an objective is usually engraved on the objective and is related to the angular aperture, AA (Figure 15). by the formula’ AA N.A. where n = the lowest index in the space between the object and the objective. Figure 15 Mgular opertize ANGULAR APERTURE OF MICROSCOPE OBJECTIVE 3—16 ------- Optics a i the Microscope I Maximum useful magnification A helpful rule of thumb lb that the use- ful magnification will not exceed 1.000 times the numerical aperture of the objective (see Tables 3 and 4). Although somewhat higher magnifiLatton may be used in specific cases, no additional detail will be resolved It is curious, considering the figures in the table, that most, if not all, manu- facturers of microscopes furnish a lOX eyepiece as the highest power. A lOX eyepiece is useful but anyone interested in critical work should use a l5-25X eye- piece. the 5- lox eyepieces are best for scanning purposes. 2 Abbe’s theory of resolution One of the most cogent theories of resolution is due to Ernst Abbe, who suggested that microscopic objects act like diffraction gratin (Figure 16) and that the angle of diffraction, therefore, increases with the fineness of the detail. He proposed that a given microscope objective would resolve a particular detail if at least two or three transmitted rays (one direct and two diffracted rays) entered the objective. In Figure 16 the detail shown would be resolved in A and C but not In B. This theory, which can be borne out by simple experiment, is useful in showiig how to improve resolu- tion. Since shorter wavelengths will give a smaller diffraction angle, there is more chance of resolving fine detail with short wavelengths. Also, since only two of the transmitted rays are needed, oblique light and a high N.A. condenser will aid in resolving fine detail. 3 Improving resolving power The following list summarizes the practical approaches to higher resolu- tion with the light mi roscope a The speLimen ‘,hould be illuminated by either critical or Kohler illumination. 11111 p ‘‘I’ll Uilllhl b Eigure 16 ABBE THEORY OF RESOLUTION b The condenser should be well- corrected and have a numerical aperture as high as the objective to be used. c An apochroraa tic oil-immersion objective should be used with a corn- pensatuig eyepiece of at least 15X magnification. The immersion oil should have an index close to 1. 515 and have proper dispersion for the objective being used. d Immersion oil should be placed between the condenser and slide and between cover slip ar objective. The preparation itself should be surrounded by a liquid having a refractive index of 1. 515 or more. e The illumination should be reasonably monochromatic and as short in wave- length as possible. An interference filter transmitting a wavelength of about 480-500 millimicrons is a suitable answer to this problem. Ideally, of course, ultraviolet light should be used to decrease the wave- length still further. The practical effect of many of these factors is critically discussed by Loveland( 2 ) in a paper on the optics of object space. 3—17 ------- Optics and the Microscope IV PHOTOMICROGRAPHY A Introduction Photomicrography, a ’i distinct from micro- photography. is the art of taking pictureb through the microscope. A mit rophoto- graph is a small photograph, a photomicro- graph Is a photograph of a small object. Photomicrography 18 a valuable tool in recording the results of microstopi al study. It enables the microseopist to 1 describe a microscopic field objectively without resorting to written descriptions. 2 rccord a particulac field for IUIUI ’L reference, 3 make particle size counts and counting analyses easily and without tying up a microscope, 4 enhance or exaggerate the visual micro- scopic field to bring out or emphasize certain details not readily apparent visually, 5 record images in ultraviolet arid infra- red microscopy which are otherwise invisible to the unaided eye. There are two general approaches to photo- rntcrography, one requires only a plate or film holder supported above the eyepiece of the microscope with a light-tight bellows. the other utilizes any ordinary camera with its own lens system, supported with a light- tight adaptor above the eyepiece. It is best, in the latter case, to use a reflex camera so that the image can b carefully focused on the ground glass. Photonu- crography of this type can be regarded simply as replacing the eye with the camera lens system. The camera should be focused at infinity, just as the eye 18 for visual observation, and it should be positioned close to and over the eyepiece. The require ments for photomic rography, however, are more rigorous than those for visual work. The eye can normally compensate for varying light intensities, curvature of field and depth of field. The photographic plate, however, lies in one plane, hence the greatest care must be used to focus sharply on the subject plane of interest and to select optics to give minimum amounts of field curvature and chromatic aberrations. With black and white film, color filters may be used to enhance the contrast of some portions of the specimen while mini- mizing chromatic aberrations of the lenses. In color work, however, filters cannot usually be used for this purpose and better optics may be required. Photornicrographic cameras which fit directly Onto the microscope are available in35 -mrnorupto3-114X4-1!4 inch sizes. Others are made which accommodate larger film sizes and which have their own support independent of the microscope. The former, however, are preferred for ease of handling and lower coat. The latter system is pre- ferred for greater flexibility and versatility and lack of vibration. The Polaroid camera has many applications In microscopy and can be used on the microscope directly but, because of its weight, only when the micro- scope has a vertically moving stage for focusing rather than a focusing body tube. B Determination of Correct Exposure Corruct exposure determination can be accomplished by trial and error, by relating new conditions to previously used successful onditions and by photometry. With the trial and error method a series of trial exposures is made, noting the type of subject, illumination, filters, objective, eyepiece, magnification, film and shutter speed. The best exposure is selected. The following parameters can be changed and the exposure time adjusted accordingly: I Magnification. ExpOsure time varies as the square of the magnification. Example Good exposure was obtained with a 1/10-second exposure and a magnification of IOOX If the magnification is now 3—18 ------- ptics and the Microscope 200X, the correct expobure is calculated as follows new exposure time old exposure time > ( new magnificauon)2 i/ (200)2 old magnification 100 4/10 or, say, 1/2 second. It should be noted, however, that th above calculation can he made only when there has been no change in the illumi- nation system including the condrnser or the objective. Only changes in magni- fication due to changing eyepiece’i or bellows extension distance can be hand- led in the above manner. 2 Numerical aperture. Exposure time varies inversely as the square of the smallest working flume rical aperture of the condenser and objective. Example Good exposure was olitarned at 1/10 second with the lOX objective, N. A. 0. 25. at full aperture. With a 20X objective, N. A. 0. 25. at full aperture and the same final magnification, what is the correct exposure time? new exposure time old exposure time X ( Old N.A . )2 1/10 (2.:_ )2 1/40 or, newN A. 0.50 say, 1/50 second. It is seen that more light reaches the photographic film with higher numeri- cal apertures at the same magnification. 3 Film. Exposure time varies inversely with the American Standards Association speed index of the film. Example A good picture was obtained with Eastman Tri-X film at 1/100 second. What is the correct exposure for Eastman Kodachrome II Type A. The A.S.A. speed for Tri-X is 400 and for Kodachrome II Type A Professional is 40. new exposure time old exposure time A. S. A. of old film 1/100(400/40) A. S. A. of new film 10/100 or 1/10 second. 4 Other parameters may be varied but the prediction of exposure time cannot be made readily. Experience and photo- electric devices are the best guides to the proper exposure. Photoelectric devices are excellent for determining correct exposure. Since ordinary photographic exposure meters are not sensitive enough for photomi- crography, more sensitive instruments, having a galvanometer or electronic mph.fying circuit, are required. Some photosensitive cells are inserted in the body tube in place of the eyepiece for light intensity readings. This has the advantage of detecting the light level at a point of high intensity but does not take into account the eyepiece, the distance to the film or the film speed. The cell may be placed just above the eye- piece so that it registers the total amount of light leaving the eyepiece. Again, the effects of film speed and the projection distance are not accounted for. The prin- cipal drawback with the total light measuring photometer is the difficulty of taking into account the area of field covered. Take, for example, a bright field in which only a few crystals appear, perhaps 1 per- cent of the light entering the field of view is scattered by the crystals and the photometer shows close to a maximum reading. Now assume that everything remains constant except the number of crystals and, conse- quently, the amount of light scattered. The photometer reading could easily drop by 50 percent, yet the proper exposure is unchanged. The situation is similar for photornicrography with crossed polars since the photometer reading depends on the intensity of illumination, on the bire- fringence and thickness of the crystals and 3—19 ------- 9p ç S on the number and size of the crystals in the field or, alternatively, on the area of the field covered by birefringent rystalb. One of the best solutions to this problem is to measure the photometer reading with no preparation on the stage. A first-order red compensator or a quartz wedge is in- serted when crossed polara are being used to Illuminate the entire field. An alternative is to place the cell on the ground glass where the film will be located. However, although all variables except film speed are now taken into account, measurements in the image plane have the disadvantage of requiring a more sensitive electronic’ photoelectric apparatus. No matter what method is used for placing the photocell, the exposure time can be determined by the general formula exposure time k meter reading The constant k will depend on the physical arrangement and film used. To determine k for any particular system, first set up the microscope to take a picture. Record the meter reading and take a series of trial exposures. Pick out the best exposure and calculate k. Then the k which was determined holds as long as no change is made in the light path beyond the photocell. !..k changing to a faster film or changing the projection distance. Thus the objective. condenser position or illuminator may be changed without affecting k if the cell is used as described above. Example With one particular arrange- ment of photocell and film, the meter reading is found to be 40. A series of photographs are taken at 1/2, 1/5, 1/10, if 25 and 1150 seconds. The photomicrograph taken at 1/5 second is judged to be the beat, hence k Is calculated as follows k meter reading X exposure time 40X 1/5 c 8. Assume now that a new picture is to be taken at another magnification (but with the same film and projection distance) and that the new meter reading is 16, therefore. exposure time k/meter reading 8/16 = 1/2 second. A Particle Size Determination Linear distances and areas can be measured with the microscope. This permits determination of particle size arid quantitative analysis of physical mixtures. The usual unit of length for microscopical measurements 18 the micron (1 )< 10 3 mm or about 4 X 10 5 inch). Measuring particles in electron microscopy requires an even smaller unit, the ml iii- micron (1 X l0 micron or 10 Angstrom units). Table 6 shows the approximate average size of a few common airborne materials. Table 6. APPROXIMATE PARTICLE SIZE OF SEVERAL COMMON PARTICULATES Ragweed pollen 25 micronB Fog droplets 20 microns Power plant flyash (after precipitators) 2-5 microns Tobacco smoke 0. 2 micron (200 mjllimicroris) Foundry fumes 0. 1 - 1 micron (100-1000 mlllimicrons) The practical lower hmit of accurate particle size measurement with the light microscope is about 0. 5 micron. The measurement of a particle smaUer than this with the light microscope leads to errors which, under the best circurn- stances, increase to about + 100 percent (usually i ). One of the principal uses of high resolving power is in the precise measurement of V MICROMETRY 3—20 ------- Ontics and the Microscone particle size There are, however, 8 variety of approximate and useful pro e- dures as well. 1 Methods of particle size measurement a Knowing the magnification of the microscope (product of the magni- fication of objective and eyepioLe), the size of particles can be esti- mated. For example, with a lox eyepiece and a 16-mm (or lOX) objective, the total magnification is 100X. A particle that appears to be 10-mm at 10 inches from the eye has an actual size of 10 mm divided by 100 or 0. 10 mm or 100 microns This is in no sense an accurate method, but it does permit quick estimation of particle size, the error in this estimation is usually 10-25 percent. b Another approximate method is also based on the use of known data. If we know approximately the diameter of the microscope field, we can estimate the percentage of the diameter occupied by the object to be measured and calculate from these figures the approximate size of the object. The size of the micro- scope field depends on both the objec- tive and the ocular although the latter is a minor influence. The size of the field should be determined with a millimeter scale for each objective and ocular. If this is done, esti- mation of sizes by comparison with the entire field diameter can be quite accurate (5-10%). c The movement of a graduated mechan- ical stage can also be used for rough measurement of diameter8 of large particles. Stages are usually gradu- ated (with vernier) to read to 0. 1 millimeter, or 100 microns. In practice, the leading edge of the particle Is brought to one of the lines of the cross hair in the eyepiece and a reading is taken of the stage position. Then the particle is moved across the field by moving the mechanical stage in an appropriate direction until the second trailing edge just touches the cross-hair line. A second reading is taken and the difference in the two readings is the distance moved or the size of the particle. This method is especially useful when the particle is larger than the field, or when the optics give a distorted image near the edge of the field. d The above method can be extended to projection or photography. The image of the particles can be projected on a screen with a suitable light source or they may be photographed. The final magnification, M, on the projection surface (or film plane) is given approxi- mately by M DX 0. M. X E. M. /25 where 0. M. objective magnification E. M. eyepiece magnification D = projection distance from the eyepiece in centimeters. The image detail can then be measured in centimeters and the actual size com- puted by dividing by M. This method is usually accurate to within 2-5 percent depending on the size range of the detail measured. e The stated magnifications and/or focal lengths of the microscope Optics are nominal and vary a bit from objective to objective or eyepiece to eyepiece. To obtain accurate measurements, a stage micrometer is used to calibrate each combination of eyepiece and objective. The stage micrometer is a glass microscope slide that has, accurately engraved in the center, a scale, usually 2 millimeters long, divided into 200 parts, each part repre- senting 0. 01 millimeter. Thus when this scale is observed, projected or photographed, the exact image magni- fication can be determined. For example, if S spaces of the stage micro- meter measure 6 millimeters when projected, the actual niagrn.fication is 3—21 ------- Optics and the Microscope 5 (0. 01) 120 times. This magnification figure can be used to improve the accuracy of method 4 above. f The simplest procedure and the most accurate is based on the use of a micrometer eyepiece. Since the eyepiece magnifies a real image from the objective, it is possible to place a transparent scale in the same plane as the image from the objective and thus have a scale superimposed over the image. This Is done by first placing an eyepiece miciometer sale dibc in the eyepiece. The eyepiece micrometer has an arbitrary scale and must be cali- bra ted with each objective used. The simplest way to do this is to place tJ stage micrometer on the stage and note a convenient whole number of eyepiece micrometer divisions. The value in microns for each eye- piece micrometer division Is then easily computed. When the stage micrometer is removed and replaced by the specimen, the superimposed eyepiece scale can be used for accu- rate measurement of any feature in the specimen by direct observation, photography or projection. 2 Calibration of eyepiece micrometer Each micrometer stage scale has divisions lOOu (0. 1 mm) apart, one or two of these are usually subdivided into lO (0. 01-mm) divisions. These form the starr:lard against which the arbitrary divisions in the micrometer eyepiece are to be calibrated. Each objective must be calibrated separately by noting the correspondence between the stage scale and the eyepiece scale. Starting with the lowest power objective focus on the stage scale, arrange the two scales parallel and in good focus. It should be possible to determine the number of eyepiece divisions exact.ly equal to some whole number of divisions of the stage scale, a distance readily expressed in microns. The calibration consists, then, of calculating the number of microns per eyepiece scale division. To make the com!arison as accurate as possible, a large part of each scale must be used (see Figure 17). Let’s assume that with the low power 16-mm objective 6 large divisions of the stage scale (s. m. d.) are equal to 38 divisions of the eyepiece scale. This means that 38 eyepiece micrometer divisions (e.rn. d.) are equivalent to 600 microns. Hence 1 e.m.d. 600/38 l5.8 . COMPARISON OF STAGE MICROMETER SCALE WITH EYEPIECE MICROMETER SCALE Thus when that micrometer eyepiece is used with that 16-mm objective each division of the eyepiece scale is equivalent to 15. 8 a, and it can be used to make an accurate measurement of any object on the microscope stage. A particle, for example, observed with the 16-mm objec- tive and measuring 8. 5 divisions on the eyepiece scale is 8. 5 (15. 8) or l35 in diameter Each objective on your microscope must be calibrated an this manner. A convenient way to record the necessary data and to calculate ,j!emd Is by means of a table. FIgure 17 3—22 ------- Optics and the Microscope Table 7 Objective No smd no. emd no emd I emd 32-mm 18 44 1800 44 40.9 16-mm 6 = 38 600 38 15 8 4-mm I = 30 100 = 30 3 33 3 Determination of particle size distribution The measurement of particle size can vary in complexity depending on parti- cle shape. The size of a 8phere may be denoted by its diameter. The size of a cube may be expressed by the length of an edge or diagonal. Beyond these two configurations, the particle ‘size” must include information about the shape of the particle in question, and the expression of this shape takes a more comi,llcated form. Martin’s diameter is the simplest means of measuring and expressing the dia- meters of irregular particles and is sufficiently accurate when averaged for a large number of particles. In this method, the horizontal or east-west dimension of each particle which divides the projected area thto halves is taken as Martin’s diameter (Figure 18). The more particles counted, the more accurate will be the average particle size. Platelike and needlelike particles should have a correction factor applied to account for the third dimension since all such particles are restricted in their orientation on the microscope slide. When particle size is reported, the general shape of the particles as well as the method used to determine the “diameter” should be noted. Particle size distribution is determined routinely by moving a preparation of particles past an eyepiece micrometer scale in such a way that their Martm’ diameter can be tallied. All particles whose centers fall within two fixed divisions on the scale are tallied. Move- ment of the preparation is usually accomplished by means of a mechanical stage but may be carried out by rotation of an off-center rotating stage. A sample tabulation appears in Table 8. The eye- piece and objective are chosen so that at least six, but not more than twelve, size classes are required and sufficient particles are counted to give a smooth curve. The actual number tallied (200 - 2, 000) depends on particle shape regularity and the range of sizes. The size tallied for each particle is that number of eyepiece micrometer divisions most closely approximating Mirtin’s diameter for that particle. 4 Calculation of size averages The size data may be treated in a variety of ways, one simple, straightforward treatment is shown in Table 9. For a more complete discussion of the treat- ment of particle size data see Chamot and Mason’s l 1andbooic of Chemical Microscopy( 3 ’ , page 26. The averages with respect to number, d 1 , surface, d 3 . arid weight or volume, d 4 , are calculated as follows for the data in Table 9. h-P-I I—p —I Figure 18 MARTIN’S DIAMETER 3—23 ------- Op ics and the Micro8cop4 TabI. 8. PARTICLE SIZE TALLY FOR A SAMPLE OF STARCH ORAHIS fl ” Nu s r of partici.. Total 1 p1 —4 -a 11 - 44 11-44 1 18 I ti-I-a re re-e4 r-t—s..a re r1- ri-i..a r ti44 1-144 re- 44 r e- I - I r 1-144 t -*-4..1 r a lii 1-1-4.1 r as 3 ri-eu ri-ia 1-144 ri - 1J 11-14 re- i.:’ 1-144 ii-’ 1-1-4.1 ti rI- I - I ri-u 44 1-1-44 j 1-1-44 f1-44 1-1-44 ti-I-I 110 4 11-44 r i— I-I ri44 11 1-1-44 1-1-44 44 1-1-44 1 1-I4 44 1144 11-44 ri44 21—44 11-4-1 ti44 11—14 1-1-44 44 11-43 1#-4 .j r1- 107 S 11 -34 1-1-44 1 2144 2144 1-1-44 1-44 2*44 £144 1-144 11-1-1 11—14 11—14 rI-44 ri—i -a 71 S 1-1-4.1 ti44 2 144 1-1-44 1-1-44 1-1-14 1 I—I.I 11-44 1-1-4.1 45 7 1-144 21—4.1 21-14 21-14 1 11 5 11 1 470 ‘md • C ieci . mtc romeier d IaIon. r nd/Ln r 1758/470 3.74 emd X 2.82* - 10. 5 i d 3 End 3 /End2 - 37440/7662 4.89 emdx 2.82 13. - End 4 /Znd 199194/37440 = 5.32 emd X 2.82 - 15. 0 *2. 82 microns per emd (determ med by calibration of the eyepiece-objective combination used for the determination). Cumulative percents by number, surface and weight (or volume) may be plotted from the data in Table 9 The calculated percentages, L : A 4X 100 for the cumulative weight or volume curve, are plotted against d. Finally. the specific surface, Sm, in square meters per gram, m, may be calculated if the density, D, is known, the surface average d 3 , is used. if D 1. 1, Sm — 6Jd 3 D - 6/13. 8(1. 1) - 0. 395m 2 1g. 3—24 ------- Optics arxl the Microscop Table 9. CALCULATIONS FOR PARTICLE SIZE AVERAGE d (Aver diam. n nd nd 2 nd 3 in emd) 2 3 4 5 6 7 8 98 196 392 784 1568 110 330 990 2970 8910 107 428 1712 6848 27392 71 355 1775 8875 44375 45 270 1620 9720 58320 21 147 1029 7203 50421 2 16 128 1024 8192 470 1758 7662 37440 199194 B Counting Analysis Mixtures of particulates can often be quantitatively analyzed by caantuig the total number of particulates from each component in a representative sample. The calculations are, however, compli- cated by three factors average particle size, particle shape and the density of the components. If all of the compon- ents were equivalent in particle size, shape and density then the weight per- centage would be identical to the number percentage. Usually, however, it is necessary to determine correction factors to account for the differences. When properly applied, this method can be accurate to within + 1 percent and. in special cases, even better. It is often applied to the analysis of fiber mixtures and is then usually called a dot-count because the tally of fibers is kept as the preparation is moved past a point or dot in the eyepiece. A variety of methods can be used La simplify recognition of the different components. These include chemical stains or dyes and enhancement of optical differences such as refractive indices, dispersion or color. Often, however, one relies on the differences in morphology. !..L counting the percent of rayon fibers in a samole of “silk”. Example 1 A dot-count of a mixture of fiberglass and nylon shows % nylon = 262/(262 + 168)X 100 = 60. 9To by number. However, although both fibers are smooth cylinders, they do have different densities and usually different diameters. To correct for diameter one must measure the average diameter of each type of fiber and calculate the volume of a unit length of each. aver. diam. volume of l- slice, 18. 5 268 fiberglass 13. 2 The percent by volume is, then. 262 X 268 % nylon (262 X 268)+(168 X 117) X 100 78. 1% by volume. Still we must take into account the density of each in order to calculate the weight percent. nylon fiberglass Therefore. 262 168 nylon 117 3—25 ------- Optics and the Microscope If the ii ,i’,itj or I I f i iiykiiu ,ig I . 2 mr glos’, then thc p (’r (nt by w(’ight i 262 X 2(8 x I I % nylon (262 x 268 X 1. h)+(lb8 x 17 X 2.2) X 100 = 72% by weight. Example 2 A count of quortz and gypsum shows quart’, gypsum 28.1 417 To ol ulate the percent by weight we must take into a ount the average partu I c size, the shape and the density of (‘a h The avcrogc parti( Ic size with rcspcLt to weight. d 4 , must be measured for each and the shape factor must be determined. Since gypsum is more plutelike than quartz each particle of gypsum is thinner. The shape factor (an be approxima ted 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 volume of the aver- age quartz particle, this is our shape factor. The final equation for the weight percent is 283X lTd 4 /6XDq %quartz 238X wd 4 /6XDq+467Xi / where Dq and Dg are the densities of quartz and gypsum respectively. 0. 80 is tha shape factor and d 4 and d are the average parti- cle sizes with respect to weight for quartz and gypsum respectively. A CKNOW LEDGM ENT This outline was prepared by the II. S. Public Health Service, Department of Health, Education and Welfare, foi use in its Training Program. REFElU ’ NCES 1 flunn, C.W. (‘rystal Giowtli from Solution. I)iscussionc. nf the [ “araday Society No. 5. 12. Gunit y ond .Jackson. l ndon. (1949). 6 X 0.80 X Dg X 100 2 Loveland, R.P., J. Roy. Micros. Soc. 79, 59. (1960). 3 Chamot, Emile Monnin, and Mason, Clyde Walter. Handbook of Chemical Microscopy, Vol. 1, third ed. John Wiley and Sons, New York (1959). 3—26 ------- STRUCTURE AND FUNCTION OF CELLS I INTRODUCTION What are cells? Cells may be defined as the basic structural units of life. The ceU has many different parts which carry on the various functions of cell life. These are called organelles (“little organs”). A The branch of biology which deals with the form and structure of plants and animals is called “Morphology.” The study of the arrangement of their several parts is called “anatomy”, and the study of cells is called “cytology”. B There is no “typical” cell, for cells differ from each other in detail, and these differences are in part responsible for the variety of ll.fe that exists on the earth. II FUNDAMENTALS OF CELL STRUCTURE A How do we recognize a structure as a cell? We must look for certain characteristics and/or structures which have been found to occur in cells. The cell Is composed of a variety of substances and structures, some of which result from cellular activities. These include both living and non-living materials. 1 Non-living components thclude a A “cell wall” composed of cellulose may be found as the outermost covering of many plant cells. b ‘Vacuoles” are chambers In the protoplasm which contain fluids of diifcrent densities (i.e., different from the surrounding protoplasm). 2 The “living” parts of the cell are called “protoplasm.” The following structures are included: a A thm “cell membrane” is located just inside the cell wall. This membrane may be thought of as the outermost layer of protoplasm. b In plant cells the most conspicuous protoplasmic structures are the “chioroplasts”, which contain highly organized membrane systems bearing the photosynthetic pigments (chlorophylls, carotenoids, and xanthophylls) and enzymes. c The “nucleus” is a spherical body which regulates cell function by controlling enzyme synthesis. d “Granules” are structures of small size and may be “living” or non -living” material. e “Flagella” are whip-like structures found in both plant and animal cells. The flagella are used for locomotion, or to circulate the surrounding medium. f “Cilia” resemble short flagella, found almost exclusively on animal cells. In the lower animals, cilia are used for locomotion and food gathering. g The “pseudopod”, or false foot, is an extension of the protoplasm of certain protozoa, in which the colloidal state of the protoplasm alternates from a “sol” to a condition from time to time to facilitate cell movement. h “Ribosomes” are protoplasmic bodies which are the site of protein synthgsls. They are too small (150 A lndiameter)to be seen with a light microscope. i “Mitochondria” are small mem- branous structures containing enzymes that oxidize food to produce energy transfer compounds (ATP). BI.CEL. la. 3. 70 4—1 ------- Structure and Function of Cells B How basic structure is expressed in some major types of orgamams. We can better visualize the variety of cell structure by considering several specific cells. 1 BacterIa have few organelles, and are so minute that under the light microscope only general morphological types (i.e.. the three basic shapes; rods, spheres, and spirals) can be recognized. The following structures have been defined: a The “capsule” is a thick protective covering of the cell exterior, con- sisting of polysaccharide or polyp eptide. b The cell wall and plasma membrane are present. c Although no well defined nucleus is visible in bacterial cells, the electron microscope has revealed areas of deoxyribose nucleic acid (DNA) concentration. This ath- stance is present within the nucleus of of higher cells, and is the genetic or hereditary material. d Some types of bacteria contain a special type of chlorophyll (bacterlochiorophyll) and carry on photosynthesis. 2 The blue-green algae are similar to the bacteria in structure, but contain the photosynthetic pigment chlorophyU a. a Like the bacteria, these forms also lack an organized nucleus (the nuclear region Is not bounded by a membrane). b The chlorophyll-bearing membranes are not localized in distinct bodies (chioroplasts), but are dispersed throughout the cell. c Gas-filled structures called “pseudiovacuoles” are found in some types of blue-greens. 3 The green algae as a group include a great variety of structural typeB, ranging from single-celled non-motile forms to large motile colonies. Some types are large enough to resemble higher aquatic plants. a The chloroplasts are modified into a variety of shapes and are located In different positions. Examples of ch]orop]ast shape and position are: 1) Parietal - located on the periphery of the cell; usually cup-shaped and may extend completely around the inner surface of the plasma membrai e. 2) Discoid - also located on the periphery of the cell, but are plate-shaped; usually many per cell. 3) AxIal - lying In the central axis of the cell, may be rthbon-lIice or star-shaped. 4) Radial - have arms or processes that extend outward from the center of the cell (radiate), reaching the plasma membrane. 5) Reticulate - a mesh-like network that extends throughout volume of the cell. b Located In the chlorop]asts may be dense, protelnaceous, starch- forming bodies called “pyrenoids”. 4 The flagellated algae possess one-to- eight flagella per cell. The chloro- plasts may contain brown and/or red pigments In addition to chlorophyll. a Reserve food may be stored as starch ( Chlamydomonas ) paralny]on or as oil. 5 The protozoa are single-celled animals which exhibit a variety of cell structure. 4—2 ------- Structure and Function of Cells a The amoebae move by means of pseudopodia, as described previously. b The flagellated protozoa (Mastigophora) possess one or more f]ageUa. c The ciliates are the most highly modified protozoans. The cilia may be more or less evenly distributed over the entire surface of the cell, or may be localized. Ifl FUNCTIONS OF CELLS What are the functions of cells and their structural components? Cellular function is called “life”, and life is difficult to define. Life is characterized by processes commonly referred to as reproduction, growth, photo- synthesis, etc. A Mlcroorganism8 living in surface waters are subjected to constant fluctuations in the physical and chemical characteristic of the environment, and must constantly modify their activities. 1 The cell requires a cource of chemical energy to carry on life processes and successfully compete with other organisms. Plant cells may obtain this energy from light, which is absorbed by chlorophyll and converted into ATP or food reserves, or from the oxidation of food stuffs. Animal cells obtain energy only from the oxidation of food. 2 Cells mu8t obtain raw materials from the environment in order to grow and carry out other Life functions. Inorganic and organic materials may be taken up by passive diffusion or by “active transport”. In the later process, energy is used to build up and maintain a higher concentration of a substance (such as phosphate) inside the cell than is found outside. Algae are able to synthesis organic matter from inorganic raw materials (carbon dioxide and water), with the aid of energy derived from light, whereas animal cells must obtain their organic matter “ready- made” by consuming other organisms. organic debris, or dissolved organics. IV SUMMARY The cell is made up of many highly special- ized substructures. The types of sub- structures present, and their appearance (shape, color, etc.) are very important in understanding the role of the organism in the aquatic community, and in classification. REFERENCES 1 Bold, H.C. Cytology of algae. In: G.M. Smith, (ed.), Manual of Phycology. Ronald Press. 1951. 2 Bourne, Geoffry H., ed. Cytology and Cell Physiology. 3rd ed. Academic Press. 1964. 3 Brachet, Jean. The Living Cell. Scientific American. 205(3). 1961. 4 Corliss, John 0. Ciliated Protozoa. Peganion. 1961. 5 Fritsch. F. E. The structure and reproduction of the algae. Cambridge Univ. Press. 1965. 6 Frobisher, M. Fundamentals of microbiology. 7th edition. W. B. Saunders Co., Philadelphia. 1962. 7 Round, F.E. The biology of the algae. St. Martin’s Press. New York. 1965. This outline origthaUy prepared by Michael E. Bender, Biologist, formerly with Training Activities, FWPCA, SEC. and revised by Cornelius I. Weber, March 1970. 4—3 ------- TYPES OF ALGAE INTRODUCTION A Algae in general may be defined as small pigmented plant-like organisms of relatively simple structure. Actually the size range is extremeS from only a few microns to over three hundred feet in length Commonly observed examples include the greenish pond scum or frog 8pittle of freshwater ponds, much of the golden brown slime covering rocks In a trout stream, and the great marine kelps and seaweeds Large freshwater forms as Nitefla and Chara or stonewort are also included B Algae approach ubiquity in distribution. In addition to the commonly observed bodies of water, certain algae also live in such unlikely places as thermal springs, the surface of melting snow, on the hair of the three toed sloth in Central America, and in con)unctton with certain fungi to form lichens. III ALGAE WILL BE GROUPED FOR THE SAKE OF CONVENIENCE INTO FOUR GENERAL TYPES: A Blue-greens (See plate: Blue-Green Algae, Cyanophyceae). This is a valid technical group. The size range is not very great, some being so small as to approach the size range of the bacteria 1 These are the only algae in which the pigments are not localized in definite bodies but dissolved through- out the cell Blue, red, or other pigments are present in addition to chlorophyll thus giving the cells a bluish green, yellow, or r d color, at least enmasec 2 ‘]‘ht. nucleus lacks a nuclear membrane. 3 Tend to achieve nuisance concentrations more frequently in the warm summer months and in the richer waters. 4 Vegetative reproduction, in addition to cell division, Includes the forma- tion of ‘hormogones,” or short specif- ically delimited sections of trichomes (filaments). 5 Spores of three types are encountered: a Akinetes are usually larger, thick walled resting spores. b Heterocysts appear like empty cell walls, but are actually filled with protoplasm, have occasionally been observed to germinate c Endospores, also called “gonidia” or conidia, are formed by repeated division of the protoplast within a given cell wall. Present in only a few genera. 6 Some common examples of blue- green algae are: Anacyatis ( Microcystis or Polycystis), Anabaena, Aphani- zomenon , and Oscillatorta B The Pigmented flagellates (in contrast to the non-pigmented or animal-like flagellates) are a heterogeneous collection of motile forms from several different algal groups (See plateS Flagellated algae). 1 There may be one, two, four, or more flagella per cell. 2 There is a well organized nucleus 3 A light-sensitive red eyespot usually present 4 The chlorophyU is contained in one or more distinctive bodies called plastids. RI MIC.cla. 19a 8 69 5- 1 ------- Types of Algae 5 Two or more cells may be associated In a colony 6 Non-motile life history stages may be encountered 7 Masses of stored starch called pyrenotd bodies are often conspicuous. 8 Some examples of pigmented flag- ellates are: Euglena, Phacus, Chiamydomonas , Gonium, Volvox, Peridinium, Ceratium Mallomonas, nura and Dinobryon . C The Non-motile green algae constitute another heterogeneous assembly of Un- related forms (See p’ate: Non-Motile Green Algae) 1 Like the flagellates they have well organized nuclei and chloroplasts. The shape of the chloroplast is often distinctive. 2 They lack flagella or any other loco- motor device. 3 There is extreme structural variation among the group. 4 Some types tend to occur as a general planktonic mass or bloom,” often in combinations of two or more species. Some examples are Sphaerocystts, Pedlastrum, Scenedesmus , and the desmid Cosmarium . 5 Threadlike (filamentous) green algae may form masses or blankets, cutting off light, and reducing water circula- tion. They also add considerably to the total mass of organic matter. Some examples of this type are Spirogyra, Hydrodictyon, Cladophora, Oedogonlum , and Chara . D The Diatoms constitute another valid technical group (See plate Diatoms- l3acUJ.ariophyceae). 1 In appearance, they are geometrically regular in shape The presence of a brownish pigment in addition to the chlorophyfl gives them a golden to greenish color. 2 Motile forms have a distinctive hesitating progression. 3 The most distinctive structural feature is the two-part shell (frustule) composed of silicon dioxide (glass). a One part fits inside the other as the two halves of a pill box, or a petri dish. b The surface of these shells are sculptured with minute pits and lines arranged with geometrical perfection. c The view from the side is called the “girdle view,” that from above or below, the “valve view.” 4 There are two general shapes of diatoms, circular (centric) and elongate (pennate). The elongate forms may be motile, the circular ones are not. 5 Diatoms may associate in colonies in various ways 6 Examples of diatoms frequently en- countered areS Stephanodiscus Cyclotelia, Astertonella, Fragilaria, Tabellaria, Synedra , and Nitzschta . This outline was prepared by H.W. Jackson, Chief Biologist, National Training Center, Water Programs Operations, EPA, Cincinnati, OH 45268 5-2 ------- Types of Algae KEY FOR IDENTIFICATION OF GROUPS OF FRESHWATER A LGAE Beginning with “in” and “lb”, choose one of the two contrasting statements and follow this procedure with the “a” and “b” state- ments of the number given at the end of the chosen statement. Continue until the name of the algal group is given instead of another key number. is. P].astld (separate color body) absent, complete protoplast pigmented, generally blue-green; Iodine starch test* negative Blue-green algae lb. Piastid or plastids present; parts of protop]ast free of some or all pigments; generally green, brown, red, etc., but not blue-green; iodine starch test* positive or negative 2 2a. Cell wail permanently rigid (never showmg evidence of collapse), and with regular pattern of fine markings (striations, etc.), plastids brown to green, Iodine starch test* negative; flagella absent; wall of two essentially similar halves, one placed over the other as a cover Diatoms 2b. Cell wall, if present, capable of sagging, wrinkling, bulging or rigi ty, depending on existing turgor pressure of cell protoplast; regular pattern of fine markings on wall generally absent; plastids green, red, brown, etc.; Iodine starch test* positive or negative; flagella present or absent; cell wall continuous and generally not of two part 3 3a. Cell or colony motile, flagella present (often not readily visible); anterior and posterior ends of cell different from one another in contents and often in shape Flagellate algae 3b. Non-motile, true flagella absent; ends of cells often not differentiated Green algae and associated forms *Add one drop Lugol’s (Iodine) solution, diluted i-i with distilled water, in about 1 minute, if positive, starch i 9tained blue and, later black. Other structures (such as nucleus, plastids, cell wall) may also stain, but turn brown to yellow. 5—3 ------- Typea of Algae CMP COMPARISON OF FOUR MAJOR GROUPS OF ALGAE Blue-Green Pigmented flagellates Greens Diatoms Color Blue-Green (Brown) Green Brown Green Brown (Light-Green) Location of pigment Throughout cell In plastida In piastids In plastids Starch Absent Present or Absent Present Absent Slimy coating Present Absent in most Absent in moat Absent in most Nucleus Absent Present Present Present Flagellum Absent Present Absent Absent Cell Wall Inseparable from slimy costing Thin or Absent Semi-rigid smooth or with spines Very rigid, with regular markings “Eye” spot Absent Present Absent Absent 5-4 ------- BLUE-GREEN ALGAE I WHAT ARE THE BLUE-GREEN ALGAE? The blue -green algae (Myxophyceae) comprise that large group of microscopic orgazusms living in aquatic or moist habitats, carrying on photosynthesis and having differentiation of cells which is a little more complex than bacteria, and simpler than aU of the other plants called algae H WHY ARE THEY CALLED BLUE-GREEN: In addition to the green photosynthetic pigment (chlorophyll -a) they always have a blue pig- ment (phyocyanin-c) which tends to give the cushions or mats they may form a blue-green tinge. III WHERE ARE THE BLUE-GREENS FOUND? Some are free floating (pelagic and planktonic), others grow from submerged or moist soil. rocks, wood and other objects in both fresh- water and marine habitats. IV WHAT ARE SOME OF THEIR GENERAL CHARACTERISTICS? Some are gelatinous masses of various shapes floating in water. Others, microscopic in size, grow in great numbers so as to color the water in which they live. Structurally their cells are similar to bacteria. Their protoplasts may be sheathed or imbedded in gelatin, making them slimy. Cells of blue- green algae are without organized nuclei, central vacuoles, or cilia and flagella. No sexual reproduction Is known. Asexual reproduction may be effected by fragmentation, in which case special separation devices are formed (dead cells, and heterocysts). Some species arc preserved over unfavorable periods by special spores (akinetes an’i endo- spores). V OF WHAT IMPORTANCE ARE BLUE- GREEN ALGAE? They have both positive and negative economic significance. Because they can convert radient energy into chemical energy, they are producers forming a first link at the base of the food chain. Because many very in- tricate nutritional relationships exist among the niyraids of organisms it is difficult to know the value of the blue-greens. However, people who know what the blue-greens can do to drinking and recreational water classify them as o! negative economic importance, because they are often nuisances when they impart color, bad odors, and fishy tastes, or toxins. Some of them can foul pipes and clog filters. VI WHEN ARE THEY MOST COMMON 9 They are widely distributed in time and space, but tend to reach nuisance concentrations more frequently in the late summer and in eutrophic waters. VII WHAT DO BLUE-GREEN ALGAE DO FOR A LIVING? The pioneer-forms are of great ecological importance because they live m habitats fre- quented by few other forms of live, synthesiz- ing organic substances and building substrata that can support other kinds of life. A Some blue-greens live in association with other organisms as symbionts. Still others are found i.n polluted waters, because they are able to exist in habitats poor in oxygen. The growth of these kinds of algae under such conditions tends to make a pol- luted condition worse. B On the other hand some species should be promoted because they provide oxygen and food through photo- synthesis. The first evident product of photosynthesis is glycogen, and is the cause of the brown coloration with the iodine test. Some of the glycogen is used to produce glycopro- teins. The gelatinous sheath is com- posed of pectic substances, cellulose and related compounds. BI. MIC. cia. iCa. H. 69 6-1 ------- Blue-Green Algae C When blue-green8 mat at the surface of the water the increased lighting may oe too strong, resulting in a kill. At this time they may turn from a blue-green to a yellow-green color. Here they decompose In mass. The resulting Intermediate products of decomposition may be highly undesirable, because of bad looks, four odors, bad tastes and toxins. Under these conditions the BOD may produce conditions not unlike raw sewage. VIII WHAT DO BLUE-GREEN ALGAE LOOK LIKE UNDER THE MICROSCOPE’ A A cross section of a typical cell would show an outside nonliving gelatinous layer surrounding a woody cell wall, which is bulging from turgor pressure from the cell (plasma) membrane, pushing the wall outward- ly. The protoplasm, contained with- in the plasma membrane, is divided into two regions. The peripheral pigmented portion called chroma- toplasm, and an inner centroplasm, the centroplasm contains chromatins, which is also known as in incipient nucleus or central body, containing chromosomes and genes. Structures (chromatophores or plastids) con- taining pigments have not been found in the blue-greens. The photosyn- thetic pigments are dissolved in the peripheral cytoplasm, which is known as the chromatoplasm. B A simple way to understand the cross section would be to compare it with a doughnut, with the hole represent- ing the colorless central body or incipient nucleus, which houses the chrornatoplasm, having the charac- teristic blue-green color from its dissolved photosynthetic pigments. DC WHAT CAUSES THESE FOUL-TO-SMELL UNSIGHTLY BLOOMS? When the protoplasts become aick or old they may develop a great number of “pseudovac- uoles’ filled with gas. These gas bubbles make the algae buoyant in such a way that they may “flower” or bloom by rising to the surface (planktonic, healthy blue-greens normally possess pseudovacuoles, which are here excepted). Soon they begin to stink because of the odors produced from putrefaction. The lack of dissolved oxygen during this period may effect other organisms. X ARE ALL BLOOMS PUTREFACTWE? No. Healthy blooms are produced by myraids of cells living near the surface of the water at times when cnvironmental conditions are especially favorable for them. Putrefactive blooms are usually from masses of algae undergoing degradation. XI WHAT ARE SOME OF THE MAJOR KINDS OF BLUE-GREENS? Most species of blue-greens may be placed into two major groups: the nonfilamentoug (coccoid) forms, and the filamentous forms. See the set of drawings following this treat- ment to get a graphic concept of the two groups. XII WHAT ARE SOME OF THE MORE DISTINCTIVE FEATURES OF BLUE- GREENS? A In comparing the blue-greens with other algae it is easier to tell what they do not possess than what they do. They do not have chromatophores or plastids, cilia, flageUa, organized nuclei, gametes, central vacuoles, chlorophyll-b. or true starch. B Many of the filamentous forms, es- pecially the Oscillatorlaceae, exhibit an unexplained movement. When the filamentous forms are surrounded by a gelatinous sheath the row of cells inside is called a trichome , and the trichome with its enclosing sheath is called a filament. There may be more than one trichome within a sheath. 6-2 ------- Blue-Green Algae Tru branching occurs when a cell of t}u series divides lengthwise and the oiit€ r-forrned cell add cells to form a true branch. However, two or more trichomes within a single sheath may be so arranged that though they appear to be branches, their cells actually have all divided in the same plane, and the trichomes have pushed out from growth to form false branch- as in Tolypothrix . C An occasional reticulated or bubbly appearance is referred to as pseudov- acuolation, and en mass imparts a pale, yellowish color to the algae. Under low powers these vacuoles appear dark, under higher magnifi- cations they are reddish. D Vegetative reproduction in addition to cell division for the unicellular forms, is by special kinds of frag- mentation. This includes the for- mation of hormogones , which are specifically delimited sections of trichomes, and are characteristic of some taxonomic entities. E Spores of three types are encountered. 1 Akinetes are usuaUy larger, non- motile, thick-walled resting spores. 2 Heterocysts appear like empty cell walls, but are filled with colorle8s protoplasm and have been occasion- ally observed to germinate. 3 Endospores, also called gonidla, are formed by a repeated division of the protoplast within a cell wall container. XIII WHAT A 1E SOME EXAMPLES OF BLUE- GREEN ALGAE A Anacystis ( Microcystis ) is common in hard waters. I Colonies are always free floating. 2 ‘I ’heir shapes may be roughly .phcrical or irregular, micro- scopic or macroscopic. 3 ‘ [ ‘he gelatinous matrix may be extremely transparent, easily broken up on preservation. 4 They frequently contain pseudov- acuoles. B Anabaena is an example of a fila- rnentous form. 1 Filaments may occur singly or in irregular colonies, and free floating or in a delicate nucous matrix. 2 Trichomes have practically the sanie diameter throughout, may be straight, spiral, or irregularly contorted. 3 Cells are usually spherical, or barrel shaped, rarely cy- lindrical and never discoid. 4 Heterocysts are usually the same shape but are slightly larger than the vegetative cells. 5 Akinetes are always larger than the vegetative cells, roughly cylindrical, and with rounded ends. 6 It may be readily distinguished from Nostoc by the lack of a firm gelatinous envelope. 7 It may produce an undesirable grassy, moldy or other odor. C Aphanizomenon is a strictly plank- tonic filamentous form. 1 Trichomes are relatively straight, and laterally joined into loose macroscopic free-floating flake- like colonies. 2 Cells are cylindrical or barrel shaped, longer than broad. 3 Heterocysts occur within the filament (i. e., not terminal). 4 Akinetes are cylindrical and relatively long. 6-3 ------- Blue-Green Algae SOME BLUE-GR EN 1. NP ittamontoi1i(cOccoid) hum Green Al ae: I : Anacy (Chr4 occus) X600. fl. Fitam.ntous blue-green algal: Trichornes of p! rulina . (X600). Trichomes of Arthrospi (X66Ur Jveg.tative )b.t.rocyst rn .‘ akinete J (spore) Anabaena (X825). ALGAE i 1 Ti Phornildium (with sheath) (X825). False branching Tolypothrlx (X375) Oscillatoria (without sheath) (X825) Hapalosiphon Prepared by Louis C. Williams (X375J Aquatic Biologist. Basic I ta, SEC. Coccochioris (Closocapsa) x600. Microcystis (x600). Polycysti . (X825) 6-4 ------- Blue-Green Algae 5 Often imparts grassy or nastur- tium-like odors to wa’ter. D Oscillatoria is a large and ubiquitous genus. 1 Filaments may occur singly or interwoven to form mats of indefinite extent. 2 Trichomes are practically the same diameter throughout. 3 Sheaths are usually distinct, fairly firm, and with a single trichome. REFERENCES 2 Trichomes are unbranched, cy- lindrical, and practically with- out sheaths. 3 Species with narrow trichomes have long cylindrical cells while those with broader tn- chomes have short broad cells. 4 No heterocysts or akinetes are known in Oscillatoria . It re- produces by fragmentation from hormongonia only. 5 Live species exhibit “osciliatorta” movements, which are oscillating. 6 Species of Oscillatoria may be readily distinguished from Lyngbya by the absence of a sheath. E Nodularia is an occasional producer of blooms. 1 Vegetative cells, heterocysts, and even the akinetes are broader than long. 1 Bartach, A. F. (ed.) En rironmental Requirements of Blue-Green Algae. FWPCA. Pacific Northwest Water Laboratory, Corvallis, Oregon. 111 pp. 1967. 2 Desikachary, T. V. Cyanophyta, Indian Council Agric. Res. New Delhi. 1G 9. 3 Drouet, Francis. Mxyophyceae. Chapter 5 in Edmondson. Freshwater Biology. p. 95-114. Wiley. 1959. 4 Drouet, Francis. Revision of the Classifi- cation of the Oscillariaceae. Monograph 15. Acad, Nat. Sci. Phil. 370 pp. 1968. 5 Jackson, Daniel F. (ed.) Algae, Man, and the Environment. Univ. Syracuse Press. 554 pp. 1968. This outline was prepared by L. G. Williams, Formerly Aquatic Biologist, Aquatic Biology Activities, Research and Development, Cincinnati Water Research Laboratory, FWPCA. 6—5 ------- GREEN AND OTHER PIGMENTED FLAGELLATES I INTRODUCTION A A flagellate is a free swimming cell (or colony) with one or more flagella. B Motile flagellated cells occur in most (not all) great groups of plants and animals. C Out main concern will be with “mature” flagellated algae. Ii THE STRUCTURE OF A PIGMENTED OR PLANT-LIKE FLAGELLATE A There is a well organized nucleus. B The flagellum is a long whip-like process which acts as a propeller. 1 It has a distinctive structure. 2 There may be one or several per cell. C The chlorophyll is contained In one or more chioroplasts. D Two or more cells may be associated In a colony. E Non-Motile Life history stages may be encountered. F Size is of little use in identification. G Pyrenoid bodies are often conspicuous. m The Euglenophyta or Euglena-Like algae (Figures 1-4) are almost exclusively Single celled free swimming flagellates. Nutrition may be holophytic, holozoic, or saprophytic, even within the same species. Referred to by zoologists as mastigophora, many animal like forms arc parasitic or commensalistic. Food reserves of plant-like forms are as paraniylin (an insoluble carbohydrate) and fats (do not respond to starch test). Thick walled resting stages (cysts) are common. “Metabolic movement” characteristics of some genera ( Euglena) . Eyespot usually present In anterior end, rarely more than one flagellum. A Euglena is a large genus with pronounced metabolic movement (Figure 1). 1 Cells spindle shaped 2 Single flagellum 3 Eye8pot usually present 4 Chioroplasts numerous, discoid to band shaped 5 E. sanguinea has red pigment. 6 E. viridis generally favors water rich lii organic matter. 7 E. gracilis 18 less tolerant of pollution. B Phacus cells maintain a rigid shape (Figure 2). 1 Often flattened and twisted, with pointed tip or tail end. 2 Cell wall (periplast) often marked with fine ridges. 3 P. pyrurn favored by polluted water. 4 P. pleuronectes relatively intolerant of pollution. C Trachelomonas cells surrounded by a distinct shell (lorica) with flagellum sticking through hole or collar (Figure 4). 1 Surface may be smooth or rough 2 Usually brown in color 3 Some species such as T. cerebea known to clog filters BI.MIC.cla.6c. 3.70 7-1 ------- Green and Other Pigmented Flagellates D Lepocincils has rigid naked cells with longitudinal or spiral ridges (Figure 3). 1 Cells uncompressed, elipsoidal to oval (in contrast to phacus) 2 Only two species with pointed tails 3 L te,cta often associated with waters of high organic content IV The Qilorophyta or grass green algae (Figures 5-9) are the largest and most varied group. Non-flagellated forms predominate but many conspicuous flagellates are included. Food reserves are usually stored as starch which is readily Identified lth Iodine. Usually two flagella of equal length are present. More planktonic forms are included than ui any other group, predominating In the late spring and early autumn. The cell Is typically surrounded by a definite wall and usually has a definite shape. Cell pigments closely resemble those of higher plants, but some have accessory pigments and a few forms have little or none. The ch]oroplaats always have a shape charac- teristic of the genus. The flagellated chiorophyta are contained in the Order Volvocales, the Volcocine algae. All are actively motile during vegetative phases. May be unicellular or colonial. AU have an eyespot near the base of the flagella. Colonies may range from a simple plate ( Gonium sociale ) to a complete hollow sphere ( Volvox spp ). A Chiamydomonas is a solitary free swimming genus (Figure 5). 1 SpecIes range from cylindrical to pearshaped. 2 Some apecle8 have a gelatinous sheath. 3 There are two flagella inserted close together. 4 Generally favored by polluted watera. B Carteria resembles Chiamydomonas very closely except that it has four flagella Instead of two. Generally favored by polluted water (FIgure 7). C Phacotus usually has free swimming blflageflate cells surrounded by biconcave envelopes resembling two clam shells. These are usually sculptured, dark colored, and Impregnated with calcium carbonate. 1 The eyespot ranges from anterior to posterior. 2 Several daughter cells may be retained within the old envelopes of the parent cell. 3 A clean water indicator. D Chiorogonjum Is a distinctive genus In which the cell Is fusiform, the tail end pointed, and the anterior end slightly blunt (FIgure 6). 1 The two flagella only about half as long as the cell. 2 The cell wall is rather delicate. 3 An eyespot usually present near the anterior end. 4 Favored by pollution. E Gonium colonies typically have 4 to 32 cells arranged in a plate (FIgure 8). 1 The cells are Imbedded In a gelatinous matrix. 2 Sixteen celled colonies move through the water with a somersault-like motion. 3 Four and eight celled colonies swim flagella end first. 4 Gonium pectorale is typically a plankton form. F Pandorina colonies range up to 32 celia, usually roughly spherical (Figure 9). 7—2 ------- Green and Other Pigmented Flagellates 1 Cells arranged in a hoLlow sphere within a gelatinous matrix. 2 Often encountered especially in hard- water lakes, but seldom abundant. 3 P. morurn may cause a faintly fishy odor. G Eudorina has up to 64 ceUs in roughly spherical colonies. 1 The cells may be deeply imbedded in a gelatinous matrix. 2 Common In the p]ankton of soft water lakes. 3 E. elegans is widely distributed. 4 May cause faintly fishy odor. H Pleodorina has up to 128 celle located near the surface of the gelatinous matrix. It is widespread in the United States. I Volvox rarely has less than 500 cells per colony. 1 Central portion of the mature colony may contain only water. 2 Daughter colonies form inBide the parent colony. 3 V. aureus imparts a fishy odor to the water when present In abundance. J Chiamydobotrys has “mulberry shaped” colonies, with biflagellate cells alternately arranged in tiers of four each. ( Spon iylomnorum has quadriflagellate cells). 1 There is no enveloping sheath. 2 c. stellata is favored by pollution. V The Pyrrhophyta includes principally the armored or dinoflagellates (Dlnophyceae) (Figures 14-16). This group is almost exclusively flagellated and is characterized by chromnatophores which are yellow-brown in color. Food reserves are stored as starch or oil. Naked, holozoic, and saprozoic representatives are found. Both “unarmored”, and “armored” forms with chromatophores are found to ingest solid food readily, and holozoic nutrition may be as important as holophytic. The great majority have walls of cellulose consisting of a definite number of articulated plates which may be very elaborate In structure. There is always a groove girdling the cell In which one flagellum operates, the other extends backward from the point of origin. Most of the dm0-flagellates are marine and some are parasitic. There are six fresh water genera of importance in this country. A Gymnodinum species are generally naked except for a few freshwater species. 0. brevis (marine) is a toxic form considered to be responsible for the “red tide” episodes in Florida and elsewhere. B Species of Gonyau].ax ( catanel]a and tamarensis ) are responsible for the paralytic shellfish poisoning. C Ceratlum is distinctive in that the anterior and posterior ends are con- tinued as long horns (Figure 16). 1 Seasonal temperature changes have a pronounced effect on the shape of the cells of this species. 2 c. hlrudinel]a in high concentration is reported to produce a “vile stench”. 7-3 Protect ,ø, Agency Corvalh Enviyo 90 1 Raseard L ab 2003 W 35thStr Cnr ia j ,g Oregon 9733Q ------- Green and Other Pigmented Flagellates I) I’urlcllriiuixi is a circular, oval, or ..tngular form, depending on the view (Figure 15). 1 Cell wall is thick and heavy. 2 Plates are usually much ornamented. 3 P. cinctum has been charged with a fishy odor. VI The Division Chrysophyta contains two classes which include flageUates, the Xanthophyceae or Heterokontae (yellow- green algae) and the Chrysophyceae (golden- green algae) (Figures 10-13). The third class, the diatoms (Bacillarieae or BaciUariophyceae), is not flagellated. A None of the Xanthophyceae are included in the present discussion. B The Chrysophyceae possess chroma- tophores of a golden brown color, usually without pyrenoids. Food reserves are stored as fats and leucosin. One or two flagella, if two, they may be of equal or unequal length. Internal silicious cysts may be formed. Tend to occur In relatively pure water. Both holozoic and holophytic types of nutrition are found. CertaIn minute forms considered to be highly sensitive to pollution. 1 Mallomonas is a solitary, free swimming genus with one flagellum (Figuze 13). a Covered with silicious plates, many of which bear long sillcious spines. b Tends to inhabit clear water lakes at moderate depths. c M. caudata imparts a fishy odor to the water. 2 Chrysococcue cells are minute, with two yellowish brown chromatophores and one flagellum. a Droplets of stored oil present b Lorica distinct c C. rules ceus a clean water form 3 C’iromullna has a single flagellum, may accumulate single large granule of leucosin at posterior end of cell (Figure 10). C. rosanoffil is a clean water indicator. 4 Synura is a bif]agellate form growing in radially arranged, naked colonies (Figure 11). a Flagella equal in length b Cells pyriform or egg shaped c S. uvella produces a cucumber or muskmelon odor 5 Uroglenopsia forms free swimming colonies of approximately spherical biflagellate cells embedded near the periphery of a roughly spherical gelatinous matrix. a Flagella are unequal in length. b U. americana may range up to .5 mm in diameter, and contain 1000 or more cells. c U. am. also causes strong fishy odor. 6 Dinobryon may be solitary or colonial, free floating or attached. Colonie8 are arborescent (Figure 12). a Cells attached to bottom of open roughly cylindrical lorica or sheath. b Two flagella of unequal length. c Conspicuous eyespot usually present. d Taxonomy of the group is involved. e D. sertularla may clog filters. f D. divergens may cause a fishy odor. 7-4 ------- ireen anu titner k’1 zinented k’Ia ellateS (fIg 1 - 13 !‘rom Lackey and Callaway) pf 1 2 Phacus 7 Lepoc inc 1 is GREEN EUGLENOIDS 9 Pandorina GREEN PHYTOVONADS Ch lorogonium - 10 11 Chromul ma 13 DInob yon Mallosonas YELLOW CHRYSOMONADS 14 Massart in 15 16 Cerat ium Per idinium YELLOW-BROWN DINOPLAGELLATES - Euglena Trachelomonas / Carter Ia ( Chiasydosonas 5 6 8 Gonius 7-5 ------- Green and Other Pigmented Flagellates PLANT FLAO LLATES (i HYTOMAST GINA) fLAGELLATES (MAsTIG0PHORA) Figure 17 Phylogenetic Family Tree of the Flagellates (from CaJaway and Lackey) V I I There are two distinctive groups whose systematic position is uncertain, the chioro- monads and the cryptomonads. Only one genus of the latter group is included here. A Rhodomonas may range from bright red through pale brown to olive green. 1 Celia compressed, narrow at the posterior end REFERENCES I Calaway, Wilson T. and Lackey, James B. Waste Treatment Protozoa Flage].lata. Series No. 3. Univ. Fla. 140 pp. 1962. 2 Gojdlcs, M. The Genus Euglena. Univ. of Wisconsin Press, Madison. 1953. 2 Two flagella of unequal length 3 R. lacustris a small form intolerant of pollution This outline was prepared by H. W. Jackson. Chief Biologist, National Training Center, MDS, Water Programs Operations, EPA, Cincinnati, OH 45268. CIIRYSOMONADINA CRYPTOMONADIN P HYTOMO 1ADINA ANIMAL FLA ELLATES (z OOMAS I ON 4 RHIZoMAsri IN P ROTC MON A DI NA POLYMASTIGINA EUGLENOIDThIA 7-6 ------- FILAMENTOUS GREEN ALGAE 1 I MANY OF THESE FORMS ARE VISIBLE C Specialized structures are present in TO THE UNAIDED EYE Borne filaments. A They may be several inches or even a foot 1 Some filaments break up into PIHU or more in length. In many cases they arc sections. not found as isolated filaments but develop in large aggregations to form floating or 2 Apical caps are present In others. attached mats or tufts. The attached forms are generally capable of remaIning 3 Replicate end walls are present In alive after being broken away from the some. substrate. 4 Some filaments are overgrown with a B Included in the group are some of the most cortex. common and most conspicuous algae In freshwater habitats. A few of them have 5 Attached filaments have the basal cell been given common names such as pond developed into a “hold fast cell” silk, green felt, frog-spawn algae, and (hapteron). stoneworts. III REPRODUCTION MAY TAKE PLACE U CHARACTERISTICS OF FILAMENTOUS BY SEVERAL METHODS ALGAE A Cell division may occur in all cells or A These algae are In the form of cylindrical in certain selected ones. cells held together as a thread (“filament”), which may be In large clusters or growing B Spores called akinetes may be formed. separately. Some are attached to rocks or other materials while others are free. C Zoospores (motile) and aplanospores They may be unbranched (“simple”) or (non-motile) are common. branched, the tips are gradually narrowed (“attenuated”) to a point. Some are D Fragmentation of filaments may occur. surrounded by a mucilaginous envelope. E Many kinds reproduce sexually, often B Each cell is a short or long cylinder with with specialized gamete forming cells. a distinct wall. The protoplast contains a nucleus which is generally inconspicuous. IV EXAMPLES OF FILAMENTOUS GREEN 1 ‘I’he plastid or chloroplast is the ALGAE ARE prominent structure. It contains chlorophyll and starch centers A Unbranched forms (“pyrenoids’), and varies in size, 5 Spirogyra shape, and number per cell. It may be pressed against the wall (“parietal”) 5 Mougeotia Zygnema or extend through the central axis of the cell (“axial”). Ulothrix Microspora 2 Clear areas of cell sap (“vacuoles”) are Trthonema generally present In the cell. Desmidium Oedogonium 1 Including a few yellow-brown and red algae. *Planktonic or occasionally planktonlc BI.MIC.cla. 14b.3.70 8-1 ------- Fi]amentous Green Algae H Branched forms E Together with other algae, they release oxygen required by fish, and for sell- Cladophora purification of streams. Pithopora Stigeoclonjum F They may produce a slime which inter- Chaetophora feres with some Industrial uses of water Draparnaldia such as In paper manufacture and in Rhizoclonium cooling towers. Audouthefla Bu]bochaete Nitelin VU CLASSIFICATION C Specialized and related forms A Ulotrichaceae Schizomerjs Ulothrix, Microspora, Horniidjum Comsopogon Batzacitospermurn B Cladophoracese Chara Lemariea C]adophora Pithophora, Rhizoclonjum Vaucheria C Chaetophoraceae V Habitats include the planktonic growths as Chaetoph , Stigeoclonjum, Draparnalcjja well as surface mats or blankets and benthic attached forms on rocks in riffles of streams, D Oedogeniaceae at the shoreline of lakes and reservoirs, concrete walls, etc. Oedogonjurn, Buibochaste A Attached forms may break ]oose to E Schizomeridaceae become mixed with plankton or to form floating mats. 1 Schizomerjs B Cladophora mats are a nuisant e on many F Ulvaceae beaches on the Great Lakes. Enteromorpha, Monostroma VI IMPORTANCE OF FILAMENTOUS G Zygnemataceae GREEN ALGAE Z nema. pirogyra, Mougeotla A They may cause clogging of sand filters, intake screens, and canals. H Desmidiaceae B They may produce tastes and odors In Desmidium i yalotheca water or putrid odor (also producing H 2 S which damage painted surfaces) when I Tribonemataceae washed ashore around lakes and reservoirs. Tribonema, BumUlerja C They may cause unsightly growths or interfere with fishing and swimming in J Characeae recreation areas. Chara, Nitella, To ypeUa D Some are useful as indicators of water quality in relation to pollution. 8-2 ------- 1 i. GkthJ N , F ILAMENTOUS OCXSONI 1•• ,i • i /(. j ?Ip l *1• I J. V as. 8-3 ------- Fi]amentoua Green Algae VIII IDENTIFICATION A Branching and attenuation are of primary importance. B P]astids shape, location and number per cell are essential. C Other characteristics include grouping of filaments, gelatinous envelope and special features such as “H” shaped fragments. REFERENCES 4 Pal, B. P., Kundu, B.C., Sundarathagam, V. S., and Venicataraman, G. S. Charophyta. Indian Coun. Agric. Res., New Delhi. 1962. 5 Soderatrom, J. Studies in Cladophora . Almquist, Uppsala. 1963. 6 Tilden. J. The Myxophyceae of North America. Minn. Geol. Surv. (Reprinted 1967, J. Cramer, Lehre, Germany) 1910. 7 Transeau, E.N. The Zygnernataceae. Ohio State Univ. Press. 1951. 1. Collins, F.S. 1909. The green algae of North America. Tufts College Studies, Scientific Series 2:79—480. Reprinted Hafner Pubi. Co., 1928 (Reprinted. 1968) Lew’s Books, San Francisco. 2 Farldi, M. A monograph of the fresh- water species of Dadophora and Rhizoclonlum . Ph.D. Thesis. University Microfilms, Ann Arbor. 3 Him, K. E. Monograph of the Oedogonlaceae. Hafner Pubi., New York. 1960. 8 Van der Hoek, C. Revision of the European species of Cladophora . Brili Publ, Leiden, Netherlands. 1963. 9 Wood, R. D. and Imahari, K. A revision of the Characeae. Volume I. Monograph (by Wood). Vol. II, Ionograph (by Wood & Imahari). 1964. This outline was prepared by C. M. Palmer, Former Aquatic Biologist, In C2iarge , Interference Organisms Studies, Micro- biology Activities, Research and Development, Cincinnati Water Research Laboratory, FWPCA. 8-4 ------- COCCOID GREEN ALGAE 1 I INTRODUCTION For the sake of convenience, the non-motile green algae are to be discussed in two sectiona’ those that tend to live as relatively discrete or free floating planktonic units, and those that tend to grow in masses or mats of material, often fi]amentous in nature, attached or free floating. II The green or “grass green” algae is one of the most varied and conspicuous groups with which we have to deal. The forms mentioned below have been artificially grouped for convenience according to cell shape. Botanists would list these genera in several different categories In the family “Chioro- phyceae.” These algae typically have a relatively high chlorophyll content, and the food reserves accumulated are typically starch. Thus these forms will usually give a typical black or deep purple color when treated with iodine. A Individual cells of the following genera are perfectly round, or nearly so. The first does not form organized colonies. In the next two the colonies themselves tend to be round, and in the last, the colonies are triangular or irregular, and the cells bear long slender spines. 1 Chiordlla cells are small and spherical to broadly elliptical. They have a single parietal chlorop]ast. This is a very large genus with an unknown number of similar appearing species. living In a great variety of habitats. Although often accumulating In great numbers, organized colonies are not formed. 1 Including miscellaneous yellow-brown algae. a Chiorella e].lipsoides in reported to be a common plankton form. b Chiorella pyrenoidosa and Chiorella vulgaris are often found in organically enriched waters. Indeed a dominance of Chiorella species is considered In some placçs to be an indication that a sewage stabilization pond is func- tioning to maximum capacity. c Chlore]la pyrenoidosa is reported as a filter clogger in water treat- ment plants. 2 Sphaerocystis colonies are free floating and almost always with a perfectly spherical, homogeneous gelatinous envelope. Up to 32 spherical cells may be included. Sphaerocystis scheoeteri , the only species, is of wide occurrence In the plankton of lakes and reservoirs. 3 Coe]astrum forms coenobial* colonies of up to 128 cells. Generally spherical or polygonal in shape--both cells and colony. Cells connected by protoplasmic processes of varying length. Coe]astrum microporurn is often reported in the plankton of water supplies. Not surrounded by gelatinous envelope as in Sphaerocystis . 4 Micractinium . The cells of this alga are spherical to broadly ellipsoidal and are usually united In irregular 4-celled coenobes. These in turn are almost always united with other coenobes to form multiple associations of up to 100 or more cells. The free face of *A coenobe is a colony in which the number of cells does not increase during the life of the colony. It was e8tablished by the union of several independent swimming cells which simply stick together and Increase in size. B!. MIC. cia. 1k. 3.70 9- 1 ------- Coccoid Green Algae each cell in a coenobe bear8 from one to seven very long slender setae or hairs. Micractinium pusillum. This is a strictly planktonic genus. B Individual cells of the following genera are elongate. In the first two they are relatively straight or Irregular and pointed. The next two are also long and pointed. but bent into a tight “C” shape (one in a gelatinous envelope, one naked). The last one ( Actinastrum ) La long and straight, but with blunt ends, and with the cells of a coenobe attached at a point. 1 Ankistrodesinus cells are usually long and slender, tapering to sharp point at both ends. They may be straight, curved, or twisted into loose aggregations. Anicistrodesmus falcatus is often found in the plankton In water supplies and is considered to be one of the forms Indicative of clean water. 2 Schroederia is a solitary, free floating alga. Cells are long and pointed at both ends. May be bent in various ways. Terminal points are continued as long slender spines which may be forked and bent back, or end as a plate. Of the three species reported in this country, Schroederia setigera has been reported in water supplies. 3 Selenautrum cells are pointed at both ends, and bent so that their tips approach each other. They tend to occur in groups of 4, 8, or 16, which may be associated with other groups to form masses of a hundred or more cells. There is no gelatinous envelope. Selenastrum gracile occurs in the plankton of water supplies. 4 KirchncrieLla . The cells of this genus are gcncraUy relatively broad, tapering to a sharp or rounded point at each end, and the whole cell bent into a C-shape. They usually occur In groups of four to eight in a broad, homogeneous, gelatinous matrix. Klrchneriella lunaris is known principally from the plankton. 5 Actlnastrum colonies or “coenobes” are composed of 4, 8, or 16 elongate cells that radiate in all directions from a common center. Actinastrum is a widely distributed plankton organism. There are two species: Actinastrum graciflirnum and Actthastrwn Hantzschil differ only in the sharpness of the taper toward the tips of the cells. The former has relatively little taper, and the latter, more. C Cells of the following genera are associated in simple naked colonies. The first has elongate cells arranged with their long axes parallel (although some cells may be curved). The last two are flat plate-like coenobes. Crucigenla has four-celled coenobes while Pedlastruni coenobes may be larger, appear plate-like, and are much more ornate. 1 Scenedesmus is a flat plate of elliptical to double ended pointed cells arranged with their long axes parallel. Coenobe consist of up to 32, but usually 4 to 8 cells. The number of cells in a coenobe may vary from mother to daughter colony. The appearance of cells may vary considerably with the species. a Scenedesmus blj iga, S. dimorphu 1 and S. guadricauda are common planktonic forms. b Scenedesinus guadricauda is also common in organicaUy enriched water, and may become dominant. c Scenedemus abundans is reported to impart a grassy odor to drinking water. 0-2 ------- Coccoid Green Algae 2 Crucigenia forms free f1oatin g four- celled coenobes that are solitary or joined to one another to form p]ate- like multiple coenobes of 16 or more cells. The cells may be elliptical. triangular, trapezoidal, or semi- circular in surface view. Cruci enia guadrata is a species often reported from water supplies. 3 Pediastrum . Colonies are free floating with up to 128 polygonal cells arranged in a single plane. There may or may not be open spaces between the cells. The exact arrangement of the cells seems to depend largely on the chance distribution of the original motile swarming zoaspores at the time the coenobe was formed. Peripheral cells may differ in shape from interior cells. a Pediastruni boryanum and , duplex are frequently found in the plankton, but seldom dominate. b Pedlastrum tetras has been reported to impart a grassy odor to water supplies. D Cells of the following Genera are slightly elongated. 1 Oocystis . The cells of Oocystis may be solitary, or up to 16 cells may be surrounded by a partially gellatinized and greatly expanded mother cell wall. Cells may be ellipsoidal or almost cylindrical, ceLl wall thai, no spines or other ornamentation. Oocystis borgei , for example, is of frequent occurrence ui the.plankton. 2 Dimorphococcus cells are arranged in groups of four, and these tetrads are united to one another in irregularly shaped free floating colonies by the branching remains of old mother-cell walls. Two shapes of cell are normally found in each tetrad (hence the name), two longer ovate cells end to end, and a pair of slightly shorter, C-shaped cells on either side. Dimorphococcus lunatus is a widely distributed plankton organism, sometimes reported In considerable numbers. E A distinctive group of green algae characterized by a median constriction dividing the cell into two geometrically similar halves is known generally as the “desmids.” ( Closterlum and Penium do not have this construction). Each half of the cell is known as a “semicell.” The nucleus lies in the “isthmus.” Extremes of ornamentation and structural, variety exist. Most are unicellular, but a few are filamentous or have the cells associated In shapeless colonies. They are found sparingly In the plankton almost everywhere, but predominate In acid waters. 1 Closterium is one of the exceptional genera without a median constriction. The cells are elongate, attenuated toward the tips but not sharply pointed, usually somewhat bent. a Cloeterium aciculare is a p]anktonlc species. b Closterium moniliforme is reported as a filter clogging organism. 2 Cosmarium is a large, poorly defined genus of over 280 species, many of which apparently lntergrade with other genera such as Staurastrum . In general, it can be said that Cosmarium species are relatively small, with a length only slightly greater than the width, and with a deep median con- striction. Shapes of the semicells may vary greatly. Although shallow surface ornamentation may occur, long spines do not occur. a Cosmarium botrytis is reported in plankton from water supply reservoirs. b Cosmarium portlanum is said to impart a grassy odor to water. c Other species have been reported to be sufficiently resistant to chlorine to penetrate rapid sand filters and occur in distribution systems in considerable numbers. 9-3 ------- Coccoid Green Algae 3 Mlcr eterias is relatively common, ornate. 4 Euastrum cells tend to be at least twice as long as broad, with a deeply con- stricted isthmus, and a dip or incision at the tip of each semicell. The cell wall may be smooth, granulate, or spined. Euastrum oblongum is reported as a planktonic species from water reser- voirs. It has also been noted as intolerant of pollution, and hence an indicator of clean water. 5 Staurastrum is the commonest of the desmids in the plankton of fresh waters; the genus contains upwards of 245 species in the United States alone. Inter- gradation with other genera 8uch as Cosmarium make it a difficult group to define. Most of the species are radially symmetrical, and almost all have a deeply constricted isthmus. The cell wall may be smooth, orna- mented, or splned In a variety of ways. Relatively long truncated processes extending from the cell body in symmetrical patterns are common. a Staurastrum polymorphum 18 a typical planktonic form. b Staurastrum punctu]atum is reported as an Indicator of clean water. c Staurastrum paradoxicum causes a grassy odor in water. 111 A type of “green” alga known as “golden green” (Xanthophyceae) is represented in the plankton by two genera. In these a]gae there is a predominance of yellow over green pig- ments, hence frequently imparting a yellowish or golden tint to the cell. Reserve food material is stored as oil and leucosin, rather than as starch, hence giving a negative test with iodine In most cases. 1 The plant body is a free floating colony of indefinite shape, with a cartiiag- inous and hyaline or orange-colored envelope; surface greatly wrinkled and folded. 2 Individual cells lie close together, in several aggregates connected in reticu]ar fashion by strands of the colonial envelope. 3 The envelope structure tends to obscure cell structure. Considerable deep orange colored oil may collect within the envelope, outside of the cells, obscuring cell structure. B Qphiocytjum capitatum like Botryococcus, , is widely distributed, but seldom abundant. 1 Both ends of cylindrical ceU are rounded, with a sharp spine extending therefrom. 2 Many nuclei and several chioroplasts are present. REFERENCES 1 Palmer, C. M. Algae in Water Supplies. Government Printing Office. PHS Publication No. 657. 1959. 2 Smith, G. S. Phytoplankton of the Inland Lakes of Wisconsin. Part I. Bulletin No. 57, Scientific Series No. 12. 1920. A Botryococcus braunli is a widely dis- tributed plankton alga, though it is rarely abundant. This outline was prepared by H. W. Jackson, Chief Biologist, National Training Center, Water Programs Operations, EPA, Cincinnati, OH 45268. 9—4 ------- DIATOMS I GENERAL CHARACTERISTICS 4 Internal shelves (“septae”) extending longitudinally or transversely. A Diatoms have cells of very rigid form due to the presence of silica in the waU. They contain a brown pigment in addition to the II REPRODUCTION chlorophyll. Their walls are ornamented with markings which have a specific pattern A The common method is by cell division. for each kind Two new half cells are formed between the halves of the parent cell. I The ce1l often arc isolated but others are in filaments or other shapes of B Awcospores and gametes may also be colonies, formed. 2 The protoplast contains normal cell parts, the moat conspicuous being the Ill EXAMPLES OF COMMON DIATOMS: plastids. No starch is present. A Pennate, symmetrical: B Cell shapes include the elongate (“pennate”) Navicula and the short cylindric (“centric”) one view Pinnularia of which is circular, ynedra Nitzschia I Pennate diatoms may be symmetrical, Diatoma transversely unsymetrical, or longitudi- Fragilaria nally unsymmetrical, Tabeilaria Cocconels C Wall is formed like a box with a flanged cover fitting over it. B Pennate, unsymmetrical: I “Valve” view is that of the top of the Gothphonema cover or the bottom of the box. Surirella Cymbella 2 “Girdle” view is that of the side where Achnanthes flange of cover fits over the box. Asterionella Meridion 3 End view is also possible for pennate types. C Centric: D (‘oil markings include çyc lotella § phanodiscus I Raphe or false raphe extending Sira lotigit iid inally. 2 Striations whieh arc linec of pores IV Habitats include fresh ar salt water. Both xtending fiow the area of the raphe to planktonic and attached forms occur, the latter the margin. Coarse ones are “costae”, often are broken loose. They may be attached by stalks or by their slimy surface. 3 Nodules which may be terminal and central. B!. MIC.cl,i. lOa 8.69 10—1 ------- Diatoms A Many diatoms are more abundant in late autumn, winter, and early 8pring than in Synedra the warmer sea8on. Asterionelj,a B The walls of dead diatoms generally remain 2 Achnanthjneae, Group with cells undecomposed and may be common in water. having one false and one true raphe. Many deposits of fossil diatoms exist. a Representative genera: V Importance of diatoms is in part due to Cocconeis their great abundance and their rigid walls. Achnanthes A They are the most Important group of orgarnsms causing clogging of sand filters. 3 Naviculineae. True raphe group with raphe in center of valve. B Several produce tastes and odors in water, including the obnoxious fishy flavor, a Representative genera: Navicula C Mats of growth may cause floors or steps Pinnularia of swimming poois to be slippery. Stauroneis PIe u rosigma D They may be significant in determining Ampluprora water quality in relation to poUution. Gomphonema Cymbella E They release oxygen into the water. Epithemia VI Classification. There are several thou- 4 Surirelllneae. True raphe group with sand species of diatoms. Only the most corn- raphe near one aide of valve. mon of the freshwater forms are considered here, a Representative genera: A Centrales Group Nitzsclua yrnatopleura 1 Representative genera’ Surirella Campylodiscus lotella Stephanodisc us Melosira VII IDENTIFICATION OF DIATOMS Rhizosolenia Bidduiphia A Some genera are easily recognized by their distinctive shape. B Pennals Group B Many genera and most species can be determined only after diatoms are freed I Fragilarineae. The false raphe group, of their contents and observed under the high magnification of an oil immersion Representative genera lens of the compound microscope. Tabellaria C Contents of the cell are generally not Meridion used in identification. Only the char- Diatoma aracterjstjcs of the wall are used, 10—2 ------- Diatoms D For identification of genera, mQSt im- portant features include I Cell shape, and form of colony 2 Raphe and false raphe 3 Striations 4 Septa E For identification of species, measure- ments involving the number of striae per 10 microns, the direction of the striae and many other characteristics may be needed. REFERENCES I Boycr, C.S. The Diatomaceae of Philadelphia and Vicinity. J. B. Lippin- cott Co. Philadelphia. 1916. p 143. 2 Boyer, C. S. Synop8iS of North America Diatomaceae. Part8 I (1927) and II (1928). Proceedings of the Academy of Natural Sciences. Philadelphia. 3 Elmore, C. J. The Diatoms of Nebraska. University of Nebraska Studies. 21: 22-215. 1921. 4 Hohn, M. H. A Study of the Distribution of Diatoms in Westcrn New York State. Cornell University Agricultural Experimental Station. Memoir 308. pp 1-39. 1951. 7 Patrick, Ruth and Reimer, Charles W. The Diatoms of the United States. Vol. 1 Fragilariaceae, Eunotlaceae, Achnanthaceae, Na’v-iculaceae. Monog. 13. Acad. Nat. Sd. Philadelphia. 888 pp. 1966. 8 Smith, G.M. Class Bacillariophyceae. Freshwater Algae of the United States. pp 440-5 10, 2nd Edition. McGraw Hill Book Co. New York. 1950. 9 Tiffany 1 L. H. and Britton, M. E. Class BaciU.ariophyceae. The Algae of illinois. pp 2 14-296. University of Chicago Press. 1952. 10 Ward, H. B. and Whipple. G. C. Class I, Bacillariaceae (Diatoms). Fresh- water Biology. pp 17 1-189. John Wiley & Sons. New York. 1948. 1 Weber, C. I. A Guide to the Common Diatoms at Water Pollution SurvejUance System Stations. FWPCA. CincinnatI. 101 pp. 12 Whipple, G. C.. Fair, G. M., and Whipple, M.C. Diatomacene. Microscopy of Drinking Water. Chapter 21. 4th Edition. John Wiley & Sons. New York. 1948. 1966. 5 Pascher, A. BaciUariophyta (Diatomeae). Heft 10 in Die Susswasser-FlOra Mitteleuropas, Jena. 1930. p 466. 6 Patrick, R. A Taxonomic and Ecological Study of Some Diatoms from the Pocono Plateau and Adjacent Regions. Farlowia. 2 143—22 1. 1945. This outline was prepared by C. M. Palmer, Former Aquatic Biologist, In Charge, Interference Organisms Studies, Microbiology Activities, Research and Development, Cincinnati Water Research Laboratory, FWPCA. 10-3 ------- FILAMENTOUS BACTERIA I INrRODUCTION ‘I’here are a number of types of filamentous hacteria that occur in the aquatic environment. They inc]uth the sheathed sulfur and Iron bacteria such as Begg [ atoa. Crenothrix and Sphaeroiilus , the actinomycetes which are inic Uu1ar microorganisms that form chains of cells with .pccIal branchings, and ( aUioneUa , a unicellular organism that secret s a long twisted ribbon-like stalk. These tilamentous forms have at times treated serious problems in rivers, reservoirs, wells, and water distribution systems. H BEGCIATOA fleggiatoa is a sheathed bacterium that grows as a long filamentous form. The flexible filaments may be as large as 25 microns wide and 100 microns long (Figure 1) Begglatos Iba 2-iSp X up to 1.000p Transverse separations within the sheath indicate that a row of cells is included in one sheath The sheath may be clearly visible or so slight that only special staining will indicate that it is present. The organism grows as a white slimy or felted cover on the surface of various objects undergoing decomposition or on the surface of stagnant areas of a stream receiving sewage It has also been observed on the base of a trickling filter and in contact aerators. It is most commonly found in sulfur springs or polluted waters where H 2 S is present. I3eggiatoa is distinguished by its ability to deposit sulfur within its cells; the sulfur deposits appear as large refractile globules. (Figure 2) amente of Beg la1oa containing granules of sulphur Vhen H S is no longer present in the environ- ment, t e sulfur deposits disappear. Dr. Pringsheim of Germany has recently proved that the organism can grow as a true autotroph obtaining all its energy from the oxidation of H 2 S and using this energy to fI.x CO into all material It can also use cer ain organic materials If they are present along with the H 2 S Faust and Wolfe. and Scotten and Stokes have grown the organism in pure culture in this country Beggiatoa exhibits a motility that is quite different from the typical flagellated motility of most bacteria, the filaments have a flexible gliding motion. FIgure 1 )tA.Ba 12.72 11—1 ------- Vi]amentous Bacteria The only major nuisance effect of Beggtatoa known has been overgrowth on trickling filters i eceiving waste waters rich in H 2 S. The normal microflora of the filter was suppressed and the filter failed to give good treatment. Removal of the H 2 S from the water by blowing air through the water before It reached the filters caused the slow decline of the i3eggtatoa and a recovery of the normal microflora. Begglatoa usually indicates polluted conditions with the presence of H 2 S rather than being a direct nuisance. Ill ACTINOMYCETES AND EARTHY ODORS IN WATER A. tinomycetes are unicellular microorganisms 1 micron In diameter, filamentous, non- sheathed, branching monopodlaUy, and reproduced by fission or by means of special coriidia. (Figure 3) Their filamentous habit and method of sporulation is reminiscent of fungi However, their size, chemical composition, and other characteristics are more similar to bacteria. (Figure 4) dull end powdery smooth and mucoid Plgur. 4 Egg albumin leolation plate A’ an actinomycete colony. and B’ a bacterial colony These organisms may be considered as 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 caune of earthy odors in some drinking waters (Romano and Safferman, Silvey and Roach) and in earthy smnel]lng substance has been isolated from several members of the group by Gerber and Lechevalier. Safferman and Morris have reported on a method for the “Isolation and Enumeration of Actinomycetes Related to Water Supplies.” But the actino- mycetes are primarily soil microorganisms and often grow in fields or on the banks of a river or Jake used for the water supply. Although residual chlorination will kill the organisms in the treatment plant or distribution U C Appesranc. Appearance Figurs 3 PlIsminte of Actlnomycet•s 1 1..2 ------- ruamentous tiacteria ysteni, the odors often are present before the watt r enters the plant. Use of perman- gana e oxidation and activated carbon filters have been most successful of the methods tried to remove the odors from the water. Control procedures to prevent the odorous material from being washed into the water supply by rains or to prevent possible develop- ment of the actinomycetes in water rich in decaying organic matter is still needed. IV FILAMENTOUS IRON BACTERIA The filamentous iron bacteria of the Sphaerotilus- Leptothrlx group, Crenothrlx , and Ga]lionel]a have the ability to either oxidize rnanganous or ferrous ions to manganic or ferric salts or are able to accumulate precipitates of these compounds within the sheaths of the organisms. Extensive growth. or accumulations of the empty, metallic encrusted sheaths devoid of cells, have created much trouble in welle or water dis- tribution system.. Pumps and back surge valves have been clogged with muses of material, taste and odor problem . hive 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 it. special morphology. Dr. Wolfe of the University of Illinois has published photomicrographs of the organism. (Figure 5) Organisms of the Sphaerotilus- L.eptothrix group have been extensively studied by many investigators (Donderoet. al., Dondero, Stokes, Waltz and Lackey, Mulder and van Veen, and Ambcrg and Cormack.) Under different environmental conditions the mor- phological appearance of the organism varies. 1’he usual form found in polluted streams or bulked activated sludge ii Sphaerotilus natans . (I”igure 6) Pigurs 5 Chr.mothrix polyspora cells are very vsriabl in size trorn small cocci or polyspores to cells 3x12 a 3-8 X 1.2 - i.ep cells Figur. S Sphaerotlius natans 11”3 ------- F’ilavncntous Bacteria l hib is a sheathcl bacterium consisting of long, unhranched Illaments, whereby individual od-shaped bacterial cells are enclosed in a linear order within the sheath. The individual cells arc 3-8 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, or milk processing plants. In these conditions, it appears 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 some rivers large masses of Sphaerotilus break loose and clog water Intake pipes or foul fishing nets. When the cells die, taste and odor problems may also occur in the water Amberg, Cormack, and Rivers and McKeown 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 such an extent that the growth of Sphaerotilus is no longer favored in the river. Sphaerotflus grows well at cool temperatures and slightly low DO levels in streams receiving these wastes and domestic sewage. Growth is slow where the only nitrogen present is inorganic nitrogen, peptones and proteins are utilized preferentially GaLlioneUa is an iron bacterium which appears as a kidney-shaped cell with a twisted ribbon- like stalk emanating from the concavity of the cell. Gallionefla obtains its energy by oxidizing ferrous iron to ferric Iron and uses only CO 2 and inorganic salts to form all of the cell material, it is an autotroph. Large masses of Gallionella may cause problems in wells or accumulate in low-flow low- pressure water mains. Super chlorination (up to 100 ppm of sodium hypochiorite for 48 hours) followed by flushing will often remove the masses of growth and periodic treatment will prevent the nuisance effects of the extensive masses of Gallionella . (Figure 7) REFERENCES Begglatoa 1 Faust, L. and Wolfe, R. S. Enrichment and Cultivation of Begglatoa Ala. Jour. Bact , 81:99-106. 1961. 2 Scotten, H. L. and Stokes, J. L. Isolation and Properties of Begglatoa . Arch Fur Microbiol. 42:353-368. 1962. 3 Kowallik, U. and Pringsheim, E.G. The Oxidation of Hydrogen Sulfide by Beggiatoa . Amer. Jour of Botany. 53•80l805 1986. Actlcwmycetes and Earthy Odors 4 Silvey, J.K.G. et.al. Actinomycetes and Common Tastes and Od3rs. JAWWA, 42•1018-1026, 1950 5 Safferman, R. S. and Morris, M E. A Method for the Isolation and Enumeration of Actinomycetes Related to Water Supplies. Robert A. Taft Sanitary Engineering Center Tech. Report W-62-10. 1962. Figure 7 GellonellA forrugIr ea 05 X 07 -1 Celle 11—4 ------- Filamentous Bacteria 6 Gerber, N.N. and Lechevaller, H.A. Geosmin, an Earthy-Smelling Substance Isolated from Actinomycetes. Appi Microbiol. l3 135-938. 1965. Filamentous Iron Bacteria 7 Wolfe. R.S. Cultivation, Morphology, and Classification of the Iron Bacteria. JAWWA. 501241-1249. 1958. 8 Kucera, S. and Wolfe, R. S. A Selective Enrichment Method for GaU.tonella ferruginca . Jour. f3acteriol. 74344- 349. 1957. 9 Wolfe, I L S. Observations and Studies of Crenothrix polyspora . JAWWA, 52915-918. 1960. 10 Wolfe, R. S. Micrqbiol. Concentration of Iron and Manganese in Water with Low Concentrations of these Elements. JAWWA. 52 1335- 1337. 1960. 11 Stokes, J. L. Studies on the Fi]amentous Sheathed Iron Bacterium Sphaerotilus natans. Jour. Bacteriol. 67:278-291. 1954. 12 Waita, S. and Lackey, J. B. Morphological and Biochemical Studies on the Organism Sphaerotilus natans . Quart. Jour. Fin. Acad. Sci. 21(4):335-340. 19 ’ 8. 14 Don:Iero, N. C. Sphaerotilus , Its Nature and Economic Significance. Advances Appl. Microbiol. 377-107. 1961. 15 Mulder, E.G. and van Veen, W. L. Investigations on the Sphaerotilus- Leptothrix Group. Antonie van Leewenhoek. 29:121-153. 1963. 16 Amberg, H.R. and Cormack, J.F. Factors Affecting Slime Growth in the Lower Columbia River and Evaluation of Some Possible Control Measures. Pulp and Paper Mag. of Canada. 6l:T70-T80. 1960. 17 Amberg, H.R., Cormack, J.F. and Rivers, M.R. Slime Growth Control by Intermittarit Discharge of Spent Sulfite Liquor. Tappi. 45:770-779. 1962. 18 McKeown, J.J. The Control of Sphaerotilus natans . md. Water and Wastes. 8:(3) 19-22 and 8:(4)30-33. 1963. 13 Dondero, N.C., Philips, R.A. and Itenkelkian, H. Isolation and Preservation of Cultures of Sphaerotilus . Appi. Microbiol. 9:219-227. 1961. This outline was prepared by R. F. Lewis, Bacteriologist, Advanced Waste Treatment Research Laboratory, NERC, EPA, Cincinnati, OH 45268. 11—5 ------- i’UNG1 AND THE “SEWAGE FUNGUS” COMMUNITY I INTRODUCTION III ECOLOGY A Description Fungi are heterotrophic achylorophyllous plant-like organisms which possess true nuclei with nuclear membranes and nu- cleoli. Dependent upon the species and in some instances the environmental conditions, the body of the fungus, the thallus, varies from a microscopic single cell to an extensive plasrnodium or mycolium. Numerous forms produce macroscopic fruiting bodies. B Life Cycle The life cycles of fungi vary from simple to complex and may include sexual and asexual stages with varying spore types as the reproductive units. C Classification Traditionally, true fungi are classified within the Division Eumycotina of the Phylum Mycota of the plant kingdom. Some authorities consider the fungi an essentially monophyletic group distinct from the classical plant and animal kingdoms. II ACTIVITY In general, fungi possess broad enzymatic caparities. Various species are able to actively degrade such compounds as complex polysaccharides (e. g., cellulose, chitin, and glycogen), proteins (casein, albumin, keratin), hydrocarbons (kerosene) and pesticides. Most species possess an oxidative or microaerophilic metabolism, but anaerobic catabolism is not uncommon. A few species show anaerobic metabolism and growth. A Distribution Fungi are ubiquitous in nature and mem- bers of all classes may occur in large numbers In aquatic habitats. Sparrow (1968) has briefly reviewed the ecology of fungi in freshwaters with particular emphasis on the zoosporic phycomycetes. The occurrence and ecology of fungi in marine and estuarine waters has been examined recently by a number of in- vestigators (Johnson and Sparrow, 1961; Johnson, 1968; Myers, 1968; van Uden and Fell, 1968). Wm. Bridge Cooke, in a series of in- vestigations (Cooke, 1965). has estab- lished that fungi other than phycomycetes occur in high numbers in sewage and polbitedwaters. His reports on organic pollution of streams (Cooke, 1961; 196?) show that the variety of the Deuteromy- cete flora is decreased at the immediate sites of pollution, but dramatically in- creased downstream from these regions. Yeasts, in particular, have been found in large numbers in organically enriched waters (Cooke, et al. , 1960, Cooke and Matsuura , 1963; Cooke, 1965b; Ahearn. et a!. . 1968). Certain yeasts are of special interest due to their potential use as “Indicator” organisms and their abihty to degrade or utilize proteins, various hydrocarbons, straight and branch chained alkyl-benzene sulfonates, fats, metaphosphates, and wood sugars. B Relation to Pollution BI. FLJ.6a.5.71 12—1 ------- Fungi C “Sewage FUngUS Community (Plate I) A few microorganlbrns have long been termed sewage fungi. “ The most common microorganisms inc’uded In this group are the iron bacterium Sphaerotilus natans and the phycomy- cete Leptomitus lacteus. 1 Sphaerotllus natans is not a fungus; rather it is a sheath bacterium of the order chiamydobactertales. This polymorphic bacterium occurs commonly in organically enriched streams where It may produce extensive slimes. a Morphology Characteristically. S. natans forma chains of rod shaped cells (1. 1 -2. x 2.5 - l? ) within a clear sheath or tn- chome composed of a protein- polysacchartdae-lipid complex. The rod cells are frequently motile upon release from the sheath; the flagella are lopho- trichous. Occasionally two rows of cells may be present in a single sheath. Single tn- chomes may be several mm in length and bent at various angles. Empty sheaths, ap- pearing like thin cellophane straws, may be present. b Attached growths The trichomea are cemented at one end to solid substrata such as stone or metal, and their cross attachment and bending gives a superficial similarity to true fungal hyphae. The ability to attach firmly to solid substrates gives S. natans a selective advantage in the population of flowing streams. For more thorough reviews of S.natans see Prigsheim(1949) arid Stokes (1954). 2 Leptomttus lacteus also produces extensive a limes and fouling flocs in fresh waters. This species forms thalli typified by regular constrictions. a Morphology Cellulin plugs may be present near the constrictions and there may be numerous granules in the cytoplasm. The basal cell of the thallus may possess rhizoids. b Reproduction The segments delimited by the partial constrictions are con- verted basipetally to aporangia. The zoospores are dip]anetic (t. e., dimorphic) and each possesses one whiplash and one tinsel flagellum. No sexual stage has been demonstrated for this species. c Distrthution For further information on the distribution and systematics of L. lacteus see Sparrow(1960), Yerkes (1966) and Emerson and Weston (1967). Both S. riatans and L. lacteus appear to thrive in organically enriched cold waters (5°-22°C) and both seem incapable of extensive growth at temperatures of about 30°C. d Gross morphology Their metabolism is oxidative and growth of both species may appear as reddish brown flocs or stringy slimes of 30 cm or more in length. e Nutritive requirements Sphaerotilus natans is able to utilize a wide variety of organic compounds, whereas L. lacteus does not assimilate simple 12—2 ------- Fung PLATE I “SEWAGE FUNGUS” COMMUNITY OR “SLIME GROWTHS” (Attached “filamentous” and slime growths) Zooiloea phaeroti1ua natans S. 4 Beggiatoa alba 3 BACTERIA 7 Leptomitus lacteus ‘a’. FUNGI ri /DO ic- PROTOZOA 5 Fuaariurnagueducturn Epistylia 8 9 Carchesium 10 ç percu1aria 12—3 ------- PLATE II IU PRESENTATIVE FUNGI . Figure - Fusarium equ .aeJ.ic uw ’. [ LU (Rluid.er an ’ 1 Ralusiltorat ) Sactailo Microconidia (A) produced from phialides a. in CepIsoit ’- aponism, remaining in slime ball.. Macroconidis (B). with to ,.cvrraj cru,.. walk.. produced from collared phial- idea. I)r.wn from culture. FIgure 3 C., 0 srtrhiun ,andi4um link a Persoon Myrelium with short cells and artbro.porra. ‘Young by- pbs (A): and mature .rthro- spore, (B). Drawn from rul- lure. FIgure 5 Arhiva ame r i cana humphrey Ooogonium with three no- spores (A); young soospor- .ngtum with ileilmited zoo- spec. (B); and ,o.aporangia (C) with released inoapoava that remain encysted in dun- tori at the mouth of the dl i. charge tube. Drawn from cal- tuft. FIgurs Z Leptomise. Igcteui (Roth) Agardh Calls ad the byphac slow- ing constrictions with c.llulin plug.. In one cell large zos spore. have loan daRseltuL Redrawn from Color, 192& F lgit r s4 8. Zo.pisggaa injidianj Mycellum with hyphal pegs (A) on which rotiferi will become impaled; gcmmae (B) produced as oceidia on abort hyphal branches; and roth or impaled on hyphal peg (C) from which hypbae have pown Into the rotifer whose shell will be discarded after the contents are consumed. Drawn from cultuEe. Ikruiuey, 1i liuin nuiriun to H 6.JB. Fioune 7 I!aplonporidinm costnI . A—mnture .po ,i-; l3—..——cnrly l)I%St11O(lItIlt. Figures 1 through 5 from Cooke; F’igures 6 and 7 from Gaitsoif. 12—4 ------- Fun f sugars and grows most luxuriantly in the presence of organic nitrogenous wastes. 3 Ecological roles Although the “sewage fungi” on occasion attain visually noticeable con’entrations , the less obvious populations of deuteromycete8 may be more important in the ecology of the aquatic habitat. Investigations of the past decade Indicate that numerous fungi are of primary importance in the mineralization of organic wastes; the overall significance and exact roles of fungi in this process are yet to be estal,lished. D Predacious Fungi 1 Zoophagus insidians (Plate II, Figure 4) has been observed to impair 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 predaclous upon nernatodes. Loops rather than “pegs” are used In snaring nematodes. IV CLASSIFICATION In recent classification schemes 1 classes of fungi are distinguished primarily on the basis of the morphology of the sexual and zoosporic stages. In practical schematics, however, numerous fungi do not dernon8trate these stages. Classification must therefore be based on the sum total of the morphological and / or physiological characteristics. The extensive review by Cooke (1963) on methods of isolation and classification of fungi from sewage and polluted waters precludes the need herein of extensive keys and species illustrations. A brief synopsis key of the fungi adapted in part from Alexopholous (1962) is presented on the following pages. This outline was prepared by Dr. Donald G. Ahearn, Professor of Biology, Georgia State College, Atlanta, GeorgIa 30303. PLATE II (Figure 4) 12—5 ------- Fungi si V 10 liii t 1A (( lit I A\A ur IIJN(,l I), I no . II alli lacking oi,aio phase a free living Plasmodiunt ‘.uh-phyiurii Myxomycotma (true slime molds) Class Myxomycetes I tl Is II ‘ma I ly well ii .- lined soniat ic phase n0 a free - living Plaimodium (true ungi) Sub-phylum Eumycotina Hyphal filaments usually coenoclytic. rarely septate, sex cells when present forming nosporeli or /ygospores. aquatic species propagating asexually by zoospores. terrestrial speci es by Loospores, sporangiospores conidia or conidia-like sporangia ‘Phycomycetes” 3 I he phycomycetes Ire generally considered to include the most primitive of the true fungi As a whole they encompass a wide dIversity of forms with some showing relation- ships t . lb. flagellates, while others tiosely resemble colorless algae, and still others ar , t rue molds 1 he vegetative body Phallus) may be non-specialized and entirely con- V. rh .1 intO a reproductive organ (holocarpic) or it may bear tapering rhizoids, or be itivi dial and very extensive The outstanding characteristics of the thallus is a tendency Ii, Ii . nonseptate and in most groups. multinucliate, cross walls are laid down in vigorously ing material only to delimit the reporductive organs. The spore unit of nonsexual re- ductaon 16 borne in a sporangium, and, in aquatic and semiaquatic orders, is provided iih a single post. rii,r or anterior flagellum or two laterally attached ones Sexual activity in the phycotnyretce characteristically results in the formation of resting spores I) ilyplial filaments i hen present septate, o Ithout Loospores, with or without sporangla. usually iIh conida. sexual reproduction absent or culminating in the formation of asci or hasiitia . 8 ( l l lagettat, it .. its characteristically produced 4 P Flag. bird .-, (Is lacking or rarely produced 7 4 (3) Motile ells tiniliagellat. 5 4’ Motil, elI.. t)iflagellate 6 S 4) /o.ispi.res post. riorly uniflagellate. formed inside the sporangiurn class . Chytridiomycetes The Chyti idiomycetee produce asexual zoospores with a single posterior whiplash flagellum The thallue Is highly variable the most primitive forms are unicellular and holocarpic anti in their early stages of development are plasmodial (lack cell walls), more advanced forms develop rhizoids and with further evolutionary progress develop mycelium The principle chemical component of the cell wall is chum, but cellulose is also present (‘hytrids a. typically aquatic organisms but may be found in other habitats Some species are i hitinolytii- and/or keratinolytic Chytridi may be isolated from nature by baiting (e g h,’in 1 , 6, mIs or pine pollen) Chytrids occur both in marine and fresh water habitats and are of ifliflC c coiiolFiic importance due to their parasitism of algae and animals The genus D.riiiuey..tt,liiirn niny he provisionally grouped with the chytrids Species of this genus cause si I i O i i’ i I pideinks of oysters and marine and fresh water fish 5’ Loospores anteriorly uniftagellate, formed inside or outside the sporangium , class Hyphochyt ridiomycetes These fungi are aquatic (fresh water or marine) chytrid.like fungi whose motile cells possess a single anterior flagellum of the tinsel type (feather-like) They are parasitic on algae and fungi or may be saprobte Cell walls contain chitin with some species also demon- sI rating cellulose etintent Little information is available on the biology of this class and at present it is muted to less than LO species 4’) Flagella flea ny • qual. one whiplash the other tin..el class Oomycetes A numb. r of representatives of the Oomycetes have been shown to have cellulosic cell walls 1 he n.yceliom is coenocytic. branched and well developed in most cases I he sexual pro. cas i esuli .i in the formation of a resting spore of the oogamous type i e . a type of fe rtm Ii, at ion in who h two hete rogametangia come in contact and fuse their contents through a pore or tub. I he thalli in this class range from unicellular to profusely branched fil.,mrntous types Must forms are eucarpic, oospores are produced throughout the class ear. p1 in the more highly advanced species Certain species are of economic Importance due to their destruction of food crops (potatoes and grapes) while others cause serious diseases of fish I c g Saprol gina parasitica ) Members of the family Saprolegniaceae are the common 12—6 ------- Fungi taMer m,,tda anal are .ainuui th, no.et ubiquitous fungi in nature Th order Lagentdiales ma tail, only a less sp cia ’s Satlia Ii arc parasitic- ian algae small animals and other aquatic tile I Ii, a .oaI l .llic St ructcit c ’s tat llama acon arc holocarpic and c’ndobmotir rh sewage fungi nra’ classitmed in the order Leptomitales Fungi of this order are characte rized by the formation ‘1 r, fractile constrictions a .ellulin plugs occur throughout the thalli or. at least at iii. ha’o ‘ ‘I hyplmac or to cut oil reproductive structures Lcptomltun lacteus may produce rather extensive fouling (Inca or slime. in organically enriched tcateru. Flagella of unequal size 1)0th uhaplash class Plasmodiophoromycetes Members of this class are obligate t’ndoparasites of vascular plants. algae, and fungi The thallu. onsists of a plasmodmum ahmch deva lops taithmn the host cclii Nuclear division at some stages ol the life cycle is of a type found in no other fungi but known to occur in protn,oa /ouspcarangia which arise directly from the plasmodium bear zoospores with two unequal .ini, nor lalgella The ci II walls of the fungi apparently lack cellulose 7 II’ ) Mainly saprohic sex cell when present a zygospore class 7 gatmycetes ‘1 his a laip has acell d vcloped mycelium with septa developed in portion, of th older hyphaa actively growing hyphae are normally non-aeptate The aiexual spores am non’ntotilc sporangiospores taplanosporesl Such spores lack flagella and are usually aeriaty allas,’mlnated S xuaI reproduction is initiated by the fusion of two gametangia with r.’qultant loritcatlon of a thick-wailed, resting spore. the zygospore In the more advani a at species the sporangia or the sporangiospores arc conidia-like Many of the Zygornycetes are of economic Importance due to their ability to synthesize commercially valuable organic acid, and alcohol,, to transform steroids such as cortisone, and to para.itiee and destroy food crops A few species are capable of causing disease in man and animals zygiamycosl,l 7’ Obligate conmmensal. of arthropods. zygo.pores usually lacking . . class Trichornycetes TIta Trichomyceta’. are an Ill -studied group of fungi which appear to be obligate comma nsal. of arthrupods The trichomycete. are associated with a wide variety of Insects dipiopods, and crustacea of terrestrial and aquatic (fresh and marine) habitats None of the members of this class have been cultured in vitro for continued period, of times with any success Asexual reproduction a by means of eporangiosporea Zygoapores have been observed in species of scv.ral order,. 8 (LI Sexual spores borne In asci . . ciass . Ascomycetes In the Ascomycetes the products of meiosis, the aacospores. are borne in sac like structures te mcccl aset 1 he a.cus usually contains eight ascospores, but the number produced may vary with the species or strain. Moat species produce extensive septate nayca, huna 1 hi’ large class is divided into two subclasses on the presence or absence of an .ascacarp [ he Ilemiascomycetidae lack an ascocarp and do not produce ascogenous hyph.i. , this ‘cubclasi includes the true yeasts The Euascon,ycetidae usually are divided Into three serIes (Plertomycetes, Pyrenomycetes, and Diecomycetes) on the basis of ascoc-arp ‘.t ructure 14 Sexual spores borne on basidia . class Basidiomycetes The Ilasidiomycetes gene raliy are considered the most highly evolved of the fungi Karyoganay and nceiosls occur in the baaidium which bears sexual exogenous spores. hasidiocipores The mushrooms toadstool., rust., and smuts are included in this class It ,esu,,l stage lacking .Form class Imperfecti ) Deuteromycetes I h.’ U. Ut, romycetea is a form class for those fungi (with morphological affinities to the Ascomyceies or Basidiomycete.) which have not demonstrated a sexual stage The gent-rally employed classification scheme for the.e fungi is based on the morphology and color of the asexual reproductive stages This scheme ,s briefly outlined below Newer oncepts of the classification based on conidium development after the classical acork of S I Ilughes (1953) may eventually replace the gross morphology system (see Itairron 191,111 12—7 ------- Fungi KEY TO THE FORM-ORDERS OF THE FUNGI IMPERFEC’li Reproduction by mean. of conidia, oidia. or by budding 2 I ’ No reproductive structure, present . Mycelia Sterilia 2 (I) Reproduction by mean, of conidia borne in pycnidla Spharrop .idale . 2’ Conldi&, when formed, net in cycnidia. 3 3 (2) Conldla borne in acervulj . . . . Melanconiales 3’ Conidia borne otherwise, or reproduction by oldie or by budding. . .. Monihales KEY TO THE FORM-FAMILIES OF THE MONILTALES Reproduction mainly by unicellular budding, yeast-like; mycelial phase. if present. secondary. srthro.pores occasionally produced. manifest melanin pigmentation lacking 2 I’ Thallus mainly filarnentous, dark melanin pigments sometime, produced . .. 3 2 W liallistospore. produced Sporobolomyceteccac 2’ No ballistospores Cryptococcaceae 3 Conidiophores, it present, not united Into sporodochia or synnemata 4 3 Sporoctochia present Tuberculariaceae 3’ Synnemata present Stilbellaeeae 4 (3) ConidIa and conidiopbore. or oidia hyaline or brightly colored Monhliaceae 4’ Conidia and/or conidiophores, containing dark melanin pigment Dematiaceac 12—8 ------- Fungi SELECTED REFERENCES Ahearn. D.G., Roth,F.J. Jr , Meyers. S.P. Ecology and Charact erization of Yeasts from Aquatic Regions of South Florida. Marine Biology 1 291-308. 1968 Barron, G. L. The Genera of Hyphomycetes from Soil. Williams and Wilkins Co., Baltimore. 364 pp. 1968 Cooke, W. H. Population Effects on the Fungus Population of a Stream. Ecology 421-18. 1961 __________ A Laboratory Guide to Fungi in Polluted Waters, Sewage, and Sewage Treatment Systems. U. S. Dept. of Health, Education and Welfare, Cincinnati, 132 pp. 1963 __________ Fungi in Sludge Digesters. Purdue Univ. Proc. 20th Industrial Waste Conference, pp 6-17. 1965a __________ The Enumeration of Yeast Populations in a Sewage Treatment Plant. Mycologia 57 696-703. 1965b __________ Fungul Populations in Relation to Pollution of the Bear River, Idaho-Utah. Utah Acad. Proc. 44(l) 298-3l5. 1967 __________ and Matsuura, George S. A Study of Yeast Populations in a Waste Stabilization Pond System. Protoplasma 57:163-187. 1963 ___________ Phaff, H. J., Miller, M. W., Shifrine, M , and 1 iapp, E. Yeasts in Polluted Water and Sewage. Mycologia 52 210-230. 1960 Emerson, Ralph .ind Weston, W. H. Aguahndereli.a fermentans Gen. et Sp. Nov., A Phycomycete Adapted to Stagnant waters. I. Morphology and Occurrence in Nature. Amer. J. Botany 54:702-719. 1967 Hughes, S. J. Conidiophores, Conidia and Classification. Can. J. Bot.31:577- 659. 1953 Johnson, T. W., Jr. Saprobic Marine Fungi. pp. 95-104. InAinsworth, G.C. and Sussman, A . S. The Fungi. III. Academic Press, New York. 1968 and Sparrow, F.K., Jr. Fungi in Oceans and Estuaries. Weuul&eiiu, Germany. 668 pp. 1961 Meyers, S. P. Observations on the Physio- logical Ecology of Marine Fungi. Bull. Misaki Mr. Biol. Inst. 12:207-225. 1968 Prigsheim, E.G. Iron Bacteria. Biol. Revs. Cambridge Phil. Soc. 24:200-245. 1949 Sparrow, F. K., Jr. Aquatic Phycomycetes. 2nd ed. Univ. Mich. Press, AnnArbor. 1187 pp. 1960. __________ Ecology of Freshwater Fungi pp. 41-93. InAinsworth, G.C. and Suegman, A.S. The Fungi, III. Acad. Press, New York. 1968 Stokes, J. L. Studies on the Filamentous Sheathed Iron Bacterium SphaerotiLlus natans . J. Bacteriol. 67:278-291. 1954 van Uden, N. and Fell, J.W. Marine Yeasts. pp. 167-201. In Droop, M.R. and Wood, E. J. F. Advances in Microbiology of the Sea, I, Academic Press, New York. 1968 Yerkes, W. D. Observations on an Occurrence of Leptomitus lacteus in Wisconsin. Mycologia 58976-978. 1966 Cooke, William B. and Ludzack, F. J. Predacious Fungus Behavior in Activated Sludge Systems. Jour. Water Poll. Cont. Fed. 30(12)’1490-1495, 1958. A lexopoulos, 2nd ed. 613 pp J. C. Introductory Mycology. John Wileyand Sons, New York, 1962 12-9 ------- PROTOZOA, NEMATODES, AND ROTIFERS I GENERAL CONSIDERATIONS A Microbial quality constitutes only one aspect of water sanitation; microchemicals and radionudlides are attracting increasing amount of attenticn lately. B Microbes considered here include bacteria, protozoa, and microscopic metazoa; algae and fungi excluded. C Of the free- Uvlng forms, some are members of the flora and fauna of surface waters; others was ied into the water from air and soil; still others of wastewater origin, nematodes most commonly from sewage effluent. D Hard to separate ‘native” from “foreign” free-living microbes, due to close association of water with soil and other environments; generally speaking, bacteria adapted to water are tho8e that can grow on very low concentrations of nutrient and zoomicrobes adapted to water are those that feed on algae, and nematodes, especially bacteria eaters, are uncommon in water but In large numbers In sewage effluent. E More species and lower densities of microbes in clean water and fewer species and higher densities in polluted water. F Pollution-tolerance or nontolerance of microbes closely related to the DO level required in respiration. C From pollution viewpoint, the following groups of microbes are of importance: Bacteria, Protozoa, Nematoda, and Rotifera. II BACTERIA A No ideal method for studying distribution and ecolo ’ of bacteria in freshwater. B According to Coil ns, 9 Pseudomonas, Achrombacter , Alcaligenes, Chromobac- terium, Flavobacterium , and Micrococcus are the most widely distributed i i ay be considered as indigenous to natural waters. Sulfur and iron bacteria are more common In the bottom mud. C Actinomycetes, Bacillus ap. Aerogenes ap., and nitrogen-fixation bacteria are primarily soil dwellere and may be washed into the water by runoffs. E Nematodes are usually of aerobic sewage treatment origin. D E. coil, streptococci, and Cl. perfrlngens are true indicators of fecal pollution. Ill PROTOZOA A Classification 1 Single-cell animals In the most primitive phylum (Protozoa) In the animal kingdom. 2 A separate kingdom, Protista, to in- clude protozoa, algae, fungi, and bacteria proposed in the 2nd edition of Ward-W ipple’s Fresh-Water Biolo r 0 ’ 3 Four subphyla or classes: a Mastigophora (flagellates)- Subclass phytomastigtha dealt with under algae; only subclass Zoomastigina included here; 4 orders: 1) Rhizomastiglna - with flagellum or flagella and pseudopodla 2) Protomonadjna - with 1 to 2 fl lj most free-living many 3) Polymastiglna - with 3 to 8 flagella;’ mostly parasitic In elementary tract of animals and man 4) Hypermastigtha - all inhabitants of alimentary tract of insects. W. BA. 45c. 10.72 13—1 ------- Protozoa,_Nernatodes, and Rotifers b Ciliopliora or infusoria (ciliates) — no pigmented members, 2 classes: 1) Ciliata - cilia present during the whole trophic life, containing majority of the ciliatee 2) Suctoria - cilia present while young and tentacles during trophic life. c Sarcodina (amoebae) - Pseudopodia (false feet) for locomotion and food— capturing, 2 subclasses 1) Rhizopoda - Pseudopodia without axial filaments. 5 orders: a) Proteomyxa - with radiating pee udopodia, without test or shell b) Mycetozon - forming plasmodium; resembling fungi in sporangium formation c) Amoebina - true amoeba - forming lobopodia d Teetacea - amoeba with single teat or shell of chitinous material e) Foraminifera - amoeba with 1 or more shells of calcareous nature, practically all marine forms d Sporozoa - no organ of locomotion, ainoeboid in asexual phase, all parasitic B General Morphology 1 Zoomastigina. Relatively small size (5 to 40 ), with the exception of Rhizomastigina, the body has a definite shape (oval, leaf- like, pear-like, etc.), common members with 1 or 2 flagella and some with 3, 4, or more; few forming colonies, ej .:tome present in many for feeding. 2 Ciliophora: Most highly developed protozoa; with few exceptions, a macro and a micro- nucleus; adoral zone of membranellae, mouth, and groove usually present in swimming and crawling forms, some with conspicuous ciliation of a disc-like anterior region and little or no body cilia (stalked and shelled forms); Suctoria nonmotile (attached) and with- out cytostome cysts formed in most. 3 Sarcodina: Cytoplasmic membrane but no cell wall, endoplasm and ectoplasm distinct or in- distinct, nucleus with small or large nucleolus, some with test or shell, moving by protruding pseudopodia, few capable of flagella transformation, fresh- water actinopods usually sperical with many radiating a.xopodia, some Testacea containing symbiotic algae and mistaken for pigmented amoebae; cysts with Biflgle or double wall and 1 or 2 nuclei. 4 Sporozoa: to be mentioned later. C General Physiology 1 Zoomastiglna: Free-living forms normally holozic, food supply mostly bacteria in growth film on surfaces or clumps relatively aerobic, therefore the first protozoa to disappear in anaerobic conditions and re-appearing at recovery, reproduction by simple fission or occasionally by budding. 2 Ciliophora: Holozoic; true ciliates concentrating food particles by ciliary movement around the mouth part, suctoria sucking through tenacles; bacteria and small 13—2 ------- Protozoa, Nematodes, and Roti.fers algae and protozoa constitute main food under natural conditions, some shown in laboratory to thrive on dead organic matter and serum protein, not as aerobic as flagellates — some surviving under highly anaerobic conditions, such as Metopus , reproduction by simple lisa ton, conjugation or encyatment. 3 Sarcodina: Holozoic, feeding through engulfing by pseudopodia; food essentially same as for ciliates, DO requirement somewhat similar to cillates - the small amoebae and Testacea frequently present in large numbers in sewage effluent and polluted water, reproduction by simple fission and encystation. IV NEMATODES A Classification 1 All in the phylum Nemata (nonsegment- ed round worms); subdivided by s e authors into two classes: Secernentea - 3 orders: (phasmids) Tylenchida, Rhabditida, Strongylida, and Teratocephalida, with papillae on male tail, caudal glands absent. Adenophora - 6 orders: (aphasmids) Araeolaimida, Dorylaimida, Chromdorida, Monhysterida, Enoplida, and Trichosyringida no papillae on male caudal glands absent. 2 Orders encountered in water and sewage treatment - Free-living forms inhabitat- ing sewage treatment plants are usually bacteria-feeders and those feeding on other nematodes; those inhabitating clean waters feeding on plant matters; they fall into the following orders: 3 Tyleachida - Stylet in mouth; mostly plant parasites; some feed on nematode such as Aphelenchoides . 4 Rhabthtida - No stylet in mouth or caudal glands In tail; mostly bacteria-feeders; common genera: Rhabiditia, D plogaster, Diplogasteroides, Monochoidéi , 1 elodera, Panagrellus , and Turbatrix . 5 Dorylaimida - Relatively large nematodes; stylet in mouth; feeding on other nematodes, algae and probably zoomlcrvbes; Dorylaimus common genus. 6 Chromadorida - Many marine forms, some freshwater dwellers feeding on algae, characterized by strong orna- mentation of knobs, bristles or punetations in cuticle. 7 Monhysterida - Freshwater d l1ers, esophago-intestinal valve spherical to elongated; ovaries single or paired, usually straight; common genus in water - Monhyatera . 8 Enoplida - Head usually with a number of setae; Cobb reported one genus, Mononchulus , in sand filters in Washington, D. C. B General Morphology Round, slender, nonsegmented (transverse markings in cuticle of some) worms, some small (about mm long, as Ti -i- cephalobus) , many ito 2 mm long ( Rhabditis, Dipjo aater . and Diplogasteriodes i or instance), and some large (2 to 7 mm, such as Dorylalmus) , sex separated but few parthenogenetic, complete alimentary canal; with elaborate mouth parts with or without stylet, complete reproductive system in each sex, no circulatory or respiratory system, complex nervous system with conspicuous nerve ring across oesophagus. C General Physiology 1 Feeding - Most sewage treatment plant dwellers feeding on bacteria, others preying on protozoa, nematodes, rotifers, 13—3 ------- Protozoa, Nematodes, and Ftotifers etc., clean-water species apparently vegetarians, those with stylet in mouth use the Latter to pierce the body of aqimal or plant and suck contents, metabolic waste mostly liquid containing ammonium carbonate or bicarbonate, enteric pathogens swallowed randomly with suspending fluid, hence remote possi- bility of sewage effluent-borne nematodes being pathogen-carriers. 2 Oxygen requirement - DO apparently diffused through cuticle into body. DO requirement somewhat similar to protozoa. Rhabditis tolerating reduced DO better than other Rhabditida members; all disappear under sepsis in liquid; some thrive in drying sludge 3 Reproduction - Normal life cycle requires mating, egg with embryo formation, hatching of eggs inside or outside femal8, 4 larval stages, and adult, few repro- duce in the absence of males. V RO’ I’IFERS A Classification 1 Classified either as a class of the phylum Aschelminthes (various forms of worms) or as a separate phylum (Rotifera); com- monly called wheel animalcules, on account of apparent circu]ar movement of cilia around head (corona); corona con- tracted when crawling or swimming and expanded when attached to catch food. 2 Of the 3 classes, 2 (Seisonidea and Bdelloidea) grouped by some authors under Digononta (2 ovaries) and the other being Monogononta(lovary); Seisonidea containing mostly marine forms. 3 Class Digononta containing 1 order (Bdelloida) with 4 families, Ph.ilodinedae being the most important. 4 Class Monogononta comprising 3 orders: Notommatida (mouth not near center of corona) with 14 families, Floscularida Me]icertida(corona with two wreaths of cilia and furrow between them) with 3 families, most import genera included in the order Notommatida: Brachionus , Keratella, Monostyla, Trichocerca, A splanchna, Polyarthra, Synchaeta, Microcodon ; common genera under the order Flosculariaceae Floscularia , and Atrochus . Common genera under order Melicertida: Ltmnias and Conochilus. 5 Unfortunately orders and families of rotifers partly based on character of corona and trophi(chewing organ), which are difficult to study, esp. the latter; the foot and cuticle much easier to study. B General Morphology and Physiology 1 Body weakly differentiated into head, neck, trunk, and foot, separated by folds, in some, these regions are merely gradual changes in diameter of body and without a separate neck, segmentation external only. 2 Head with corona, dosal antenna, and ventral mouth; mastax, a chewing organ, located in head and neck, connected to mouth anteriorly by a ciliated guilet and posteriorly to a large stomach occupying much of the trunk. 3 Common rotifers reproducing partheno- genetically by diploid eggs; eggs laid in water, cemented to plants, or carried on female until hatching. 4 Foot, a prolongation of body, usually with 2 toes, some with one toe, some with one toe and an extra toe-like structure (dorsal spur). 5 Some, like Philodina , concentrating bacteria and other microbes and minute particulate organic matter by ciliary movement on corona larger microbes chewed by mastax; some such as Monostyla feeding on clumped matter, such as bacterial growth, fungal masses, etc. at bottom; virus generally not ingested - appar ntly undetected by cilia. 6 DO requirement somewhat similar to protozoa, some disappearing under reduced DO, others, like PluLodina , surviving at as little as 2 ppm DO. 13—4 ------- Protozoa, Nematodes , and Rotifers VI SANITARY SIGNIFICANCE A Pollution tolerant and pollution non- tolerant species - hard to differentiate - requiring specialist training in protozoa, nematodes, and rotifers. B SAgnificant quantitative difference in clean and polluted waters - clean waters con- taining large variety of genera and species but quite low in densities. C Aerobic sewage treatment processes (trickling filters and activated sludge processes, even primary settling) ideal breeding grounds for those that feed on bacteria, fungi, and minute protozoa and present In very large numbers; effluents from such processes carrying large nurn- bers of these zoomicrobes; natural waters receiving such effluents showing significant increase in all 3 categories. D Possible Pathogen and Pathogen Carriers 1 Na ia causing swimming associated menlngto encephalitis and Acanthameoba causing nonswimmlng associated cases. 2 Amoebae and nematodea grown on pathogenic enteric bacteria in lab; none aUve in amoebic cysts; very few alive in nematodes after 2 days after ingestion; virus demonstrated in nematodes only when very high virus concentrations present, some freeliving amoebae parasitizing humans. 3 Swimming cLliatea and some rotifers (concentrating food by corona) ingesting large numbers of pathogenic enertic bacteria, but digestion rapid; no evidence or concentrating virus; crawling dillates and flagellates feeding on clumped organisms. 4 Nematodes concentrated from sewage effluent in Cin lnnati area showing liveE. coil and streptococci, but no human eiiertic pathogens. VII I D(AMrNATION OF WATER FOR MICROBES A Bacteria - not dealt here. B Protozoa and rotifers - should be included in examination for planktonic microbes. C Nematodes D Laboratory Apparatus 3 1 Sample_Bottles - One-gallon glass or plastic bottles with metal or plastic screw caps, thoroughly washed and rinsed three times with distilled water. 2 C ptilary Pipettes and Rubber Bulbs - t ng (9 In.) Pasteur capillary pipettes and rubber bulbs of 2 ml capacity. 3 Filtration Unit - Any filter holder assembly use cbin bacteriological examination. The funnel should be at least 650 ml and the filter flask at least 2 liter capacity. 4 Filter Membranes - Millepore SS ‘SS 047 MMltype membranes or equivalent. 5 Micro e - Binocular microscope with lox eyepiece, 4X, lOX, and 43X objectives, and mechanical stage. E Collection of Water Samples Samples are collected in the same mannef 1 ) as those for bacteriological examination, except that a dechiorinating agent is not needed. One-half to one gallon samples are collected from raw water and oae-gallon samples from tap water. Refrigeration is not essential and samples may be transported without it unless examination is to be delayed for more than five days. F Concentration of Samples 1 One gallon of tap water can usually be filtered through a single 8-u membrane withIn 15 minutes unless the water has high turbidity. At least one gallon of sample should be used in a single examina- tion. Immediately after the last of the water Is disappearing from the membrane, the suction line is disconnected and the membrane placed on the wall of a clean 50 to 100 ml beaker and flushed repeatedly with about 2-5 ml of sterile distilled water 13—5 ------- Protozoa, N’matodes, and itotifers with the aid of a capillary pipette and a rubber bulb. The concentrate Is then pipetted into a clean Sedgewick-Rafter Counting Cell and is ready for examina- tion. 2 In concentration of raw water samples having visible turbidity, two to four 8-micron membranes may be required per sample, with filtration through each membrane being limited to not more than 30 minutes. Samples ranging from 500 ml to 2 lIters may be filtered with one membrane, depending on degree of turbidity. After filtration the membranes are placed on the walls of separated beakers and washed as above. To prevent the particulates from obscuring the nematodes, the washing from each filter is examined in a separate counting chamber. G Direct Microscopic Examination Each counting chamber containing the filter concentrate is first examined under a 4X objective. Unless the concentrate contains more than 100 worms, the whole cell area is surveyed for neinatodes, with respect to number, developmental stage, and motility. When an object having an outline resembling that of a nematode is observed, it is re-examined under a lox objective for anatomical structures, unless the object exhibits typical nematode move- ment, which is sufficient for identifying the object as a neniatode. When the concentrate contains more than 100 worms, the worm density can be estimated by counting the number of worms in representative micro- scopic fields and multiplying the average number of worms per field by the number of fields In the cell area. The nematode density may be expressed as number of worms per gallon with or without differenti- ation as to adult or larval stages or as to viability. H General Identification of Nematodea I While actively motile nematodes can be readily recognized by any person who has some general concept of micro- scopic animals, the nonmotile or sluggishly motile nematodes may be confused with root fibers, plant fila- ments of various types, elongated ciliates such as Homajpzppn vermi- ia1are , or segments of appendages of small cruetacea. To facilitate a general identification of nematodes, the gross morphology of three of the free- living nematodes that are frequently found in water supplies is shown in the attached drawing. The drawing provides not only the general anatomy for recogni- tion of neinatodes but also most of the essential structures for guidance to those who want to use the “Key to Genera” in chapter No. 15 on Nemata by B. G. Chitwood and M. W. Allen in the book, Fresh Water Biology . (10) 2 Under normal conditions, practically all nematodes seen in samples of finished water are in various larval stages and will range from 100 to 500 microns in length and 10 to 40 microns in width. Except in the fourth (last) stage, the larvae have no sexual organs but show other structural characteristics. 3 If identification of genera is desired, the filter washings are centrifuged at 500 rpm for a few minutes. The supernate is discarded, except a few drops, and the sediment is resuspended in the remaining water. A drop of the final suspension is examined under both lox and 43X objectives for anatomical characteristics without staining, and for supplementary study of structures the rest is fixed in 5% formahn or other fixation fluid and stained according to iistructions given in Chitwood and Allen’s Chapter on Nemata,( ) Goodey’s Soil and Freshwater Nema- todes(l1) or other books on nematology. VIII USE OF ZOOMICROBES AS POLLUTION INDEX A Idea not new, protozoa suggested long ago, many considered impractical because of the need of identifying pollution-intolerant and pollution-tolerant species - proto- zoologist required. Method also time consuming. 1 —6 ------- Protozoa, Nematodes, and Roti.fers B ( ‘ iri tuti Lit nt Ott a lu.trbLittLtiv . battis — n4 mt1to(I4 II. rtnd nrinplgm’ntr d In ()to7.oa prFHlnt In Hn a11 numbei s in tI ..tn w iti. Nunthi i H grtatly increased whri polluh with filuent from aerobic tteatment plant or recovering from sewage pollution, no significant error introduced when clean-water members Included in the enumeration if a suitable method of com- puting the pollution index developed. C Most practical method involves the equation: A+B + 1000 C Z. P.!., A where A number of pigmented protozoa, B non pigmented protozoa, and C nematodes in a unit volume o sample, arid Z. P.1. • zoological pollution index. For relatively clean water, the value of Z.P.I. close to 1, the larger the value above 1, the greater the pollution by aerobic effluent (see attached report on zoomicrobial indicator of water pollution). D C CONTROL A Chlorination of effluent B Prolongation of detention time of effluent C Elimination of slow sand filters in nematode control. LIST OF COMMON ZOOLOGICAL ORGANISMS FOUND IN SEWAGE TREATMENT PROCESS - TRICKLING FILTERS Sarcodina - Amoebae Amoeba p teus, Aradiosa Hartm anne 1 Ia Arcella Vu garis Noegleria gruberi Actinoph — FLAGJTh LA TA Hodo caudatus Pleuromonas j _ aculans Oikomonas terrno Cercomonas longicauda Peranema trichophorium Swimming type Cillophora. Colpidium colpoda poda cuculus Glaucoma pyriformis Paran-iecium candatum , P bursaria Stalked type Qpercularia ep. (short stalk dichotomous) Vorticella sp. (stalk single and contractile) ‘ jpUcati.us (like opercularla, more colonial, stalk not contractile) Carcheejuin sp. (like vorticel]a but colonial, — individual zoolds contractile) Zoothamniuin sp. (entire colony contracts) Crawling type Euplotes ateUa Sty chia mylitus ! ! 2 t 8p. sp. Diplogaster sp. Doryl mus sp. Monocholdes sp. Chlindrocorpussp. g teroldes sp. Cephalobus sp. Rhabditia ep. Rhabditolaimug sp. sp. p hyst sp. Aphelenchoides sp. Trilobus sp. PROTOZOA NE MA TODA 13—7 ------- Protozoa. Nematodes. and Rotifers ROTA TORIA i2 & r lonoatyla r th_ Phi lodtha Keratella Brachinnus OLIGOCHAETA (bristle worms) he cnp rich! Aul phorus limos a Tubifex tubifex Lumbricillus Uneatua INSECT LARVAE Chlronomus Psychoda ep. (trickling filter fly) ARTHROPODA Lessertia sp. Po:rhomma sp. Achoratus subuiaticus (collemb3la) Foleornia sp. (collembola) Tomocerus ap. (collemb.,la) REFERENCES 1 American Public Health Association 1 American Water Works Association and Water Pollution Control Federation. Standard Methods for the Examination of Water and Wastewater , 13th ed. New York. 1971. 2 Chang, S. L., et al. Survey of Free- Living Nematodes and Amoebas In Municipal Supplies. J.A.W.W.A. 52: 613-618. 3 Chang, S. L. Interactions between Animal Viruses and Higher Forms of Microbes. Proc. Am. Soc. Civ. Eng. .fl. San. Eng. 96:151. 1970 4 Chang, S. L. Zoomicrobiai Indicators of Water Pollution presented at the Annual Meeting of Am. Soc. Microbial 1 Philadelphia, April 23-28, 1972. 5 Chang, S. L. Pathogenic Free-Living Amoebae and Recreational Waters. Presented at 6th International Confer- ence of Water Pollution Research Association, Jerusalem, Israel, June 19-24, 1972. 6 Chang, S. L. Proposed Method for Examination of Water for Free-Living NematodeB. J.A.W.W.A. 52:695-698. 1960. 7 Chang, S. L., et al. Survival and Protection Against Chlorination of Human Enteric Pathogt ns in Free- Living Nematodes Isolated from Water Supplies. Am. Jour. Trop. Med. and Hyg. 9:136-142. 1960. 8 Chang, S. L. Growth of SmaU Free- Living Amoebae in Bacterial and Bacteria-Free Cultures. Can. J. Microbial. 6:397-405. 1960. 9 Chang, S. L. and Kabler, P. W. Free- Living Nematodes In Aerobic Treatment Plant Effluents. J.W.P.C.F. 34:1256-2161. 19 3. 10 Chitwood, B. G. and Chitwood, M. B. An Introduction to Nemato . Section I: Anatomy. 1st ed. Monumental Printing Co. Baltirmre. 1950. pp 8-9. 11 Cobb, N. A. Contributions to the Science of Nematciogy VII. Williams and Willdns Co. BaltImore. 1918. 12 CollIns, V. G. The Distribution and Ecology of Bacteria In Freshwater, Pts. I & II, Proc. Soc. for Water Treatment and Exam. 12:40-73. 1963. (England) 13 Edmondson, W.T., et al. Ward-Wh pp1e’s Fresh Water B1o y. 2nd ed. Joon Wiley & Sons, New York. 1958. pp 368- 401. ------- ______ Protozoa, Nematodes, and Rotifers 14 Goodey, T. Soil and Freshwater Nematodes . (A Monograph) let ed. Methuen and Co. Ltd. London. 1951. This outline was prepared by S. L. Chang, Chief, Etiology, Criteria Development Branch, Water Supply Research Laboratory, NERC, EPA. Cincinnati, OH 45268. I —3 ------- Protozoa, Nematodes, and Rotifera -1 Sewage Food Chain In Aerobic Sewage Treatment Processes 13—10 ------- FREE- LIVING AMOEBAE AND NEMATODES I FREE-LIVING AMOEBAE A Importance of Recognizthg Small, Free- Living Amoebae In Water Supplies 1 Commonly found in soil, aerobic sewage effluent and natural, fresh waters - hence, frequently en- countered In examination of raw water. 2 Cysts not infrequently found in municipal supplies - not pathogen carriers. 3 Flagellate-amoebae Naegleria Involved In 50 some cases of mentngoencepha].ttis, about half In the U.S.; associated with swimming in small warm lakes. Acanthamoeba rhy des parasitizing hymen throats and causIng (3 cases) nonewimmlng- associated menthgo- encephalitis. 4 Cysts not to be confused with those of Endamoebahistoly ica in water- borne epidemics. B Classification of Small, Free- Living Amoebae 1 Recognized classification based on characte -istics in mitosis. 2 Common species fall into the following families and genera: Family Schizopyrenidae: Genera Naegler1a Dldascalus and Sch jrenus - first two being flagellate amoebae. Family Hartmanndllidae: Genera a J ( ) 3 How to prepare materials for studying mitosis - Feulgen stain C Morphological Characteristics of Small, Free-Living Amoebae 1 Morphology of Trophozoites - Ectop]asm and endoplasm usually distinct; nucleus with large nucleolus. 2 Morphology of cysts - Single or double wall with or without pores D Cultural Characteristics of SmaU, Free- Living Amoebae 1 How to cultivate these amoebae - plates with bacteria; cell cultures, axenic culture. 2 Growth characteristics on plate, cell, and axenic culture 3 Complex growth requirements for most of these amoebae E Resistance of Amoebic Cysts to Physical and Chemical Agents FREE- LIVING NEMATODES A Classification of Those Commonly Found in Water Supplies 1 Phasmidia (Secerneutes): Genera Rhabditis , Di plogaster Dipl g gteroides , heilob , Panagro]aimus 2 Aphasmidia (Aderiop’ioro): Genera Monhyst he1enchus, Turbatrix (vinegar eel), Dorylaimus, and Rhabdolatmug B Morphological Features 1 Phasmlds: paplua on tail of males, mouth adapted to feed on bacteria, few exceptions. 2 Aphasmids: no papilla on male tail; glandular cells in male. BI. AQ. 14b. 10. 72 14—1 ------- Amoebae and Nernatodea in Water SUDDIIeS C Life Cycle 1 Methods of mating 2 Stages of develop nent 3 Parthenogenesis D Cultivation 1 Bacteria-fed cultures 2 Axenic cultures E Occarren e in Water Supplie8 1 Relationship between their a earance in fthi hed water and that In raw water. 2 Frequency of occurrence In different types of raw water and sources. 3 Szrvival of human enteric path- ogeriic bacteria and viruses In nematodea. 4 Protection of human enteric pathogenic bacteria and viruses in -iematode-carrjers. F Control 1 ChlorInation of sewage effluent 2 Flocculation and sedimentation of water 3 chlorination of water 4 Other methods o destruction REFERENCES Amoebae 1 SLngh, B. N., “Nuilear Division In Nine Species of Small, Free- Living Amoe- bae and its Bearing on the Classifica- tion of the Order Amoebida”, Ph11o . Tra’is. Royal Soc. London, Series B, 236:405-461, 1952. 2 Chang, S. L., et al. “Survey of Free- Living Nematodes and Amoebas In Mw iclpal Supplies”. J. A. W . W. A. 52:613-618, 1960. 3 Chang, S. L., “Growth of Small Free- Living Amoebae In Various Bacterial and In Bacteria-Free Cultures”. Can. Jour. Mlcrobiol. 6:397-405, 1960. Ne rnatodes 1 Goodey, T., “Freshwater Nematodes”, 1st. Edition, Methuen & Co., London, 1951. 2 Edmondson, W.T., Ed., Ward & Whipple’s “Fresh-Water Biology” 10th Edition, page 397, 1955. 3 Chang, S. L., et al., “Occurrence of a Nematode Worm in a City Water Supply”. J.A.W.W.A., 51:671-676, 1959. 4 Chang, S. L., et al., “Survival, and Protection Against Chlorination, of Human Enteric Pathogens In Free- Living Nematodes Isolated From Water Supplies”. Am. Jour. Trop. Medicine & Hygiene, 9:136-142, 1960. 5 Chang, S. L., et ai., “Survey of Free- Living Nematodes and Amoebas In Municipal Supplies”. J.A.W.W.A., 52:613-618, 1960. 6 Chang, S. L., “Proposed Method for Exa n1nation of Water for Free- Living Nematodes”. J. A. W. W. A., 52:695-698, 1960. 7 Chang. S. L., “Viruses, Amoebas, and Nematodea and Public Water Supplies”. J.A.W.W.A., 53:288-296, 1961. 8 Chang, S. L., and Kahier, P. W., “Free- Living Nematodes In Sewage Effluent from Aerobic Treatment Plants”. To — be published . _____ _____ This outline was prepared by Shlh L. Chang, M. D., Chief, Etiology, Criteria Development Branch, Water Supply Research Laboratory, NERC, EPA, Cincinnati. 3H 45268. 14—2 ------- SUGGESTED CLASSIFICATION OF SMALL AMOEBAE Suhphylunv Sarcoclina Hertwig and Lesser Class Rhizopoda von Siebold S bc1ass Amoebaea i3utschli Order: Amoebida Calldns and Ehrenberg Superfamily Amoebaceae - free-living (Endamoebaceae - parasitic in animals) FaJTuly: Schizopyrentdae - active limax form common; transcient flagellates present or absent; nucleonus-origin of polar masses, polar caps and interzonal bodies present or absent Genus: Sch yrenus - no trariscient flagellates, single-walled cysts; no polar cape or fnterzonal bodies in mitosis Species S. thaenusa - reddish orange pigment formed in agar cultures with gram-negative bacillary bacteria S. r isselJi - no pigment produced in agar cultures Genus: Didascalus- morphology and cytology similar to Schiz!pyrenus but small numbers of transcient flagellates formed at times Species: D. thorntoni- only species described by Slngh (1952) Genus: Na 1eria Alexe eff - double-walled cysts; transcient flagellates formed readily; polar caps and interzonal bodies present In mitosis Species N. Lruberi (Schardinger) - only species established; Slngh (1952) disclaimed the N. solihe described In 1951 Family Hartmannellidae - no transcient flagellate formed; motility sluggish; no lirnax form, nucleolus dlsappearing probably forming spindle In mitosis, no polar caps or masses 1 aster and cehtrosome not known Genus HartrnanneUa - ectoplasm clear or less granular than endoplasm, single- walled cysts; single vacuole Species: H. bae - clear ectoplasm H. g ico1a - ectoplasm less granular than endoplasm Genus Acanthamoeba - filamentous processes from ecto- or endoplasm, growing axenically in flutd bacteriological n ,edla 14—3 ------- S iggc sted Classification of Small Amoebae Species: A. j sode8 Genus: 1n he],la - double-walled cysts; ecto- and endoplasm 1ndistingu ahable; many vacuoles Species: Sth ea1eptomemus 14—4 ------- ANIMAL PLANKTON I i .JTHODUCTi()N A Flanktonic animals or zooplankton are found in nearly every major group of animals. I Truly planktonic species (euplankton) spend all or most of their active life cycle suspended in the water. Three groups are predominantly involved in fresh water; the protoroa, rotifers, and microcrustacea. 2 Transient planktonic phases such as floating eggs and cysts, arid larval stages ocrur in many other groups. B Many forms arc strictly seasonal in occurrence. C C’crtairi rare forms c,ccur in great numbers at unpredictable Intervals. D Techniques of collection, preservation, and identification strongly influence the species reported. E In oceanographic work, the zooplankton is considered to include many relatively large animals such as siphonophores. ctenophores, hepteropods, pteropods, nrrowworms, and euphausid shrimp. F The plant-like or phytoplankton on the other hand are essentially similar in all waters, and are the nutritional foundation for the animal community. U PHYLUM PROTOZOA A The three typically free living classes. Mastigophora, Rhizopoda, and Ctllophora, all have planktonic representatives As a group however, the majority of the phylum is benthic or bottom-loving. Nearly any of the benthic forms may occasionally be washed up into the overlying waters and thus be collected along with the cuplankton B Class mastigophora, the nonpigrnented zooflagellates. These have frequently been confused with the phytomastigina or plant-like flagellates. The distinction is made here on the basis of the presence or absence of chlorophyll as suggested by Palmer and Ingram 1955. (Note Figure: Nonpigmented, Non-Oxygen Producing Protozoan Flagellates In the outline Oxygen Relationships.) 1 Commonly encountered genera Bodo Peranema 2 Frequently associated with eutrophic conditions C Class Rhizopoda - arnoeboid protozoans I Forms commonly encountered as plankton: Chaos Pt rcella Difflugia E uglypha (Amoeba) Centropyxis Helio oa 2 Cysts of some types may be encountered in water plants or distribution systems; rarely in plankton of open lakes or reservoirs. D Class Cihophora 1 Certain Tattachedh forms often found floating freely with plankton: Vorticella C arc he slum 2 Naked, unattached dilates Halteria one of commonest in this group. Various heavily ciliated forms (holotrich ) may occur from time to time such as Colpidlum, Enchelys , etc. 3 Ciliates protected by a shell or test (teataceous) are most often recorded from preserved samples. Particularly common in the experience of the National Water Quality Sampling Network are: Codonella fluviatile Codonella cratera Tintinnidlum (usually with organic matter) Tintinno ig BI.AQ.20e.4 70 15 —I ------- Animal l’lankton 111 PHYLUM flUTII’El A A Some forms such as Anuraea cochlearis and p anchr1a pridóiita tend to be present at alTUines oUthe year. Others such as Notholca striata, N. longispina and Poly- arthra pIatyp1 Fä are reported to be eases- ti&LLy wInter forms. B Specie8 in appro .mate order of descending frequency currently recorded by National Water Quality Sampling Network are: Keratella cochlearis Polyarthra vulgaris Synchaeta pectinata Brachionu8 guadridentata Trichocerca longiseta r l a p. Filinia longiseta Kellicottia longispina Pornpholyx sp . C Benthic species almost without number may be collected with the plankton from time to time. IV PHYLUM ARTHEOPOD.A A Class Crustacea 1 The Class Crustacea includes the larger common freshwater euplankton. They are also the greatest planktonic consum- ers of basic nutrients in the form of phytoplankton, and are themselves the greatest planktonlc contribution to the food of fishes. Most of them are herb- ivorous. Two groups, the cladocera and the copepods are most conspicuous. 2 Cladocera (Subcla8a Branchiopoda, Order Cladocera) or Water Fleas a Life History 1) During most of the year, eggs which will develop without fertil- ization (parthenogenetic) are deposited by the female in a dorsal brood chamber. Here they hatch into minature adults which escape and swim away. 2) As unfavorable conditions develop, males appear, and thick-walled sexual eggs are enclosed in egg cases called ephippia which can often endure freezing and drying. 3) Sexual reproduction may occur at different seasons in different species. 4) Individuals of a great range of sizes, and even ephippia, are thus encountered in the plankton, but there is no “larval” form. b Seasonal variation - Considerable variation may occur between winter and summer forms of the same species in some cases. Similar variation also occurs between arctic and tropical situations. c Forms commonly encountered as open water plankton include: Bosmina_longirostris and others Daphn.ta galeata and others Other less common genera are: Diaphanosoma, Chydorus, Sida, Acroperus, ceriodaphnla, flio- trephes, and the cariii örous L ra and Polyphemus . d Heavy blooms of Cladocerans may build up in eutrophic waters. 3 The copepods (order Copepoda) are the perennial microcrustacea of open waters, both fresh and marine. They are the most ubiquitous of animal plankton. a Cyclops is the genus most often T und by the National Water QuaUty Sampling Network activities. clops, Paracyclops , Diaptomus, Canthocamptus. F ptschura , Limnocatanus are other forms reporTed to be planktonic. b Copepods hatch into a minute char- acteristic larvae called a nauplius which differs considerably from the adults. After five or six moults, the copepodid stage is reached, and after six more moults, the adult. These larval stages are often encountered and are difficult to identify. 15—2 ------- Animal Plankton B Class Inserta 1 Only a single species of insect can be ranked as a true plankton, this is the midge fly Chaborus (approx. 8 app, formerly C orethra) . 2 The larva of this insect has hydrostatic organs that enable it to remain perman- ently suspended in the water. 3 It is usually found in the depths during the daytime, but comes to the surface at night. V OCCASIONAL PLAN} TERS A While the protozoa, t-otlfers, and micro- crtistacea ninke up the bulk of the plankton. there are many other groups as mentioned above that may also occur. Locally or periodically these may be of major import- ance. Examples are given below. B Phylum Coelenterata 1 Polyps of the genus Hydra may become detached and float about hanging from the surface film or floating detritus. 2 The freshwater medusa Craspedacusta occasionally appears in lakes or reser- voirs in great numbers. C Phylum Platyhelininthes 1 Minute Turbellaria (relatives of the well known Planaria ) are sometimes taken with the plankton in eutrophic conditions. They are readily confused with ciliate proto7oa. 2 Cercaria larvae of Trematodes (flukes) parasitic on certain wild animals, frequently appear In great numbers When trapped in the droplets of water on a swimmer’s skin, they attempt to bore in. Man not being their natural host, they fail. The resultant irritation is called “swimmer’s itch”. Some can be identified, but many unidentifiable species may be found. 3 In many areas of the world, cercaria larvae of human parasites such as the blood fluke Schistosoma japonicum may live as plankton, and penetrate the human skin directly on contact. D Phylum Nemathelminthes 1 Nematode (or nemas) or rouridworras approach the bacteria and the blue-green algae in ubiquity. They are found in the soil and In the water, and in the air as dust. In both marine and fresh waters and from the Arctic to the tropics. 2 Although the majority are free living. some occur as parasites of plants. animals, and man, and some of these parasites are among out most serious. 3 With this distribution, it is obvious that they will occasionally be encountered as plankton. A more complete discussion of nematodes and their public health implications in water supplies will be found elsewhere (Chang, S L ). E Additional crustacean groups sporadically met with in the plankton include the following: 1 Order Anostraca or fairy shrimps (formerly included with the two following orders in the Euphyllopoda) primarily planktonic in nature. a Extremely local and sporadic, but when present, may be dominating. b Arteniia , the brine shrimp, can tolerate very high saitnities. c Very widely distributed, poorly understood. 2 Order Notostraca, the tadpole shrimps. Essentially southern and western in distribution. 3 Order Conchostraca, the clam shrimps. Widely distributed, sporadic in occur- rence. lvlany local species. 4 Subclass Ostracoda, the seed shrimps. Up to 3 in. in length. Essentially benthic but certain species of Cypris , and Notodromas may occur in consid- erable num Th as plankton at certain times of the year. 5 Certain members of the large subclass Malacostraca are limnettc, and thus, planktonic to some extent. a The scuds, (order Amphipoda) are essentially benthic but are sometimes collected In plankton samples around 15—3 US i. vlron ,00fltdI Protection Aqency Corvallis Environmen i Ressarc 14b. 200 3 W 35th Street Cor ajh 9 Oregon 97330 ------- Anunal hriktøn w d hd o: J .ar Hhu . Nc kto— phnkton c. form s include Pontoporela and some species of Gamn-iarus . b The mysid, or opossum shrimp8 are represented among the plankton by M _ ysls relicta , which occurs in the deeper waters, large lakes as far north as the Arctic Ocean. F The Class Archnoidea, Order Hydracarina (or Acari) the mites. Frequent in plankton tows near shore although Unionlco],a crass- Ipes has been reported to be virtually planktonlc. U The phylum Mollusca is but scantily represented in the freshwater plankton, in contrast to the marine situation. Glochldia (ciliated) larvae are occasion- ally collected, and snails now and then glide out on a quiet surface film and are taken in a plankton net. An exotic bivalve Corbicula has a planktotrophlc veliger stage. H Eggs and other reproductive structures of many forms including fish, insects, and rotifers may be found in plankton samples. Special reproductive structures such as the statoblasts of bryozoa and sponges, and the ephippia of cladocerans may also be included. I Adventltjou and Accidental Planicters Many shallow water benthic organisms may become accidentally and temporarily incorporated into the plankton. Many of those in the preceding section might be listed here, In addition to such forms as certain free living nematodea, small ollgochaetes, and tardigrades, Collembo]a and other surface film livers are also taken at times but should not be mistaken for plankton. Fragments and molt skins from a variety of arthropods are usually observed. Pollen from terrestrial or aquatic plants Is often unrecognized, or confused with one of the above. Leaf hairs from terrestrial plants are also confusing to the uninitiated, they are sometimes mistaken for fungi or other organisms (and vice versa). In flowing waters, normally benthic (bottom living) organisms are often found drifting freely in the stream. This phenomenon may be conatant or periodic. When included In plankton collections, they must be reported, but recognized for what they are. REFERENCES 1 Edmondson, W.F., ed. Ward and Whlpples’s Freshwater Biology, 2nd Edition, Wiley & Sons, Inc., New York. 1959. 2 Hutchlnso; G. Evelyn. A Treatise on Limnology. Vol. 2. Introduction to Lake Biology and the Liinnoplankton. Wiley. 1115 pp. 1967. 3 Lackey, J. B. Quality and Quantity of Plankton In the South End of Lake Michigan in 1952. JAWWA. 36:689—74. 1944. 4 McGauhey, P.H., Elch, H.F., Jackson, H.W., and Henderson, C. A Study of the Stream Pollution Problem In the Roanoke, Virginia, Metropolitan District. Virginia Polytech. Inst., Engr. Expt. Sta. 5 Needham, J. G. and Lloyd, J. T. The Life of Inland Waters. Ithaca, New York, Cornstock Publishing Co., Inc 1937. 6 Newell, G. E. and Newell, R. C. Marine Plankton. Hutchinson Educ. Ltd. London. 2 2lpp. 1963. 7 Palmer, C.M. and Ingram, W.M. Suggested Classification of Algae and Protozoa in Sanitary Science. Sew. & lad. Wastes. 27:1183-88. 1955. 15—4 ------- Animal Plankton 8 Pennak, R.W. Freshwater Invertebrates 10 Welch, P.S. Limnology, McGraw-Hill of the United States. The Ronald Press, Book Co., Inc., New York. 1935. New York. 1953. 9 Sverdrup, H.W., Johnson, M.W., and FlemIng, Ft. H. The Oceans, Their _______________________________________ Physics, Chemistry and General This outline was prepared by H. W. Jackson, Biology. Prentice-Hall, Inc., New York. thief Biologist, National Training Center, OWP, 1942. EPA, Cincinnati, OH 45268. 15—5 ------- P lua PROTOZOA Prei Livii g Representatives I. Pla ell*t.d Protozos, Class sstigopbora A thoDhy ,U Pollution toll.rsnt Pollution tollsraftt 19 p II . Ameboid Protozoa, Class Saroodina ;: PMtir.smo.. Pollution toll.rsnt lO-5O,ii J. 2 Ler.LI zeport.d to be intollorent of pollution, 45,11. III. Ciltatod Protozoa, Class Ciliophora aolothrva , r.portod to be intollorant of pollution, 35 i LW. Jackson 3/4 ao1 of ____________ Poll*tion toll.rant, 35 ,4* 6 o..5ooj Colnodj Pollution tollorunt 2Ol2o LL zA&z , pollution toll.rant. Colonies often nasroasopic. 15—6 ------- PLANKTONIC PROTOZOA Animal Plankton Peranema trichophorum Actinosphaerium Codonella cratera Tint&nnidiurn fluviatile Top Side Chaos Arcella vulgar is Vorticella lS”7 ------- Animal Plankton PLANKTONIC Various Forms of ROTIFERS Keratella co hleari . y chaeta pectinata ! y arthra Jaris Brachionus quadridentata Rotaria ap 15 .8 ------- SOME PLANKTONIC CRUSTACEANS CRUSTACEANS A Nauplius larva of a Copepod Copepod; Cyc oj Order Copepoda 2-3 mm Water Fles t ptn1a OSTRA CODE 1-5 mm 2-3 mm Order Ciadocera Left: Shell closed 1-2 mm Appendages extended 15 -9 ------- Animal Plankton P LA NKTONIC ARTHROPODA Chaoborus inidge larva In sect 0. Aspects In the life cycle of the human tapeworm Diphyllobothrium laturn , class Cestoda. A a< as In human Intestine; B. procercoid larva In copepod; C. plerocercoid larva in flesh of pickerel (X-ray view). H.W. Jackson A mysid shrimp - crustacean A water mite - arachnid 15—10 ------- LABORATORY. IDENTIFICATION OF DIATO I OBJECTIVES A To become familiar with important structural features of diatoms. B To learn to recognize some common forms at eight. C To learn to identify lees common forms using technical keys. II PROCEDURE A Transfer a drop of the water sample con- taining diatoms to a microscope slide. Cover with cover glass and observe under low power (lOX) of microscope. 1 Do all of the diatom cells appear to have the same shape? Do Borne have square ends and some rounded ends? Touch the cover glass with your pencil several times as you observe through the microscope and note the relationship of the two types of ends to one another. 2 Find a place where a round-ended and a square-ended cell are close together and observe these under oil power (90X). The round-ended view Is that of the top or bottom of the diatom cell and is called the “valve” view. The square ended view is that of the side of the cell and is called the “girdle” view. 3 In the valve view note the cross lines in the wall. In this diatom there are many fine lines and a smaller number of coarse lines. The former are present in all diatoms and are called “striae” or “striations.” The latter are present only in some genera of diatoms and are called “septa,” or in other genera, “costae.” Which of the two types of lines arc continuous from side to side? The space left in the center by the in- terrupted lines is known as a “false (pseudo) raphe.” 4 What is the predominant color of the diatom? How many plant id a? In diatoms, the identification is based al- most entirely on the characteristics of the cell wall. 5 Make an outline drawing, at least 3 inches long, of a valve view and a girdle view of the diatom. Show the markings in the upper third of each. Label the striae, septa, and falee-raphe. Make a drawing of what you image an end view or cross(transverse) section view would be like. 6 Using the key, identify your specimen, listing the alternatives selected. B Use the key to identify other unknowns as far as possible, listing the alternatives selected in the key. Make a sketch of Navicula and Cyclotella if you identify these forms. Ill IMPORTANT TERMS Capitate - having a known-Uke end. Costae - coarse transverse ribs in wall. False raphe - (see pseudoraphe) Frustule - the wall of the diatom. Girdle view - the side view, in which the diatom appears to have square or blunt ends. Nodule - a lump-like swelling in the center or ends of the valve. Pseudoraphe - a clear space extending the length of the diatom and bordered on both sides by striae. Punctae - the dots which comprise the st riae. SI. MIC. cla. lab. 16—1 ------- Laboratory: Identification of Diatoms Raphe - a longitudinal line (cleft) bordered on both sides of striae. Septa - a self-Like partition in the diatom, appearing often as a coarse line. Striae - fine transverse lines especially evident in the valve view. Valve view - the top or bottom view, in which the diatom has rounded ends, or is circular in outline. REFERENCES 1 Boyer. C.S. The Diatomaceae of Philadelphia and Vicinity. J. B. Lippin- cott Co. Philadelphia. 1916. p 143. 2 Boyer. C.S. Synopsis of North America Diatomaceae, Parts 1(1927) and U (1928). Proceedings of the Academy of Natural Sciences, Philadelphia. 3 Elmore, C. J. The Diatoms of Nebraska. University of Nebraska Studies, 2 1:22- 215. 1921. 4 Hohn, M. H. A Study of the Distribution of Diatoms in Western New York State. Cornell University Agricultural Experi- mental Station. Memoir 308, pp 1-39. 1951 5 Pascher, A. Bacillariophyta (Diatomeae). Heft 10 in Die Suaswasser-Florg Mitteleuropas, Jena. 1930. p466. 8 Patrick, R. A Taxonomic and Ecological Study of Some Diatoms from the Pocono Plateau and Adjucant Regions, Farlowia. 2:143-221. 1945. 7 Smith, G.M. Class Baci1larioph yceae. Freshwater Algae of the United States, McGraw-Hill Book Co. New York. pp 440-5 10 2nd Ed. 1950. 8 Tiffany, L. H., and Britton, M.E. Class Baclllariophyceae. The Algae of Illinois, University of Chicago Press. pp 214-296. 1952. 9 Ward, H. B., and Whipple, G.C. Class I, Bacillariaceae (Diatoms). Freehwuier Biology, J. Wiley & Sons. New York. - pp 17 1-189. 1948. 10 Weber, C.!. A Guide to the Common Diatoms at Water Pollution Surveillance System Stations. USD1. FWPCA, Cincinnati, OH. 1966. 11 Whipple, G. C., Fair. G.M. • and Whipple, M.C. Diatomaceae. Microscopy of Drinking Water. Chapter 21, 4th ed. J. Wiley and Sons, New York. 1948. This outline was prepared by L. G. Williams, Aquatic Biologist, Formerly with Research and Development, Cincinnati Water Research Laboratory, FWPCA, SEC. 16—2 ------- PREPARATION OF PERMANENT DIATOM MOUNTS I The identification of many diatoms to genus and all diatoms to species requires that the celia be free of organic contents. This Ia necessary because the taxonomy of the diatoms is based on the structure of the frustule ( helis) of the organisms and many features are masked by the presence of organic materials which may remain inside. It is also necessary that at least 1000X magnification (oil immersion) be used to detect the structural features used In identification. No simple procedure for the accurate routine counting of diatoms has yet b een developed. II MATERIALS NECESSARY A Sample Concentration 1 Centrifuge (such as Universal DU) 2 100 ml centrifuge tubes 3 Membrane filter apparatus 4 Vacuum B Slide Preparation 1 Slides, 1 X 3 inch, frosted-end 2 Cover glasses, circular #1, 18 mm, 0. 13 - . 18 mm thick 3 Resinous mounting medium (such as Harleco microscope mounting medium) 4 Hot plates a 180°F b 700° F 5 DIsposable pipettes 6 3 X6 X 1/4 inch steel plate Il PROCEDURE A The volume of sample needed will vary according to the density of diatoms and alit, and only with experience can the correct sample size be determined. In most cases, 100 ml will be sufficient. 1 Spin 100 ml at 1000 G for 20 mInutes. 2 Withdraw the supernatant liquid with an aspirator, being careful not to disturb the concentrate at the botton of the centrifuge tube. (Draw off all but 2-3 ml.) 3 Transfer the concentrate to a labelled 10 ml disposable vial. Label the vial with a magic marker, diamond pencil, or “time” label. 4 If the sample has been preserved with formalin, or contains more than 1.0 gram per liter dissolved solids, it will be necessary to wash the concentrate with distilled water. In this case, transfer the entire concen- trate to a 15 ml centrifuge tube. Dilute to 15 ml with distilled water, making certain that the sample is well mixed. Spin for 10 minutes at full speed in a clinical centrifuge. With- draw the Bupernatant liquid, and refill with distilled water. Spin again for 10 minutes. Withdraw the supernatant liquid as before, return the concentrate to the rinsed vial in 2-3 ml of distilled water and proceed with the mounting. 5 II more than 200 ml of sample must be centrifuged to obtain sufficient material to prepare a diatom slide, concentrate the diatoms by filtering the sample through a 1. 2 micron pore diameter membrane filter. Transfer the filter to a 15 ml centrifuge tube, and dissolve with 90% acetone. Centrifuge 10 minutes (full speed) and decant with an aspirator. Refill with 90% acetone, B!. MIC. enu. lab. Sb. 6.68 17—1 ------- Preparation of Permanent Diatom Mounts agitate, and spin again for 10 minutes. Repeat until three fresh acetone washes have been used. Replace the acetone with 2-3 ml of distilled water and transfer to a labelled vial as described In #4. B If the loss of minute forms in supernatant is suspected, spin 100 ml at 1000 gs in a batch centrifuge for as long as may be necessary, then proceed as below. C Mounting 1 Heat the hot plates to the prescribed temperatures. 2 Place one cover glass on the steel plate for each sample. 3 Place the steel plate on the 1800 F hot plate. 4 Transfer a drop of sample to a cover glass. 5 Allow the water to evaporate (caution: do not allow it to boil.) 6 Continue to add more sample until a thin layer of material is noticeable on the dry cover glass, or until all of the concentrate has been used. This step is especially critical, and can be learned only by trial and error. 7 Transfer the steel plate to the 700°F hot plate for 20-30 minutes. (The plate should be hot enough to incinerate paper.) 8 While the material is on the high temperature hot plate, label the microscope slides (use a #2 pencil or a fine point drawing pen); place them on the low temperature hot plato, which now has been reset to approxi- mately 275°F. 9 Place a drop of mounting resin on the n icroscope slides and allow the solvent to evaporate. 10 When the incineration of the material on the cover glasses is complete, transfer the cover glasses, while still hot, to the mounting medium. 11 Allow the resin to penetrate the frustules (1-2 minutes). 12 Remove the slide, place it on a cool desk top, and press the cover glass lightly with a pencil eraser for a few seconds. The medium will harden In 5-10 seconds. 13 Scrape off the excess resin with a razor blade. D The preparation is now ready for exam- ination under an oil immersion objective. ACKNOWLEDGEMENT: Certain portions of this outline contains training material from a prior outline by M.E. Bender. This outline was prepared by Dr. C.I. Weber, Chief, Biological Methods Section, Analytical Quality Control Laboratory, 1014 Broadway, Cincinnati, OH 45202. 17—2 ------- LABORATORY IDENTIFICATION OF ANIMAL PLANKTON I INTRODUCTION A The great majority of organisms commonly encountered in plankton analysis work are plants or at least plant-like (holophytic). Animals, however, (holozoic or nonchioro- phyli bearing forms) are an impoctax t part of the community, and the ability to recog- nize them may be quite important. B Many animals are soft bodied and so are best observed in the living condition, as Uiey shriiik arid become otherwise distorted on preservation. There are consequently many which will not be available in a suitable form for the following exercise. Only such forms will be dealt with as can readily be obtained alive, or which retain essential characteristics on preservation. II OBJECTIVES A To Study the nature and use of a key for identifying organisms B To Introduce the Beginners to the Use of thy Microscope C To Learn to Recognize Basic Animal Types D To Identify Animal Plankton Species as Available, and to Become Familiar with the Literature III PROCEDURE A The Use of the Biological Key 1 Obtain a “Basic Invertebrate Collection” from the instructor 2 Sclect a specimen designated by the instructor, and t,,rn to the “Key to Selected Larger Groups of Aquatic Animals a Examine your specimen carefully, then read the first couplet of statements in the key (la and ib). b Since the specimen is large enough to see, it obviously could not be the object of statement Ia. Therefore due to the nature of the key (as explained in the second paragraph of the introduction) the second alternative (lb) must apply This alternative instructs us to proceed to couplet 2. c From here on, follow from couplet to couplet, considering each couplet by itself, until a final selection leads to a name. If this name or couplet is, followed by another couplet number, this means that the group named is further subdivided. 3 Identify the other specimens in the Basic Invertebrate Collection in the same way. 4 Carry the identification further, to genus and species if possible, in one or more of the more detailed keys listed at the end of the “Key to Selected Larger Groups of Aquatic Animals.” B The Use of the Microscope 1 Obtain preliminary information from the instructor as to how to set up and operate the instrument. 2 Place a prepared slide of a printed letter on the stage and observe it successively under low (lOOX) and high (45X) powers. When the letter is right side up to you, how does it appear through the microscope? 3 Place a prepared slide of a micro- crustacean on the stage and identify it using the “Key to Selected Larger Groups of Aquatic Arumals.” Continue your BI. MIC. cia. lab. 5c. 10. 72 18—I ------- Laboratory Identification of Animal Plankton identilication as far as possible using Eddy and Hodson’s “Taxonomic Keys.” 4 Prepare a “wet mount” under the direction of the instructor and identify the organism. Confirm your identifica- tion in one or more of the technical reference books available. C Identify each of the specimens in the reference collection as to phylum and class, and then genus and species JJ possible (do not spend undue time on the specie8 without assistance). Make a flash caI-d sketch of at least one organism of each phylum observed as an example of a type. ED Examine the living material provided. Sketch and identify animal forms encountered as far as time permits. Can you draw any conclusions as to the types of animal Life found In the various habitats indicated? This outline was prepared by H. W. Jackson, Chief Biologist, National Training Center, DTTB, MDS, OWP, EPA, Cincinnati, 0 11 45226. 18—2 ------- ‘rECIENIQUES OF PLANKTON SAMPLING PROGRAMS I INTRODUCTION A A plan is necessary. ‘1.1 you fail to plan, you are planning to fail.” Overall objec- tives, integration with other survey units, statistical design. B A planned program of plankton analysis should involve periodic sampling at weekly intervals or more often. 1 Most interference organi8m8 are smaU, and hence have relatively short-We histories. 2 Pop ]ations of such organisms may fluctuate rapidly In response to chang- ing water, weather, or seasons. 3 Seasonal growth patterns of plankton tend to repeat themselves from year to year, thus they are relatively predictable. C A well-planned stucb’ or analysis of the growth pattern of plankton in one year will provide a basis for predicting conditions the following year. 1 SInce the seasons and the years differ, the more records are accumulated, the more useful can they become. 2 As the time for an anticipated bloom of some trouble maker approaches, the frequency of analysis may be increased. D Detection of a bloom in its early stages will facilitate more economical control. II FIELD ASPECTS OF THE ANALYSIS PROGRAM A Two general aspects of sampling are com- monly recognized quantitative and qualitative. 1 Qualitative examination tells what is present. 2 Quantitative tells how much. 3 Either approach is useful, a combination is best. B Equipment of collecting samples in the field is varied. 1 A half—liter bottle will suffice for sur- face samples of phytoplankton if carefully taken. If zooplankton also are of interest, 2 or more liters should be collected. (See be1ov . 2 Plankton nets concentrate the sample in the act of collecting, and capture certain larger forms which escape from the bottles. Only the more elaborate types are quantitative however. 3 A kernmerer-type sampler is suggested for depth samples. 4 Other methods such as the Clark-Bumpus sampler or the Juday plankton trap may be employed for special purposes. C The location of sampling points is important I Both shallow and deep samples are suggested. a “Shallow” samples should be taken at a depth of 6 inches to one foot. b “Deep” samples should be taken at such intervals as the depth of the reservoir permits. There should be at least one open water sampling point. c Each major bay or shoal area should have at least one sampling point. d Additional sampling stations should be established on the basis of ex- perience and resources. e Samples may be composited if nec- essary to give an overall summary of conditions. Such summaries are not advised and should be interpreted with care. 19-I flr ff,n — I ,, ------- Techniques of Plankton Sampling Programs A standardized vertical haul however c n be useful for routine co1nparison . III FACTORS WHICH INCLUENCE SAMPLING AND DATA COLLECTION A Physical Features 1 Temperature a Lakes are warmed In spring princi- pally by the action of wind forcing the warmer water down into the cooler water against the forces of gravity. b Thermal stratification 2 Turbidity 3 Color 4 Water movement 5 Ugit penetration a A factor of turbidity, color, biolog- ical activity, and time of day (1) EffectIve length of daylight dimenishes with the depth of the lake. 6 Wind velocity and direction 7 Bottom materials 8 Size, shape, and slope of lake basin B Chemical Factors 1 Alkalinity, pH, and dissolved minerals excluding nitrogen and phosphorus 2 Dissolved oxygen a From photosynthesis in sunlight b From contact of lake surface with the air 3 Nutrients for biological growth - especially nitrogen and phosphorus a A given body of water will produce a given quanity of aquatic life. Biological production is determ.Ined primarily by the nutrients in solu- tion in the water, and an Increase in basic fertility will increase biological activity. b Basic suppliers of nutrients include tributary streams, precip- itation from the atmosphere, and Interchange with lake bottom sedi- ments. IV FIELD PRESERVATION OF SAMPLES Provision should be made for the field stabilization of the sample until the laboratory examination can be made. Techniques and materials are listed below. No “ideal” pre- servative or technique has yet been developed. each has its virtues. A Refrigeration or icing. The container containing the sample can be cooled, but under no circumstances should ice be dropped into the sample. B Preservation by 3-5% formaline is time- tested and widely used. Formaline shrinks animal tissue, fades colors, and makes all forms brittle. C Ultra-violet sterilization is useful in the laboratory to retard decomposition of plankton. D Lugol’s solution is often used. E A special merthiolate preservative developed by the FWPCA Water Pollution Surveillance System which has proved very satisfactory and is described in reference No. 9. c Fluctuates 8easonally because of temperature and biological activity, and diurnaUy because of biological activity. 19—2 ------- Techniques of Plankton Sampling Prog am V SUMMARY AND CONCLUSIONS A The field iampling program should be carefully planned to evaluate all signifi- cant locations in the reservoir or stream, giving due consideration to the capacity of the laboratory. 8 Adequate records and notes should be made of field conditions and associated with the laboratory analyses in a permanent file. C Once a procedure for processing plankton is adopted, it should be used exclusively by all workers at the plant. D Such a procedure qhould enable the water plant operator to prevent plankton troubles or at least to anticipate them and have corrective materials or equipment stockpiled. ACKNOWLEDGMENT. Portions of this outline were prepared by K. M. Mackenthun, Bioiogist, formerly with Technical Advisory and Investigation Activities 1 FWPCA, SEC, Cincinnati 1 Ohio. REFERENCES 1 API-LA. Standard Methods for the Examination of Water and Wastewater, 13th Ed, pp 726-742, NY, 1971. 2 llutcheson, George E. A Treatise o i Limnology. John Wiley and Co. New York. 1957. 3 Jackson, I-I. W. Biological Examination (of plankton) Part Ill in Simplified Procedures for Water Examination. AWWA Manual M12. Am. Water Works Assoc., N Y. 1964. 4 Lickey, J. B. The Manipulation and Counting of River Plankton and Changes in Some Organisms Due to Forma]in Preservation. Public Health Reports, 53: 2080-93. 1938. 5 Mackenthun, K. M., Ingram, W. M., and Ralph Porges. Limnological Aspects of Recreation Lakes, DHEW, PHS Publication No. 1167, 1964. 6 Olson, Theodore A. and Burgess, Fred- erick J. Pollution and Marine Ecology. Intersclence Publishers. 364 pp. 1967. 7 Palmer, C. M. Algae in Water Supplies. U. S. Department of Health, Education, and Welfare, Public Health Service Publication No. 657, Superintendent of Documents, Washington 25, D. C. 8 Schwoerbel, J. Methods of Hydrobiology (Freshwater Biology). Pergamon Press, 1970. 9 Weber, C. I. Methods of Collection and Analysis of Plankton and Periphyton Samples in the Water Pollution Surveillance System. App. and Devel. Rep. (AQC Lab., 1014 Broadway, Cincinnati, OH 45202) 19 pp 1966. 10 Welch, P.S. Limnological Methods. The Blaldston Co., Phija. Toronto. 1948. 11 Williams, L. 0. Plankton Population Dynamics, in National Water Quality Network, Supplement 2, U.S. PHS Pub. No. 663. 1962. This outline was prepared by H. W. Jackson, Chief Biologist, National Training Center, DTTB, MDS, OWP, EPA, Cincinnati, OH 45268. ------- PREPARATION AND ENUMERATION OF PLANKTON IN THE LABORATORY I RECEPTION AND PREPARATION OF SAMP LES A Preliminary samphng and analysis 18 an essential preliminary to the establish- ment of a permanent or semi-permanent program B Concentration or sedimentation of pre- served samples may precede analysis. 1 Batch centrifuge 2 Continuous centrifuge .3 Sedimentation C Unpreserved (living) samples should be analyzed at once or refrigerated for future analysis. PREPARATION OF MERTHIOLATE PRESERVATIVE A The Water Pollution Surveillance System of the FWPCA has developed a modified merthiolate preservative’. (Williams. 1967) Sufficient stock to make an approx- imately 3 5% solution in the bottle when filled is placed In the sample bottle in the laboratory The bottle 18 then filled with water in the field and returned to the laboratory for analysi8 B Preparation of Merthiolate Preservative 1 Merthlolate is available from many chemical laboratory supply houses; one should specify the water soluble sodium salt 2 Merthiolate stock dissolve approxi- mately 1 5 gram of sodium borate (borax and approximately I gram of merthiolate in 1 liter of distilled water The amount of sodium borate and merthiolate may be varied slightly to adjust to different waters, climates, and organic contents. 3 Prepare a saturated aqueous LugoV s solution as followg a Add 60 grams of potassium iodide (KI) and 40 grams of iodine crystals to 1 liter of distilled water 4 Prepare the preservative solution by adding approximately 1. 0 ml of the Lugol’s solution to 1 liter of merthi- olate stock. III SAMPLE ANALYSIS A Microscopic examination is most frequently employed in the laboratory to determine what plankton organisms are present and how many there are: 1 Optical equipment need not be elaborate but should Include. a Compound microscope with the following equipment: 1) Mechanical stage 2) Ocular: laX, with Whipple type counting eyepiece or reticuie 3) Objectives: approx. approx. approx. approx. 1OX( 16mm) 20X(8 mm) 4QX(4 mm) 95X(1. 8 mm)(optional) A 40X objective with a working distance of 12. 8 mm and an erect Image may be obtained as special equipment A water Immersion objective (In addition to oil) might be considered for use with water mounts. Binocular eyepieces are optional, B!. MIC. enu. l f. 10. 72 20—1 ------- Preparation and Enumeration of Plankton Stage micrometer (this may be borrowed, if necessary, as it is usually used only once, when the equipment is calibrated) b Inverted microscopes offer certain advantages but are not widely available The same is true of some of the newer optical systems such as phase contrast microscopy. These are often excellent but ex- pensive for routine plant use 2 Precision made counting chambers are required for quantitative work with liquid mounts. a Sedgwick-Rafter cells (hereafter referred to as S-R cell) are used for routine counts of medium and larger forms. b Extremely small forms or t nanno plankton’ may be counted by use of the nannoplankton (or Palmer) cell, a Fisher- Ltttman cell, a hemacyto- meter, the Lackey drop method, or by use of an inverted microscope. 3 Previous to starting serious analytical work, the microscope should be cali- brated as described elsewhere Di- mensions of the S-ft cell should also be checked, especially the depth. 4 Automatic particle counters may be useful for coccoid organisms. B Quantitative Plankton Counts All quantitative counting techniques Involve the filling of a standard cell of known dimensions with either straight sample or a concentrate or dilution thereof. 2 The organisms in a predetermined number of microscope fields or other known area are then observed, and by means of a suitable series of multi- plier factors, projected to a number or quantLty per ml gallon, etc. 3 Direct counting of the unconcentrated sample eliminates manipulation, Saves time, and reduces error. If frequency of organisms is low, more area may need to be examined or concentration of the sample may be in order 4 Conventional techniques employing concentration of the sample provide more organisms for observation, but because they involve more manipulations, intro- duce additional errors and take more time. C Several methods of counting plankton are in general use. The numerical or clump count is regarded as the simplest. a Every organism observed must be enumerated. If it cannot be identi- fied, assign a symbol or number and make a sketch of it on the back of the record sheet. b Filaments, colonies and other associations of celia are counted as units, equal to single isolated ceUs. Their identity as Indicated on the record sheet is the key to the significance of such a count 2 IndivIdual cell count . In this method, every cell of every colony or clump of organisms is counted, as well as each individual single- celled organism. 3 The areal standard unit method offers certain technical advantageB, but also involves certain inherrent difficulties. a An areal standard unit Ia 400 square microns. This is the area of one of the smallest subdivided squares in the center of the Shipple eyepiece at a magnification of IDOX. b In operation, the number of areal units of each species is recorded on the record sheet rather than the number of individuals. Average areas of the common species are 20-2 ------- Preparation and Enumberation of Plankton are sometimes printed on record sheets for a particular plant to obviate the necessity of estimating the area of each cell observed individually. c The advantage of the method lies in the cognizance taken of the relative masses of the various species as indicated by the area presented to the viewer These areas, however, are often very difficult to estimate. 4 The cubic standard unit method in a logical extension of the areal method, but has achieved less acceptance. 5 Separate field count a In counting separate fields, the question always arises as to how to count organisms touching or crossed by the edge of the Whipple field Some workers estimate the proportion of the organism lying Inside the field as compared to that outside. Only those which are over bali way inside are counted. b Another system is to select two adjacent sides of the square for reference, such as the top and left boundaries Organisms touching these lines in any degree, from outside or inside, are then counted, while organisms touching the opposite sides are ignored It is important to adopt some such system and adhere to it consistently. c It is suggested that if separate microscopic fields are examined, a standard number of ten be adopted. These should be evenly spaced In two rows about one-third of the distance down from the top and one-third of the distance up from the bottom of the S-El cell 6 Multiple area count . This is an ex- tension of the separate field count. A considerable increase in accuracy ha recently been shown to accrue by emptying and refilling the S-El cell. after each group of fields are counted and making up to 5 additional such counts. This may not be practical with high counts. 7 The strip count . When a rectangular slide such as the S-R cell is used, a strip (or strips) the entire length (or known portion thereof) of the cell may be counted instead of separate Isolated fields Marking the bottom of the cell by evenly spaced cross lines as ex- plained elsewhere greatly facilitates counting. a When the count obtained is multi- plied by the ratio of the width of the strip counted to the width of the cell, the product is the esti- mated number of organisms in the cell, or per ml. b When the material in the cell is unconcentrated sample water, this count represents the condition of the water being evaluated without further calculation. 8 Survey count . A survey count is an examination of the entire area of a volumetric cell using a wide field low power microscope. The objective is to locate and record the larger forms, especially zooplankton such as copepods or large rotifers which may be present in size. Special large capacity cells are often employed for this purpose. For still larger marine forms, numerous special devices have been created. 9 Once a procedure for concentration arid/or counting is adopted by a plant or other organization It should be used consistently from then on so that results from year to year can be compared. D Differential or qualitative “counts ” are essentially lists of the kinds of organisms found 20-3 ------- Preparation and Enumeration of Plankton E I’roportional or r lattv countl-3 of spIcial groups are often very useful For cx- ampk, diatoms It is best to always count a standard numbers of cells. F Plankton are sometimes measured by means other than microscopic counts. Settled volume of killed plankton in an Imhoff cone may be observed after a standard length of time. This will evaluate primarily only the larger forms. 2 A gravimnetric method employs drying at 60° C for 24 hours followed by ashing at 800° C for 30 minutes. This Is particularly useful for chemical and radlochemnical analysis. 3 Chemical and physical evaluation of plankton populations employing various instrumental techniques are coming to be widely used. Both biomass and productivity rates can be measured Such determinations probably achieve their greatest utility when coordinated with microscopic examination. 4 The membrane (molecular) filter has a great potential, but a generally acceptable technique has yet to be perfected a Bacteriological techniques for coliform determination are widely accepted b Nematodes and larger organisms can readily be washed off of the membrane after filtration. c It is also being used to measure ultraplanktori that pass treatment plant operations d Membranes can be cleared and organisms deposited thereon observed directly, although accessory staining is desirable. e Difficulties include a prediiec- tion of extremely fine membranes to clog rapidly with silt or increase in plankton counts, and the difficulty of making observations on individual cells when the organisms are piled on top of each other. It is sometimes necessary to dilute a sample to obtain suitable distribution. IV SUMMARY AND CONCLUSIONS A The field sampling program should be carefully planned to evaluate all significant locations in the reservoir or stream, giving due consideration to the capacity of the laboratory. B Adequate records and notes should be made of field conditions and associated with the laboratory analyses in a permanent file. C Optical equipment in the laboratory should be calibrated. D Once a procedure for processing plankton is adopted, it should be used exclusively by all workers at the plant E Such a procedure should enable the water plant operator to prevent plankton troubles or at least to anticipate them and have corrective materials or equipment stockpiled. REFERENCES Ely Lilly Company. Merthiolate as a Preservative. Ely Lilly & Co. Indianapolis 6, Indiana. 2 Gardiner, A. C. Measurement of Phytoplankton Population by the Pigment Extraction Method. Jour. Marine Biol. Assoc. 25(4):739-744. 1943. 3 Goldberg, E. D., Baker, M., and Fox, D. L. Microfiltration in Oceanographic Research Sears Foundation. Jour. Mar. Res. 11:194-204. 1952. 20-4 ------- Preparation and Enumeration of Plankton 4 Ingram, W. M., and Palmer, C. M. Simplified Procedures for Collecting, Examining, and Recording Plankton in Water. Jour. AWWA. 44(7): 617-624. 1952 — 5 Jackson, H. W. Bilogical Examination (of plankton) Part III tn Simplified Procedures for Water Examination. AWWA Manual M 12. Am. Water Works Aseoc. N.Y. 1964. 6 Lund, J. W. G.. and Tailing, J. F. Botanical Limnological Methods with Special References to the Algae Botanical Review. 23(8&9):489-583. October. 1957. 7 Weber, C. I. The Preservation of Plankton Grab Samples. Water Pollution Surveillance Systems, Applications and Development Report No. 26, USD1, FWPCA, Cincinnati, Ohio. (1967) 8 Williams, L. G. Plankton Population Dynamics. National Water Quality Network Supplement 2. U. S. Public Health Service Publ. No. 663 (1962) 9 Wohiechag, D. D., and Hasler, A. D. Some Quantitative Aspects of Algal Growth in Lake Mendota. Ecology. 32(4):581-593. (1951) This outline was prepared by H. W. Jackson, Chief Biologist, National Training Center, DTTB, MDS, OWP, EPA, Cincinnati, OH 45268. 20—5 ------- CALIBRATION AND USE OF PLANKTON COUNTING EQUU’MENT I INTRODUCTION A With the exception of factory-set instruments, no two microscopes can be counted upon to provide exactly the same magnification with any given com- bination of oculars and objectives. For accurate quantitative studies, it is there- fore necessary to standardize or ‘calibrate” each instrument against a known standard scale. One scale frequently used is a microscope slide on which two millimeters are subdivided into tenths, and two addi- tional tenths are subdivided into hundredths. FIgure 3. B In order to provide an accurate measuring device in the microscope, a Whipple Plankton Counting Square or reticule (Figure 2a) Is installed in one ocular (there are many different types of reticules). This square is theoretically of such a size that with a lOX objective, a lox ocular, and a tube length of 160 mm, the image of the square covers a square area on the slide one mm on a slide. Since this objective is rarely attained however, most microscopes must be standardized or “calibrated” as described below in order to ascertain the actual size of the Whipple Square as seen through the microscope (hereinafter referred to as the “Whipple field”). This process is schematically represented in Figures 5 and 7. If the Whipple eyepiece is to be used at more than one magnification, it must be recali- brated for each. A basic type of monocular microscope is shown in Figure 1. C Microscopes with two eyepieces (binocular) are a convenience but not essential. Like modern cars they are not only great “performers,” but also complicated to service or, in this instance, calibrate. On some instruments, changing the inter- pupillary distance also changes the tube length, on others it does not. The “zoom” feature on certain scopes is also essentially a system for changing the tube length. The resultant is that in addition to calibra- tion at each combination of eyepiece and objective, any other factor which may affect magnification must also be considered. In some instances this may mean setting up a table of calibrations at a series of micro- scope settings. Arother procedure is to select a value for each of the variables involved (inter- pupillary distance, zoom, etc.) and calibrate the scope at that combination. Then each time the scope is to be used for quantitative work, re-set each variable to the value selected. A separate multipli- cation factor must be calculated for each adjustment which changes the magnification of the instrument. Since the Whipple Square can be used to measure both linear dimensions and square areas, both should be recorded on an appropriate form. A suggested format Is shown in Figure 6. (Data written in are used as an illustration and are not intended to apply to any particular microscope. An unused form is included as Figure 6-A.) U THE CALIBRATION PROCEDURE A In8talling the Whipple Square or Reticule To install the reticule in the ocular (usually the right one on a binocular microscope), carefully unscrew the upper lens mounting and place the reticule on the circular diaphragm or shelf which will be found approximately half way down inside (Figure 4). Replace the lens mounting and observe the markings on the reticule. If they are not in sharp focus, remove and turn the reticule over. On reticules with the markings etched on one side of a glass disc, the etched sur- face can usually be recognized by shining the disc at the proper angle in a light. The markings will usually be in the best focus with the etched surface down. If the markings are sandwiched between two glass discs cemented together, both sides are alike, and the focus may not be quite as sharp. B Observation of the Stage Micrometer Replace the ocular in the microscope and observe the stage micrometer as is illus- trated schematically in Figure 5: Calibration of the Whipple Square. On a suitably ruled form such as the one illustrated, Figure 6, Calibration Data, record the actual distance in millimeters subtended by the image of BI. MET.mic. Id. 4.70 21—i ------- Calibration and Use of Plankton Counting Equipment the entire Whippic field and also by each of Its subdivisions. This should be deternnned for each cigruficant settling of the interpupillary distance for a binocular microscope, and also for each combination of lenses employed. Since oculars and objectives marked with identical magnifi- cation, and since microscope frames too may differ, the serial or other identifying number of those actually calibrated should be recorded. It is thus apparent that the determinations recorded will only be valid when used with the lenses listed and on that particular microscope. C Use of the 20X Objective Due to the short working distance beneath a 46X (4mm) objective, it is Impossible to focus to the bottom of the Sedgewick- Halter plankton counting cell with this lens. A lox (16mm) lens on the other hand “wastes” space between the front of the lens and the coverglass, even when focused on the bottom of the cell. In order to make the most efficient use possible of this cell then, an objective of intermediate focal length is desirable. A lens with a focal length of approximately 8 mm, having a magnification of 20 or 21X will meet these requirements. Such lenses are available from American manufacturers and are recommended for this type of work. In CHECKING THE CELL The internal dimensions of a Sedgewick-Rafter plankton counting cell should be 50 mm long by 20 mm wide by 1 mm deep (Figure 8). The actual horizontal dimensions of each new cell should be checked with calipers, and the depth of the cell checked at several points around the edge using the vertical focusing scale engraved on the fine adjustment knob of most microscopes. One complete rotation of the knob usually raises or lowers the objective 1 mm or 100 microns (and each single mark equals 1 micron). Thu8, approximately ten turns of the fine adjustment knob should raise the focus from the bottom of the cell to the underside of a coverglass resting on the rim. Make these measurements on an empty cell. The use of a No. 1 or 1-1/2, 24 X 60 mm covergiass is recommended rather than the heavy coverglass that comes with the S-H cell, as the thinner glass will somewhat con- form to any irregularities of the cell rim (hence, also making a tighter seal and reduc- ing evaporation when in actual use). Do not attempt to focus on the upper surface of the rim of an empty cell for the above depth measurements, as the covergiasa is supported by the highest points of the rim only, which are very difficult to identify. Use the average of all depth measurements as the “true” depth of the cell. To simplify calculations below, it will be assumed that we are dealing with a cell with an average depth of exactly 1. 0 mm. l v PROCEDTJI E FOR STRIP COUNTS USING THE SEDGEWICK-R.AFTER CELL A Principles Since the total area of the c l is 1000 mm 2 , the total volume is 1000 mm or 1 ml. A “strip” the length of the cell thus constitutes a volume (V 1 ) 50 mm long, 1 mm deep, and the width oflhe Whipple field. The volume of such a strip in mm 3 is: V 1 50 X width of field X depth = 5OXwX1 = 50w In the example given below on the plate entitled Calibration Data, at a magnification of approximately 200X with an interpupillary setting of “80”, the width of the Whipple field is recorded as approximately 0. 55 mm (or 550 mIcrons). In this case, the volume of the strip is: V 1 = 50 w 50 X 0. 55 = 27. 5 (mm 3 ) B Calculation of Multiplier Factor In oi’der to convert plankton counts per strip to counts per ml, it is simply necessary to multiply the count obtained by a factor (F 1 ) which represents the number of tim s the volume of the strip examined (V 1 ) would be contained in 1 ml or 1000 mm 3 . Thus in the example given above: F = volume of cell in mm 3 I volume examined In mm - 1000 - 1000 — —vS— — = 36.36 = approx. 36 If more than one strip is to be counted, the factor for two, three, etc., strips could be calculated separately using the same relationships outlined above, changing only the measurement for the length of 21—2 ------- Calibration and Use 01 1anKtOfl Couzfling guiprneni I’ igurc I. TiLE COMPOUND MICROSCOPE A) coarse adjustment; B) fine adjustment; C) arm or pillar; D) mechanical stage which holds slides and is movable in two directions by means of the two knobs; E) pivot or joint. This should not be used or “broken” while counting pia kron; F) eyepiece (or ocular cf: fIgure 4); 0) draw tube. This will be found on monocular microscopes only (those having only one eyepiece). Adjustment of this tube is very helpful in calibrating the micA o cope for quantitative counting (Sec. 5. 5. 2. 2.). H) body tube. In some makes of microscopes this can be replaced with a body tube having two eyepieces 1 thus making the ‘scope into a “binocular.’ I) revolving nosepiece on which the objectives arc niount d; .1) through M arc objectives, any oiu of which cai be turned toward the object being studied. In this case J is a 40X, K is a 100X, L is a 20X, and M is a lox objective. The product of the magnification power of the objective being used times the magnification power of the eyepiece gives the total magnification of the microscope. Different makes of microscopes employ objectives of slightly different powers, but all are approximately equivalent. N) stage of the microscope; 0) Sedgwick-Rafter cell In place for observation; P) substage condenser; Q) mirror; R) base or atand;.note: for information on the optical system, consult reference 3. (Photo by Don Moran.). F I ______________ t 1I1 —G 21—3 ------- Calibration and Use of Plankton CounUng Equipment b T1 1llHhi Types of eyepiece micrometer discs or reticules (reticulea, graticulee, etc.). When dimensions are mentioned in the following description, they refer to the markings on the reticule diecs and not to the measurements subtended on the micro- scope slide. The latter must be determined by calibration procedures such as those described elsewhere. (a) Whipple plankton counting eyepiece. The fine rulings in the subdivided equare are sometimes extended to the margin of the large square to facilitate the estimation of sizes of organisms in different parts of the field. (b) Quadrant ruling with 8. 0 mm circle, for countint bacteria in milk smears for example. (C) Linear scale 5.0 mm divided into tenths. For measurement of linear dimensions. (d) Porton reticule for estimating the size of particles. The sizes of the series of discs is based on the square root of two so that the areas of successive discs double as they progress in size. a , ) : ) ( C d Figure 2 21-4 ------- Cahbration and Use of Plankton Countthg Equipment strip counted. Thus for two strips in the example cited above V 2 100W 100 X 0.55 55 mni 3 - 1000 - 1000 - F 2 - -v.--— -— - -18.2 F it will however be noted that F 2 = Likewise a factor F 3 for three strips F 1 would equal — - or approximately 12. etc. C An Empirical “Step-Off” Method A simpler but more empirical procedure for determining the factor is to consider that if a strip 20 mm wide were to be counted the 1en h of the cell, that the entire 1000 mm would be included since the cell 18 20 mm wide and 1 mm deep. This 20 mm strip width can be equated to 1000 mm 3 . If a strip (or the total of 2 or more strips) is less than 20 mm in width, the quotient of 20 divided by this width will be a multiplier factor for converting from count per strip(s) to count per ml. Thus in the example cited above where at an approximate magnification of 200X and with an interpupillary setting of 60, the width of the Whipple field is . 55 mm. Then: 20 F 1 — = 36.36 or approx. 36 (as above) If two strips are counted: 55 + 55 20 1:10 andF 2 =r_l. = 18.2= approx. 18,etc. This same value could be obtained without the use of a stage micrometer by carefully moving the cell sidewise across the field of vision by the use of a mechanical stage. Count the number of Whipple fields in the width of the cell. There should be approxi- mately 36 in the instance cited above. V SEPARATE FIELD COUNT USING THE SEDGEWICK-RAFTER CELL A Circumstances of Use The use of concentrated samples, local established programs, or other circumstances Figure 3. STAGE MICROMETER The type illustrated has two millimeters divided into tenths, plus two additional tenths subdivided Into hundredths. H III I I I I I I EnI&rgsm t a Mlcrometsr Scile W.t.r 21—5 ------- (alibration and Use of Ulankton Counting E uipmcnt Figure 4. Method of Mounting the Whipple Disc in an Ocular. Note the upper lens of the ocular which has been carefuUy unscrewed, held in the left hand, and the Whipple disc, held in the right hand. (Photo by Don Moran). 21—6 ------- Calibration and Use of Plankton Counting Equipment CALIBRATION OF WHIPPLE SQUARE as seen with lOX Ocular and 43X Objective (approximately 430X total m .gni.ftc*tion) \. __.Lmm -_...\ (lOO i) ——— PORTION OF MAGNIFIED IMAGE OF S ”AGE MICROMETER SCALE Figure 5 CALIBRATION OF THE WHIPPLE SQUARE The apparent relationship of the Whipple Square is shown as it Is viewed through a microscope while looking at a stage micrometer with a magnification of approximately 430X (lox ocular and 43X obj ective). subtend Whapple Square as seen through ocu1ac (“Whipple field’) “Large square” subtend. one tenth of entire Whipplo Square .026 mm or 26i micrometer \ - ‘1/ -- 01mm (iO ) 21-7 ------- Calibration and Use of Plankton Counting Equipment MIt t SrOPF (‘AIARRATION DATA Microet opr No 25 79 Apr oximnte M . ,t1ificntInn Tube Length, or Intq.rp , pt1Jnry iotung Linear dimenelona of Whipple equni 08 In tiutlinwtcrg* F’actor for Converejon to count/mi Whole [ .ar e JSmail I(JOX, obtained with (2 S-R Stripe) -—---- - - —-- -___ -_______ Serial No ‘/71a t’dthO%) 50 LIio C /1.3 & 9 and 0, ular S.rIal No 60 . h29k7 LáM) 70 f/to 0 ho 9 ,1 200 )1, obtained with ObJectiv Serial No t i 92I9f Ix) and Otular t 0 /29I,7qL6 14 70 400X, obtained with — Stripe) 0 . S 0 0.55p O P5 , 0/ 1.2 /7.9 0.59. 0.055 p -g 00/10 /J3 (Nannopiunkton) (eell-20 Objective Serial No and Ocular SorlalNo /A96? 2(’o. ) fielde 57) 0 6), 7 &-? 2 6’6’ 3 0o Z ’ ,0053 /7.2 g, /7F 70 2. 2e ,o • mm I 0 1 ) 0 ,,ti , II N Microscope calibration data. The fui iii shown is suggested for the recording of data pertaining to a particular microscope. Heathngs could be modified to suit local D I. AQ. p1. 8b. 7.66 Figure 6 situations. For example, ‘Interpupillary Setting” could be re , laced by “Tube Length” or the “ZS-R Strips could be replaced by “per field” or “per 10 fields. 21-8 ------- Approximate Magnification irneri S Calibration and Use of Plankton Counting Equipment MICROSCOPE CALIBRATION DATA Microscope No. 100X. obtained with Objective Serial No. and Ocular Serial No 200X, obtained with________ ________ (2 S-R Strips) Objective Serial No. and Ocular Serial No. . (Nannoplankton) I with (cell-20 fields 400X obtaine C Objective Serial No. and Ocular Serial No. _______ 5 lmm = 1000 microns SI. AQ. p1 8 10. 60. Figure 6-A MICROSCOPE CALIBRATION DATA Suggested work sheet for the calibration of a microscope. Details will need to be adapted to the particular instrument and situation. (2 S-R Strips) 21-9 ------- Calibration and Use of Plankton Counting Equipment Figure 7 A cube of water as seen through a Whipple square at bOX magnification in a Sedgewick-Rafter cell. The figure Is drawn as If the microscope were focused on the bottom of the cell, making visible only those organisms lying on the bottom of the cell. The little “bug” (copepod) halfway up, and the algae filament at the top would be out of focus. The focus must be moved up and down in order to study (or count) the entire cube. S-R COVER GLASS —WHIPPLE SQUARE WATER IN S-R CELL LTHKKNESS SR SLIDE 21-10 ------- Calibration and Use of Plankton Counting Equipment may make it necessary to employ the more conventional technique of counting one or more separate Whipple fields instead of the strip count method. The basic relation- ships outlined above still hold, namely: F = volume cell in mm 3 volume examined in mm 3 The volume examined in this case will consist of one or more squares the dimen- sions of the Whipple field in area and 1 mm in depth (Figure 7). Common practice for routine work is to examine 10 fields, but exceptionally high or low counts or other circumstances may indicate that some other number of fields should be employed. In this case a “per field” factor may be determined to be subsequently divided by the number of fields examined as with the strip count. The following description however is based on an assumed count of 10 fields. As stated above, the total volume represented in the fields examined con- sists of the total area of the Whipple fields multiplied by the depth. V 4 (side of Whipple field) 2 X depth (1 mm) X no. of fields counted) For example, let us assume an approxi- mate magnification of bOX (see Figures 6 and 7 and an interpupillary setting of “50”. The observed length of one side of the Whipple field in this case is 1. 13 mm. The calculation of V 4 is thus: V 4 side 2 X depth X no. of fields l.l3Xl.l3XlXlO 12.8 m m 3 The multiplier factor is obtained as above (Section IV A): volume cell in mm 3 volume examined in mm - 1000 - (approx.) 78 (If one field were counted, the factor would be 781, for 100 fields it would be 7. 8.) NANNOPLANKTON COUNTIb 1000 and F 5 = —n- = (approx.) 1850 REFERE NCES B Principles Involved For counting nannoplankton using the high dry power (lOX ocular and 43X objective) and the “nannoplankton counting cell” (Figure 9) which is 0. 4 mm deep, a minimum of 20 separate Whipple fields is suggested. The same general relationships presented above (Section IV) can be used to obtain a multiplier or factor (F 5 ) to convert counts per 20 fields to counts per ml. To take another example from Figure 4, at an approximate magnification of 400X and an interpupillary setting of 70 (see also Figure 3) we observe that one side of the Whipple field measures 0. 260 mm. The volume of the fields examined is thus obtained as follows: V 5 = side 2 X depth X no. of fields 0.26 X 0. 26 X 0.4 X 20 = . 54 mm 3 C Calculation of Multiplier Factor It should be noted that the volume of the nannoplankton cell, . 1 ml, is of no significance in this particular calculation. F 4 1 American Public Health Association, et. al. Standard Methods for the Examination of Water, Sewage, and Industrial Waste8. 13th Edition. Am. Public Health Assoc. New York. 1970. 2 Jackson, H. W. and Williams, L. G. The Calibration and Use of Certain Plankton Counting Equipment. Trans. Am. Mic. Soc. LXXXj(l):96-103. 1962. 3 Ingram, W. M. and Palmer, C. M Simphfied Procedures f or Collecting, Examining, and Recording Plankton in Water. Jour. Am. Water Works. Assoc. 44(7): 617-724. 1952. 4 Palmer, C M Algae in Water Supplies. U. S. D. H. E. W. Public Health Service Pub. No. 657. 1959. 5 Palmer, C. M and Maloney. T E. A New Counting Slide for Nannoplankton. American Soc. Limnol. and Oceanog. Special Publications No. 21. pp. 1-6. 1954. 2 1—11 ------- Calibration and Use of Plankton CountIng Eauipment Sedgewick-Rafter counting cell 8hOwiflg bottom scored across for ease in counting strips. The “strips” as shown in the illustration simply represent the area counted, and are not marked on the slide. The conventional dimensions are 50 X 20 X 1 mm, but these should be checked for accurate work. L. Figure 9 Nannoplankton cell. Dimensions of the circular part of the cell are 17. 9 mm diameter X 0. 4 mm depth. When covered with a covergiass, the volume contained is 0. 1 ml. The channels for the introduction of sample and the release of air are 2 mm wide and approximately 5 mm long. This slide Is designed to be used with the 4 mm or 43X (high dry) objective. 6 Welch, Paul S. Limnological Methods. Blakiston Company. Phila. Toronto. 1948. Drinking Water. John Wiley aid Sons. New York. 1948. 7 Whipple, C. C., Fair, 0. M., and Whipple, M. C. The Microscopy of This outline was prepared by H W. Jackson, Chief Biologist, National Training Center, DTTB, MDS, OWP , EPA, Cincinnati, OH 45268. Figure 8 21—12 ------- DETERMINATION OF ODORS I INTRODUCTION III PRECAUTIONS Odor shall be determined 8Ubstaritially as prescribed by the 11th edition of ‘Standard Methods for the Examination of Water and Sewage”, subject to certain stipulations and modifications made necessary by the Inter- national Joint Commission. The procedure and technique to be followed are described below Ii REAGENTS AND APPARATUS A Odor-free water - prepared by passing tap water through activated carbon at a slow rate of speed. Activated carbon can be placed at the bottom of a 20-liter glass bottle The bottle can be connected to the tap by rubber tubing leading to glass tubing above the water. The outlet from the bottom of the bottle should be glass tubing. A trap made of inch glass tubing filled with activated carbon is placed at the end of the outlet. B 500 ml glass-stoppered Erlenmeyer flasks, each flask with a number. Glass- ware must be thoroughly cleaned and rinsed several times with odor-free water before each use. C Chemical Thermometer (0-100°C) D 10 ml Mohr pipettes, 25 ml graduated cylinders, 50 ml graduated cylinders, 200 m.l graduated cylinders, 500 ml graduated cylinders. Other pipettes and cylinders as needed. E One liter glass-stoppered bottles to hold samples of water being examined Other glass bottles and flasks as needed. A Certain conditions are required to obtain consistent results. Considerable practice with the test is desirable to develop consistent sensitivity to the sense of smell 1 In view of the perishability of the odor test, these determinations should be made immediately after collection. 2 The prepared odor-free water shnuld be truly free of all detectable odor. 3 All glassware must be free of odor. This is accomplished by thorough cleansing followed by several rinses with odor-free water. 4 All dilutions should be compared with an odorless standard. This aids the observer in deciding whether air odor is present or not. 5 All dilutions when examined for odor should be of a uniform temperature, deviation not to exceed 1°C. 6 A sudden change in the character of the odor during the testing procedure should be considered as a warning that there may be interference from outside odors or that the diluting water may not be odor-free. The character of odor should always be recorded for future consideration. B To eliminate psychological influences, the samples should be coded and in- terni .xed so as not to suggest to the observer what odor concentration is being observed. I Bottles should be colored or covered with odor-free matezial or the observer blindfolded to eliminate auto suggestion WS. TO. lab. la. 1. 66 22—1 ------- Determination ol Odors since many samples may possess color or turbidity. 2 Test should be conducted in a room free of outside odors. The observer should be cautioned to refrain from smoking or eating for an appreciable time before taking test. Odors should be washed from the hands prior to taking test. 3 The test should not be prolonged to a point where the sense of smell becomes fatigued. IV PROCEDURE A To obtain the approximate range of odor value take 50 ml, 14 ml and 5 ml of sample and make each sample up to 200 ml with odor-free water. Compare the odor of these three with 200 ml of odor-free water. 1 Cold odor: Bring dilutions to temper- ature of 24 - 2 5°C. 2 Shake each flask uniformly before smelling for odor. Observer should characterize type of odor. 3 Note which flasks contain odor and which do not. According to result8 obtained, prepare intermediate di- lutions, in each case using sufficient odor-free water to make a total volume of 200 ml. 4 Include a flask with 200 ml of odor- free water with each series, as a blank for comparison. B Arrange flasks so that their identity is unknown and bring to desired temperature. I Observe for odor and make chart with a “plus” or “zero” for each dilution. 2 The results are reported in “threshold odor numbers”. The threshold odor number is calculated from the amount of sample in the most diluted portion which gives perceptible odor. The volume of the d.ilution(200 ml) divided oy the volume of the sample In the dilution equals the threa hold odor number. For example, if 5 ml diluted to 200 ml is the most dilute portion giving perceptible odor: 40, the threshold odor is numbered 40. C The threshold odor number shall not be confused with the “threshold odor con- centration”. The threshold odor concentra- tion is the smailest amount of odor-producing material in mg/i required to give perceptible odor. If the threshold odor concentration is known, that value multiplied by the threshold odor number will give the concentration of the odor-producing material in the sample. This outline was prepared by E. L. Robinson, Research Aquatic Biologist, Fish Toxicology Laboratory, 3411 Church Street, Newtown, OH 45244. 22-2 ------- Determinatjoii of Odors ROBERT A. TAFT SANITARY ENGINEERING CENTER AQUATIC BIOLOGY ALGAL THRESHOLD ODOR EXPERIMENT Amount of Culture ________________ ml Exp. No. ________________ Age of Culture___________________ days Temp. Tested at °C No. Cells per ml Culture Medium________ Mixed, Unialgal, Pure Date___________________ Recorder Observer No. 1 Observer 2 3 4 1 6 j 7 Flask Culture Dilution R R R R R Threshold Odor No. Description of Odor + Odor Detected 0 No Odor Detected Remarks Estunated Composite* T. 0. No. ________________________________ *Geometric average of T.O. No. of individual observers E. L. R. 1956 22-3 ------- DETERMINATION OF PLANKTON PRODUCTIVITY I INTRODUCTION IV STANDING CROP METHOD Primary production is the synthesis of organic matter from inorganic raw materials. The energy required for this process may come from light (photosynthesis), or from chemical sources (chemosynthesls). The primary synthesis of organic matter in lakes and streams is carried on by planktonic and ben- thic algae and bacteria, and aquatic macrophytes. II PHOTOSYNTHESIS The productivity of a body of water Is indicated in a general way, by the density of the plankton population. The standing crop of plankton is commonly measured by determining one or more of the following: A Dry and Ash-free Weight of Seston B Cell or Unit Counts C Cell Volume The photosynthetic process involves the up- take of CO 2 and the release of 02. The reactions are enzyme catalyzed and are af- fected by the following factors. A Temperature B Light Intensity C Light Quality D Chlorophyll E Particulate and Dissolved Carbohydrate F Particulate and Dissolved Organic Carbon Increases in the standing crop over a period of time may be used to determine productivity. However, this method provides only a rough approximation of the rate of primary production. D pH E Nutrients V OXYGEN METHOD F Trace Elements III MEASURING PRODUCTIVITY Methods employed to measure plankton pro- ductivity are: A Standing Crop B Oxygen C pH D Carbon-14 The use of dissolved oxygen to determine short-term rates of primary production was introduced by Gaardner and Gran (192?). Estimates of the amount of carbon fixed are based on the premise that one molecule of oxygen is given off for each atom of carbon assimilated. CO 2 + H 2 0 -. CH 2 O + 02 A “ Light ” and “ dark ” bottles are filled with sample and resuspended at various depth8 for 4 - 24 hours. B The concentration of dissolved oxygen is determined (using the Wink.ler Method) at B!. ECO. pro. la. 4.70 23—I ------- Determination of Plankton Productivity the beginning and end of the incubation period. The values obtained are as follows 1 Final “light” bottle 02 — initial 02 net photosynthesis 2 InItial 02 - Final “dark” bottle °2 = respiration 3 Net photosynthesis + respiration gross photosynthesis This method has some serious dlsadvantages A The bottles provide an artificial substrate for the prohferat on of bacteria which use up large amounts of °2’ resulting in erroneously high respiration and low net photosynthesis values. B The lower limit of sensitivity of the Winkler Method is 0.02 mg 02/liter. This is a serious handicap when working in oligo- tropic lakes and the open sea. VI CARBON- 14 METHOD The use of carbon- 14 for the measurement of the rate of carbon assimilation by phyto- plankton was pioneered by E. Steernann NIelsen (1952). The method is simple and very sensitive. A Carbon- 14 labelled sodium bicarbonate (4 - l0 ic/liter) is added to ‘ g t” and “ dark ” bottles, which are resuspended in the water for 4 - 24 hours. B An aliquot of the sample is passed through a membrane filter (1.2 p. pore diameter), and the filters are treated with acid to remove any inorganic labelled carbon. C The (beta) activity of the filter is deter- mined with an end-window Geiger tube, or with gas flow or liquid scintillation techniques. D The carbon fixed is determined as follows There are several important disadvantages in this method. A Some of the labelled photosynthesis pro- ducts will be broken down immediately by reapiration, and the liberated carbon-14 reused in photosynthesis. Therefore, it is generally agreed that the method mea- sures only net photosynthesis. B It has been found that the algae rapidly ex- crete up to 50% of the photosynthate In the form of organic acids, carbohydrates, and amino acids. Since these labelled materials are not retained by the filter, they escape detection. VU pH METHOD The uptake of CO 2 by the algae during photo- syntheses results in an increase in the pH of the surrounding medium. Periodic pHmeaaure— ments are made of the body of water being studied, and the carbon uptake is determined using published nomographa. Verduin (1952) used this method in a study of the productivity of Lake Erie. However the method has not gained wide acceptance because it can be used only in waters with low alkalinity. REFERENCES 1 Allen, M. B. Excretion of Organic Com- pounds by Chlamydomonas. Arch. f. Miicrobiol. 24 163-168. 1956. 2 Curl, H. Jr., and Small, L. F. Variations in Photosynthetic Assimilation in Natural Marine Photoplankton Communities. Limnol. Oceanogr. lO(Suppl.):R67-R73. 1965. 3 Gaardner, T., and Gran, H. H. Investi- gations of the Production of the Plankton in the Oslo Fjord. Rapp. et Proc. - Verb., Con. Internat. Explor. Mer. 42 1-48. 1927. Correction for carbon - activity on filter < available < isotope fixed - total activity added HCO discrimination 2 . —2 ------- Determination of Plankton Productivity 4 Goldman, C. R. Molybdenum as a Factor Limiting Primary Productivity in Castle Lake, California. Science 132: 1016-1017. 1960. 5 Kamen, M. D. Primary Processes in Photosynthesis. Academic Press, New York. 1963. 6 Marshall. S. M., and Orr, A. P. Carbo- hydrate as a Measure of Photoplankton. J. Mar. Biol. Aesoc. U.K. 42:511-519. 1962. 7 Ryther, J. H. Photosynthesis in the Ocean as a Function of Light intensity. Limnol. Oceanogr. 1:61-70. 1956. 8 Steemann Nielsen, E. The Use of Radio- active Carbon (C-14) for Measuring Organic Production in the Sea. J. Con. Internat. Explor. Mer. 18 117-14O. 1952. 10 Verduin. J. Photosynthesis and Growth Rates of Two Diatom Communities in Western Lake Erie. Ecology 33(2): 163—168. 1952. 11 Vernon, L.P. Bacterial Pbytosyntheala. Ann. Rev. Plant. Physiol. 15:73-100. 1962 12 Wetzel, R. G. A Comparative Study of the Primary Productivity of Higher Aquatic Plants, Per iphyton, and Phytoplankton in a Large, Shallow Lake. Internat. Rev. Hydrobiol. 49:1-61. 1964. 13 Yentsch, Charles S. The Measurement of Chioroplastic Pigments- Thirty Years of Progress? pp. 255-270 in Chemical Environment In the Aquatic Habftat. Proc. IBP Symposium. Amsterdam. 1967. (N.y. Noord- Holiandsche Uitgevers Maatschappij. Amsterdam, Netherlands. 8.95) 9 Strickland, J. D. H. Measuring the Production of Marine Phytop]ankton. Bull. Fish. Res. Bd. Can. No. 122: 1-172. 1960. This outline was prepared by C. I. Weber, Chief, Biological Methods Branch, Analytical Quality Control Laboratory, NERC, EPA, Cincinnati, OH 45268. 23—3 ------- LABORATORY: PROPORTIONAL COUNTING OF P LANKTON I OBJECTIVE b Move the slide at random and repeat the process. Do this for 5 or 10 To learn and practice the techniques of fields, or for one or two stripe. proportional counting of mixed plankton samples. c Tally the results and compute the percent of each type. U MATERIALS 2 Five hundred count A Several plankton samples, each containing a Moving the slide at random count a number of plankton forms, and tally all the types of plankton as before until a total of 500 cells B Class slides, cover slips, and dropping or clumps have been counted. pipets. b TaUy the results and compute the percentage of each type as before. UI PROCEDURES A Make an ordinary wet mount of the IV RESULTS sample provided. A Record your results for both methods B Scan the slide. Identify and list all types on the board. of plankton present. B Discuss the two methods and the use of C Proportional Counting (use clump count) the proportional count results. 1 Field count a Count and tally all individuals of each type present In a field. The beat way to do this is to list the _____________________________________ most common types separately and This outline was prepared by M. E. Bender, record the counts and then enumerate Biologist, formerly with FWPCA Training the other forms. Activities, SEC. BI.MIC.enu.]ab. 6a. 8.69 24—I ------- LABORATORY. CALIBRATION OF PLANKTON COUNTING EQUIP!V NT I OBJECTIVES D Record the exact dimensions of the entire field in the column marked “Whole” on the plate “Microscope Calibration Data.” A To Become Familiar with Microscope Calibration Procedures E Do the same with the 200X and 400X magnifications. B To Calibrate the Particular Equipment Assigned to you F Return the stage micrometer to the supply table. II MATERIALS G Values for the “Large” and “Small” columns may now be calculated arithmetically. There are ten large A Whipple, Plankton Counting Reticule squares across the whole field, and 5 small squares across the large square B Compound Microscope as Assigned which is subdivided 1 in the center of the field. C Stage Micrometer H Calculate the conversion factors to counts per ml according to the formulae in the UI PROCEDURE lecture entitled “Calibration and Use of Plankton Counting Equipment.” A Adjust the interpupillary distance to the position most comfortable for your eyes. and record the setting on the “Microscope Calibration Data” sheet. B Install a Whipple plankton counting reticule in the right eyepiece. This outline was prepared by H. W. Jackson C Obtain a stage micrometer and focus on the chief Biologist, National Training Center, scale at 100X magnification. DTTB, OWP, EPA, Cincinnati, OH 45288. BI. MET. mic. lab. la. 5. 70 251 ------- Laborstory Calthration of Plankton Counting Equipment and Ocular Serial No. MICROSCOPE CALIBRATION DATA Microscope No. Length, or Magnification Interpuptilary - Setting Linear dimensions of Whipple squares in millimeters* Factor for Conversion tO count/mi EEte T ube__ whole ge 5mah1 l00X obtained with (2 S-R Strips) Objective Serial No. and Ocular Serial No 200X, obtained with (2 S-R Objective Serial No. and Ocular Serial No. 400X, obtained with (Nannoplankton) (cell-20 fields Objective Serial No. •lmm 1000 microns 81. AQ. p1 8 10. 60. 25—2 ------- Laboratory: Calibration of Plankton Counting Eq pment and Ocular Serial No. MICROSCOPE CALIBRATION DATA Microscope No. Approximate Magnification Length, or Tube 1 Linear dimensions of Whipple Interpupulary aquarea in millimeters* Setting Whole Large Small Factor for Conversion to count/mi bOX, obtained with (2 S-R Strips) Objective Serial No. and Ocular Serial No 200X, obtained with________ Objectiv — (2 S-R Strips) Serial No. and Ocular Serial No. 400X. obtained with (Nannoplankton) (cell-20 fields Objective Serial No. *1mm 1000 microns BI. AQ. p1. 8 10. 60. 25—3 ------- LABORATORY FUNDAMENTALS OF QUANTITATIVE COUNTING I OBJECTIVE C Starting from one end of the S-R cell and proceding to the opposite (this is called a To learn and practice the basic techniques of strip count, begin counting (clump counts) quantitative plankton counting the plankton forms. The length of the cell may be traversed in several ways. U MATERIALS 1 Count all the forms in the Whipple square or in a portion of the square, record the count and move the slide so A Plankton Samples Containing a Variety of that the square covers the adjoining Plankton Forms area. B S-R Cells and Covergiasses , Large Bore 2 Move the slide very slowly counting 1 ml Pipettes, Whipple Di8cs. Plankton and recording the various forms as Record Form they pass the leading edge of the Whipple disc. m PROCEDURE IV RESULTS A FLU the S-R cell with sample number 1 as follows: A Using the conversion factor obtained In the previous laboratory compute the Place the covergiass diagonally across number of plankton organisms per ml. the S-R cell. This leaves the other two corners uncovered; one for putting in B Record the results on the board. the sample fluid, the other to allow air to be driven out as it is replaced c Discussion of Results by the incoming aliquot. Shake the sample to disperse the plankton. Before D Refill the slide with a fresh aflquot and settling occurs in the sample draw about recount the sample. Compare results 1-1/4 ml of the fluid into the pipette with the first count. and quickly fill the S-R cell by delivering the aliquot into one of the Open corners E Count the other samples of mixed plankton of the chamber, as assigned, following the same procedure. B Using lOOx focus on the sample. After focus has been obtained switch to 2 00x. This outline was prepared by M. E. Bender, Scan the slide and list the plankton forms Former Biologist, FWPCA, Water Pollution present. Training A ctivities, SEC. BI.MIC.enu.lab.7.6.68 26-1 ------- Laboratory Fundamentals of Quantitative Counting PIAPUTON COUNT UCO4D .ourr DO _________ nod, Dot. Cot i..tod ________________ D.t. AnsIps .d u .n.. D.ptb C0U..t,r 41 4IC .50 pI 4 4 44 io mo 4*4,0 rDO4 P PLOOrVOD DOCDODA HWJ 4, ’ . Mot•• T i) i.y. T ot al Vol Ar... 07 +f D Of Count flintfl cot ion .nltlt.otor Count r .t lit.r Group Tutu ). • TITTAL T Grist inns, Cl ff.r.nti.l 000.1 4 .. = -. .--—- - - — .- ,IiW UNtDITIDOD ii.. of O.p Air T..p 4.1.7 T.ou lottlof tOdOr P 700 100. p..m4*r To,bidltp 44ttcd T 7004 10 (1 441usd of I. ,) 1001 1001 7, 1 .1 Vol Cnil.ot.d PnUr,, lit. C.n.n.) CondIIlou’,o fIlt ..ttuo. AIf . . 0*007 Pi.nt.i Purl.,. 5000 47.4 (liii odor v not., Olt.r Ohp• irsl or C.l0.l D.tt IA A7 T AJALTIT4 N.tbod of Itoparatlon DAp.rtur. (no. 144014*, ul4lI(IC .410 Of 14041*4 ?nn ltot f4 . IdodI (SAX? 04 OfflI DATA. (04 i i Trn.Ia.At to Dunn. a .. c c i 00447 CM.ioll. RV7tAlt D I TI ’nII*4DI Toot. SM Odor 0.. floi.d 7 11 10, nun. 0*457 26-2 ------- A LGAL GROWTH POTENTIAL TEST I INTRODUCTION Dense growths of algae in surface waters are aesthetically undesirable, cause problems In water treatment, produce changes In the aquatic environment that are harmful to fish and other aquatic life, and are symptomatic of pollution. The density of phytoplankton opulationa is directly related to the concentration of nutrients. This relationship has been well documented, and is now embodied in the concept of trophic level or trophic status of surface waters. One or more of the following parameters are commonly used to descrthe the trophic status: (a) nutrient concentration - principally N and P, (b) algal count, (c) chlorophyll concentration, (d) primary productivity. (e) particulate organic matter, (f) oxygen depletion in the hypolimnion, and (g) phytoplankton species composition or Indicator species (Rawson 1956; Davis 1964; Goldman & Carter 1965, Oglesby & Edmondson 1986; Fruh, Steward, Lee & Rohltch 1986). II EUTROPHICATION Three general trophic levels now recognized, here arranged in ascending order, are: oligotrophic (low), mesotrophic (intermediate), and eutrophic (high). The addition of nutrients to surface waters raises the trophic level and results in an increase in phytoplankton density and changes in the species composition. This process, commonly referred to as eutrophication, is greatly accelerated by the discharge of nutrient-laden domestic and industrial wastes (Hasler 1947), Edznondson & Anderson 1956). III MEASUREMENTS OF TROPHIC LEVELS Although chemical analyses provide Information on the concentration of nutrients, their availability to the algae can be determined only by biological assay. Biological assays to determine the potential (algal) productivity of surface water were first used in the late twenties (Schreiber 1927) and early thirties (Strom 1933). but until recently had been used only infrequently (Potash 1956, Skulberg 1964, 1967; Shelef & Halperin 1970). In 1987, the Joint Industry-Government Task Force on Eutrophication took steps to develop a standardized algal growth potential (AGP) test. Using this test, one can: A Evaluate the effectiveness of waste treatment processes In removing elements that support or stimulate the growth of algae B Determine at what point along the time scale of progressing eutrophication the water of a given lake or stream happens to lie (trophic status). C Anticipate the effect on algal production of introducing extraneous nutrients. D Determine the extent to which nutrient levels must be reduced in a body of water to effect an acceptable remedy. IV BASIC STEPS OF ALGAL GROWTH POTENTIAL TEST A A surface (test) water sample is collected and the indigenous microorganisms are removed by filtration (0. 45 micron membrane filter at 15 inches of mercury) or ultracentrifugatlon. B The surface water and standard medium (Table 1) are inoculated with 1000 cells/mi of Selenastrum capricornutum , or 50, 000 cells/mi of Anabaena flos-aquae or Microcystis aeruginosa . C The cultures are prepared in triplicate and incubated 7-10 days at 24° C, 200 fc (blue-greens) or 400 fc ( Selenastrum ) continuous illumination, with shaking at 100 oscillatIons/mm ( ilturing may be by flask, chemostat, or in situ technique). D Algal growth is measured daily by (1) cell counts, (2) determining the B!. BlO. alg.Lb. 10.72 27—1 ------- AIKaI Growth Potential Test TABLE 1 MAY. 1970 VERSION OF PAAP NUTRIENT BASAL MEDIUM (This formula consists of 30% of the concentrations of the macroelements listed in the February, 1969, PAAP Booklet. The Na 2 CO 3 was replaced by NaHCO 3 , and the EDTA was reduced to 333 Mg/i.) MACROELEMENTS : (milligrams per liter) Compound Final Conc . Element Element Furnished Conc . NaNO 3 25.500 N 4.200 K HPO 1.044 P 0.186 2 4 K 0.469 MgC1 2 5.700 Mg 1.456 MgSO •7H 2 0 14.700 Mg 1.450 S 1.911 CaC 1 2 2H 2 O 4.410 Ca 1.202 NaHCO 3 15.000 Na 1l.OOj If the medium is to be filtered, add the following trace-element-tron-EDTA solution from a single combination stock solution after filtration. With no filtration, K HPO should be added last to avoid iron precipitation. Stock solutions of ?nd1vi ua1 salts may be made up In 1000 X’s final conc. or less. MICROELEMENTS : (micrograms per liter) H 3 B0 3 185.5 B 32.5 MnCI 2 264.3 Mn 115.4 ZnC 1 2 32.7 Zn 15.7 CoC1 2 0.780 Co 0.354 CuCl 2 0.009 Cu 0.004 Na 2 MoO 4 •2H 2 0 7.26 Mo 2.88 FeCl 3 96.0 Fe 33.05 Na 2 EDTA2H 2 O 300.0 27—2 ------- A1 ia1 Grnwth PnfPnflkl Tecit chlorophyU content, In vivo f1uorescen e, light scattering or optical density (600 nm) of the culture, (3) measurIng the C- 14 uptake, or (4) determining the dry weight of the algae at the end of the Incubation period. Regardless of the parameter used to measure growth response, the result should always be expressed in terms of the final dry weight of the culture. E The growth response of the alga in the test water is compared to its growth in the standard medium. V PHASES OF THE TEST STILL UNDER STUDY INCLUDE: A Composition of the standard growth medium. B Effects of ventilation and shaking on the growth response of batch cultures. C Techniques of measuring growth response. D Techniques of removing indigenous microorganisms from test surface waters. VI For copies of the Provisional Algal Assay Procedure and information on the availability of subcultures of the test organiazn, contact: Dr. A. F. Bartach, Chairman JTF Research Program Group Director, Pacific Northwest Water Research Laboratory Corvallis, Oregon 97330 REFERENCES _________ Provisional Algal Assay Procedure. Joint Industry-Government Task Force on Eutrophication, P.O. Box 3011, Grand Central Station, N.Y. 10017. 1969. 2 Davis, C. C. Evidence for the eutrophication of Lake Erie from phytoplankton records. Umnol. Oceanogr. 9:275. 1964. 3 Edmondson, W. T. and Anderson, G. C. Artificial eutrophication of Lake Washington. Limnol. Oceanogr. 1 (1):47—53. 1956. 4 Fruh, E.G., Stewart, K.M., Lee, G.F., and Rohlich, G.A. Measurements of Eutrophication and Trends. JWPCF 38(8):1237-1258. 1966. 5 Goldman, C.R. and Carter, R.C. An investigation by rapid C’ 4 bioassay of factors affecting the cultural eutrophication of Lake Tahoe, California. JWPCF 37:1044- 1063. 1965. 6 Hasler, A. D. Eutrophication of lakes by domestic drainage. Ecology 28(4): 383-395. 1947. 7 Oglesby, R. T. and Edmondson, W. T. Control of Eutrophication. JWPCF 38(9):1452-1460. 1986. 8 Potash, M. A biological test for determining the potential productivity of water. Ecology 37(4):631-639. 1956. 9 Rawson, D. S. Algal Indicators of lake types. Limnol. Oceanogr. 1:18-25. 1956. 10 Schrelber, W. Der Reinkultur von rnarinem Phytoplankton und deren Bedeutung fur die Erforschung der Produktions-fahigkeit des Meerwasaers. Wissensch. Meeresunters., N.F. 16:1-34. 1927. 11 Shelef, G. and Halperin. R. 1970. Wastewater nutrients and algae growth potential. In: H. I. Shuval, ed., “Developments in Water Quality Research”, Proc. Jerusalem Internat’l. Con!, on Water Quality and Poll. Rca., June, 1969. Ann Arbor-Humphrey Science Pubi., p. 211-228. 1 27—3 ------- Algal Growth Potential Test 12 Sku]berg, O.M. A].galproblems related to the eutrophication of European water 8uppUes. and a bioassay method to assess fertilizing Influences of pollution on th.land waters. In: D.F. Jackson 1 ed., “Algae and Man”, Plenum Press, N. Y. p. 262-299. 1964. 13 Skulberg, O.M. Algal cultures as a means to assess the fertilizing Incluence of pollution. In: Advances in Water Pollution Research, Volumn 1, Pergamon Press, Washington, D. C. 1967. 14 Strom, K. M. Nutrition of algae. Experi- ments upon; the feasibility of the Schrelber method in fresh waters, the relative inportance of Iron and manganese In the nutritive medium; the nutritive substance given off by lake bottom muds. Arch. Hydrobiol. 25:38-47. 1933. ADDITIONAL RECENT REPORTS: _______________ Algal Assay Procedure Bottle Test. 82 pp. Environmental Protection Agency, National Eutrophica- tion Research Program, Corvallis, Oregon. 1971. -. Inter- Laboratory Precision Test. An Eight-Laboratory Evaluation of the Provisional Algal Assay Procedure Bottle Test. 70 pp. Environmental Protection Agency, National Eutrophication Research Program, Corvallis, Oregon. 1971. 3 Berge, G., Predicted Effects of Fertilizers Upon the Algae Production in Fern Lake. Fisk Dir. Skr. Serv, Hay. Unders., 15:339-355. 1969. 4 Jonnson, J.M., T.O. Odlaug, T.A. Olson, and O.R. Ruschmeyer. The Potential Productivity of Freshwater Environ- ments as Determined by an Algal Bioassay Technique. Water Resources Research Center Bulletin No. 20, University of Minnesota, Minneapolis. 1970. 5 Maloney 1 T.E., W.E. Miller, andT. Shiroyama. Algal Responses to Nutrient Additions in Natural Waters. I. Laboratory Assays. In: Special Symposia 1:134-140. Amer. Soc. Limnol. Oceanogr. 1972. 6 MIller, W.E., andT.E. Maloney. Effects of Secondary and Tertiary Wastewater Effluents on Algal Growth in a Lake-River System. JWPCF 43(12)2381—2365. 1971. 7 Murray, S., J. Scherfig, and P.S. Dixon. Evaluation of Algal Assay Procedures- PAAP Batch Test. .JWPCF 43(10): 1991—2003. 1971. 8 Shapiro, J., and R. Riberiro. Algal Growth and Sewage Effluent In the Potomac Estuary. JWPCF 37(7): 1034-1043. 1965. 9 Toerien, D. F., C. H. Huang, J. Radimsky, E. A. Pearson, and J. Scherfig. Final Report, Provisional Algal Assay Procedures. 211 pp. Sanitary Engineer- ing Research Laboratory Report No. 7 1-6, University of California, Berkeley. 1971. This outline has been prepared by Dr. C. I. Weber, Chief, Biological Methods Section, Analytical Quality Control Laboratory, NEEC, EPA, Cincinnati, OH 45268. 1 2 27—4 ------- ALGAE AND ACTINOMYCETES IN WATER SUPPLIES I Water treatment always should include detection and control of microorganisms. A Two types of microorganisms are involved 1 Pathogenic types include such forms as the typhoid bacteria, the dysentery ameba, and the Infectious hepatitis virus. 2 Interference types include taste and odor organisms, filter-clogging organisms, pipe-infesting organisms, and others. B Water treatment practices are closely associated with these organisms. 1 For pathogens, practices include coliform tests, use of chlorine, and guarding the water supply against fecal pollution. 2 For interference organisms, practices include plankton enumeration, use of copper sulfate and the covering of reserviOrs. 3 Many of the other treatment practices have significant effects on the organisms. C This discussion will be limited to the inter- ference organisms. II EXAMPLES OF PROBLEMS CAUSED BY INTERFERENCE ORGANISMS A At Chicago, the alga Dinobryon reappears almost every year, generally In June and July in numbers sufficient to impart a prominent fishy odor to the water. In 1951, it required an estimated $70, 500 worth of activated carbon to control the odor of this organism for a period of two months. B At Indianapolis, copepods were present in parts of the distribution system In numbers sufficient to be visible in the drinking water. The eggs of the copepode were found to pass through the filters and to hatch In the distribution system. C At Oklahoma City, prominent earthy odors have appeared frequently. The organisms blamed for this trouble are the mold-like actinomycetes. D At Peoria, white wigglers up to 3/8” long were reported in the tap water, during early March, 1956. These chironomid larvae had hatched in the city’s open reservoir, requiring that the reservoir be drained, cleaned and treated with a larvicide. E At Chicago, diatoms are a very important cause of short filter runs. The one diatom TabeLlarja is considered to be more responsible than any other organism for this trouble. F In Ontario, the alga Cladophora often grows in large numbers attached to rocks on the shoreline of lakes. When the alga is broken loose it collects near the shore- line and gives rise to very offensive odors. G In a water supply impoundment in Utah the plankton algae frequently cause the pH of the water to increase to 8. 3 or higher, requiring that the water be treated with acid to obtain the desired pH of 8 or lower. H In Texas a water supply from underground sources was stored in a large open settling basin. Osciflatoria and unicellular green algae developed in large numbers in the stored water, turning it green and pro- ducing a strong odor. I Los Angeles has more than 25 open reser- voirs of various sizes and ranging in elevation from almost sea level to over BI.MIC. 12c. 3.70 28—1 ------- Algae and Actinomycetes in Water Supplies 7,000 feet. Many tons of copper sulfate are used every year in these reservoirs for rigid control of plankton, chiefly diatoms and occassionally blue-green algae. This treatment is carried out to improve the water quality including the reduction of tastes and odors. III TYPES OF PROBLEMS CAUSED BY INTERFERENCE ORGANISMS A Tastes and Odors 1 May be caused by algae, actinomycetes, crustacea, and anaerobic bacteria. 2 Common algal odors imparted to water are ones described as fish, earthy, musty, grassy, cucumber, geranium, nasturtium, and septic. 3 Common actinomycete odor is earthy. 4 Tastes produced in water by algae include sweet and bitter. 5 Other causative agents of tastes and odors may be industrial wastes, sludge, and compounds dissolved from soil and rock, and chemicals used in treatment. B Filter Clogging 1 Both rapid and slow sand filters are affected. 2 Diatoms are the organisms most frequently involved but blue-green algae, filamentous green algae and other organisms as well as silt may cause it. C Other Pro ems in the Treatment Plant 1 Algae may cause variation in the pH, hardness, color, and organic content of the water. 2 Amount of plankton organisms often influences the rate and effectiveness of coagulation. 3 Qilorlne dosage may depend upon amount of plankton organisms present. 4 Growths of algae may reduce the flow through thfluent channels and screens. 5 Organisms may be responsible for increasing the quantity of sludge to be disposed of in sedimentation basins. 6 Miorocrustacea “spot” paper in paper mill rolls. D Infestation of Distribution Systems 1 Attached organisms reduce the rate of flow in the pipes. 2 Iron and sulfur bacteria may initiate or stimulate corrosion of pipes. 3 Organisms may appear as visible bodies in tap water. 4 Tastes and odors may result from presence of organisms. S Chlorine residual is difficult to main- tain when organic matter is present. 6 Organisms could theoretically harbor and protect against chlorine certain pathogenic bacteria. E Profuse Growths of Organisms in Raw Water Supplies 1 A limited and balanced growth of various organisms is generally an asset. 2 Extensive surface mats, blooms and marginal growths often cause troubles along the shoreline and eventually in the treatment plant. 3 Some fish kills may be caused by profuse growths of algae by reducing the DO during the night. 4 Certain massive growths of blue-green algae are deadly poisonous to animals. ORGANISMS INVOLVED Animal forms include protozoa, rotifers, crustaceans, worms, bryozoans, fresh water Iv A 28-2 ------- Algae and Actinomycetes in Water Supplies sponges, water mites and larval stages of 3 By eliminating shallow marginal areas various insects. 4 By reducing the amount of fertilizing B Plant forms include algae, actinomycetes nutrients entering the reservoir. and other bacteria, molds and larger aquatic green plants. VII It is generally more satisfactory to anticipate and prevent problems due to these V IMPORTANCE OF BIOLOGICAL organisms than it is to cope with them later. PROBL EMS A Routine biological tests are essential to A The increased use of surface water supplies detect the initial development or presence increases the problems cauBed by organ- of interference organisms. isms. Biological problems are less common with ground water supplies. 1 Control measures can then be used before problem becomes acute. B Standards of water quality requested by domestic and industrial patrons are rising. 2 These tests should be applied to the raw treatment plant water supply and C Procedures for detection, control and distribution system. prevention of problems caused by organisms are improving and are receiving more B In the Reservoir or Other Raw Water Supply extensive use. 1 Routine plankton counts should be made of water samples from selected loca- VI A number of methods may be used to tions. Plankton counter should be control the interference organisms or their aware of the particular organisms products: known to be most troublesome. A Addition to water or an algicide or pesticide 2 During the warmer months routine such as copper sulfate, chlorine dioxide surveys of the reservoir, lake or or copper -chlorine-ammonia. stream should be made to record any visible growths of algae and other B Mechanical cleaning of distribution lines, organisms. settling basins, sand filters, screens, and reservoir walls. 3 Odor tests of water from several locations should be made to obtain C Modification of coagulation, filtration, advance notice of potential trouble at chemical treatment, or location of intake the treatment plant. D Use of absorbent, such as activated carbon, for taste and odor substances. C In the Treatment Plant E Modification of Reservoir to Reduce the 1 Records of plankton counts and threshold Opportunities for Massive Growths of odor between each step in treatment Algae gives data on effectivenesB of each procedure. 1 By covering treated water reservoir to exclude sunlight 2 Coagulation and filtration can be adjusted to remove up to 95% or more 2 By increasing the depth of the water in of organisms in water. reservoirs 28—3 ------- Alaae nd Actiriomycetes in Water unnH i Mi roscnpic analysis of samples of filter material for organisms can supply data useful in modifying sand filtration and treatment of finished water. D In the Distribution System With Its Finished Water 1 Open reservoirs require constant attention especially during summer. 2 Parts of the system farthest from the treatment plant or adjacent to dead ends require most frequent sampling for organisms and tastes and odors. VIII SUMMARY A Interference organisms cause problems in distribution systems, treatment plants. raw water supplies. B Organisms involved include algae, actino- rnycetes, other bacteria, and minute aquatic animals. C Control is by special chemicals, mechanical cleaning, adjustment of chemical or mechanical treatment and by modification of reservoirs, intakes, etc., for the raw water supply. D Facilities for detection of problems in their early stages are required for most efficient and satisfactory control. REFERENCES 1 Palmer, C M. Algae in Water Supplies. An Illustrated Manual on the Identification, Significance, and Control of Algae in Water Supplies. U. S. Public Health Service Publication No. 657. 1959. p. 88. 2 Palmer, C.M. and Poston, H.W. Algae and Other Interference Organisms in Indiana Water Supplies. Jour. Amer. Water Works Asen, 48:1335—1346. 1956. 3 Palmer, C.M. Algae and Other Inter- ference Organisms in New England Water Supplies. Jour. New England Water WorkeAsen, 72:27-46. 1958. 4 Palmer, C.M. Algae and Other Orga- nisms in Waters of the Chesapeake Area. Jour, Amer. Water Works Asen. 50:938-950. 1958. 5 Palmer, C. M. Algae and Other Inter- ference Organisms In the Waters of the South Central United States. Jour. Amer. Water Works Asen. 52:897- 914. 1960. 6 Siivey, J.K. and Roach, A.W. Actinomycetes May Cause Tastes and Odors in Water Supplies. Public Works 87. 5:103-106,210,212. 1956. 7 Ingram, W.M. and Bartech, A.F. Operators Identification Guide to Animals Associated with Potable Water Supplies. Jour. Amer. Water Works As n. 52:1521-1550. 1960. 8 Otto, N.E. and Bartley, T.R. Aquatic Pests on Irrigation Systems. Identification Guide. Bur. of Reclamation. USD1. 72 Pp. 1965. 9 Herbst, Richard P. Ecological Factors and the Distribution of C]adophera glomerata in the Great Lakes. Amer. Midi. Nat. 82:90-98. 1969. This outline was prepared by C.M. Palmer, formerly Aquatic Biologist, In Charge, Interference Organism Studies, Microbiology Activities, Research & Development, Cincinnati Water Research Laboratory, FWPCA. 28—4 ------- Algae and Actinornycetes in Water Supplies ALGAE IMPORTANT IN WATER SUPPLIES TASTE AND ODOR ALGAE PLATE I 28—5 ------- g e and Actinornycetes in Water Supplies FILTER CLOGGING ALGAE PLATE 2 28 —8 ------- Algae and Actinomycetes in Water Supplies POLLUTED WATER ALGAE PLATE 3 287 ------- Algae and Actinomycetes in Water Supplies CLEAN WATER ALGAE IZo 1 ,Uu CLADQPNQ A 0 API.MO1N(C( MIC OCCL(LJ$ PLATE 4 coccoNt” L tNAN(A 28-8 ------- Algae and Actinornycetes in Water Supplies SURFACE WATER ALGAE 1 I/Ifl JIJiZ’hViJh PLATE 5 28—9 ------- Algae and Actinornycetes in Water Supplic ALGAE GROWING ON RESERVOIR WALLS -F I , OToGoCCuI 4 PLATE 6 O(DOSONNId /1 c1To., o * 28-10 ------- ALGAE AS INDICATORS OF POLLUTION I UMITATIONS A Algae are only one of a number of types of organisms present which could be considered. B Forms recognized here as algae are comparatively simple, pigmented, aquatic organisms, including blue-greens, greens, diatoms and pigmented flagellates. C Various pollutants react differently on algae. Organic pollutants such as house- hold sewage will be dealt with here. D No algae are intestinal organisms. They therefore are not indicators of pollution in the same way that coliiorm bacteria are. II ALGAE AND ORGANIC POLLUTION A Heavy pollution may tend to limit various kinds of algae to certain zones in the affected area. B These zones are distinguished according to the degree of change which has occurred in the organic wastes. One set of names for these zones includes the Polysaprobic, alpha -mesosaprobic, beta - mesosaprobic and oligosaprobic. C A few “pollution” algae are common in the first two zones. Many algae are common in and often limited to one or both of the last two zones. D Some workers have listed separately those algae indicative of each of the four zones. Ill REASONS FOR SELECTIVITY OF POLLUTANTS TO ALGAE A Certain components of wastes are chemi- cain toxic to some algae but not to others. B Wastes may have physical effects on certain algae. May cause plasmolysis, change in rate of absorption of nutrienta, etc. C Wastes may reduce available light, increase the water temperature, and cover up the areas for attachment to rocks. D Wastes may prevent algal respiration at night by reducing the DO of water. E Wastes may stimulate other organisms at the expense of certain algae. F Products of waste decomposition may act as powerful growth stimulants for certain algae. IV ALGAE AS INDICATORS OF POLLUTION A Selection of list of “pollution” algae follows an evaluation of the kinds re- ported in published reports by numerous workers as relatively prominent in, or representative of, the polysaprobic and alpha-mesosaprobic zones in a stream poUuted with sewage. It includes also other conditions or areas approximating these zones. B A total list of more than 1000 kinds of algae has been compiled to date. 1 In order to tabulate the information, an arbitrary numerical value is allotted to each author’s record of each pertinent alga. 2 The algae are then arranged in order of decreasing emphasis by the authors as a whole. BI.IND. l0a.8.69 29—1 ------- Alg e as Indicators of Pollution VI SOME GENERA AND SPECIES OF ALGAE HIGH ON THE LIST ARE AS FOLLOWS A Genera’ Oscillatoria, Euglena, Navlcu]a, Chiorella, Chiamydomonas, Nitzschla, Stigeoclonium, Phormidium, Scenedesmus, Ankietrodesmus, Phacus , B Speciee: Euglena viridia, Nltzschia palea, Oscillatoria chiortha, Osciflatoria limosa, Osciliatoria tenuis Scenedesmus guadricauda, Stigeoclonium tenue Synedra ulna and Pandorina morum . VII SOME ALGAE REPRESENTATIVE OF CLEAN WATER ZONES IN STREAMS: Chry8OCOCcu8 rufeecens, Cocconets p].acentula, Entophysalis lemaniae , and Rhodomonas lacustris . VIII REUABIUTY IN USE OF INDICATORS DEPENDS IN PART UPON ACCURATE IDENTIFICATION OF SPECIMENS REPRESENTATIVE LITERATURE 1 Brinley, F. J. Biological Studies. Ohio River Pollution Survey. I. Biological Zones in a Polluted Stream. II. Plankton Algae as Indicators of the Sanitary Condition of a Stream. Sewage Works Journal, 14:147-159. 1942. 2 Butcher, R.W. Pollution and Repurification as Indicated by the Algae. Fourth International Congress for Microbiolo (held) 1947. Report of Proceedings. 1949. 3 Fjerdingatad, E. The Microflora of the River Moelleaa with Special Reference to the Relation of the Benthal Algae to Pollution. Folia Limnological Scandinavia. No. 5. 1950. 4 Fjerdingstad, E. Taxonomy and Saprobic Valency of Benthic Phytomicro- Organisms. Intern. Rev. Ges. Hydrobiol. 50:475-604. 1965. 5 Hawkes, H.A. The Biological Assess- ment of Pollution in Birmingham Streams. The Institute of Sewage Purification, Journal and Proceedings. 177-186. 1956. 6 Kolkwltz, R. Oekologie der Saprobien. Schriftenrejche des Vereins für Wasser-, Boden-, und Lufthyglene Berlin-Dahiem. Piscator - Verlage, Stuttgart. 7 Lackey, J. B. The Significance of Plankton in Relation to the Sanitary Condition of Streams. Symposium on Hydrobiology. University of Wisconsin. 311-328. 1941. 8 Liebmann, H. Handbuch der Fris hwasser - und Abwasserbiologie. R. Oldenbourg, Munchen. 9 Palmer, C.M. Algae as Biological Indicators of Pollution. In Biological Problems in Water Pollution. Trans. of 1956 Seminar. Robert A. Taft Sanitary Engineering Center. 1957. 10 Palmer, C.M. The Effect of Pollution on River Algae. Annal. N.Y. Acad. Sci. 108:389-395. 1963. 11 Palmer, C.M. A Composite Rating of Algae Tolerating Organic Pollution. Jour. Phycology 5 (1):78-82. 1969. Algae in Water Supplies States. In. Algae and Plenum Press. N.Y. 1964. 13 PatrIck, R. Factors Effecting the Distribution of Diatoms. Botanical Review, 14: 473-524. 1948. 14 Whipple, G.C., Fair, G.M. and Whlpple, M. C. The Microscopy of Drinking Water, 4th ed. J. Wiley and Sons. New York. 1948. This outline was prepared by C. M. Palmer, Aquatic Biologist, Cincinnati Water Research Laboratory, FWPCA. 12 Palmer, C.M. of the United Man,Ch 12, pp. 239-261. 29—2 ------- POLLUTED WATER CHL AMYooeOTAV LE OCINCLIS C NLO OCO CC UN LV NC B VA PH OR NIUIt)I I ALGAE NFRISMOPE A \\ 1 CARTLRIA \ \\\ If I RAEORUN LUGLENA ) SPIROGYRA CHLQROGONIUN CHLAMVDONONAS PLATE 3 29—3 ------- Indicators of Pollution. CLEAN WATER ALGAE MICROCO Lj. ) Li N PLATE 4 ,1 V tI’OC RHO 49 . t4OT H f Cf 1)1 IRP M€ RISMOPEDIA HHOMUI NA 29—4 COCCONr IS I EMANEA ------- ODOR PRODUCTION BY ALGAE AND OTHER ORGANISMS I Most biological odors present in our water supplies are derived from algae, actinomycetes, and bacteria. A The odor produced by algae and actino- mycetes is generally the result of intracellular metebolic activity while the odor caused by bacteria usually results from extracellu]ar enzymatic activity upon other organisms. B The odors produced by actinoinycetes are usually earthy while those produced by the algae are aromatic, grassy, and fishy. II SOME SPECIES OF ALGAE CAUSING ODORS A Diatoms 1 Asterionella (aromatic, fish) 2 Cyclotella (aromatic) B Pigmented Flagellates 1 Synura (cucumber) 2 Dinobryon (fishy) C Blue-green Algae 1 Anabaena (grassy, green corn, nasturtium) 2 Aphanizomenon (grassy, nasturtium) D Green Algae 1 Chlorocoecum (grassy) Ili RESEARCH ON ALGAE ODORS A Growing Algae for Odor Research 1 ObtainIng unialgal bacteria-free cultures a Plating out on semi-solid medium b Single cell isolation c Use of antibiotics d Exposure to ultra -violet light 2 Determining nutritional requirements a Inorganic salts b Organic growth factors B Methods of e ctract1ng odoriferous material from algal cultures 1 Distillation - steam and vacuum 2 Solvent extraction 3 Use of ion exchange resins 4 Freeze out methods C Some Results of Research 1 Effect of culture age upon odor production 2 Effect of pH on odor intensity 3 Comparison of odor intensity in intact and broken cells 4 Groups of chemicals which may be responsible for causing algal odors IV RESEARCH ON ACTINOMYCETE ODORS A A number of actinomycetes were isolated from water and muds of rivers and lakes. BI.MIC.to. lOc. 3.70 30-1 ------- Odor Production by Algae and Other Organisms 1 Large numbers were o nd to be present in muds, while there were relatively few in the water. 2 Most species belonged to the Streptorn es and a few to the Micromonospora. B Extraction of Odoriferous Material 1 Streptornjces oluteus was used in this work. a Cultured in a defined niedium (1) Cultures have threahhold ddor of 20, 000 to 53, 000 2 Primary eictraction was by 0 dlatl]iing the culture at 100 C at atmospheric pressure. a Distillation of 10% of the culture volume resulted In 90% odor removal. 3 Odor was further coacentrated by two methods a Ether extraction of the distilLing off of the ether in vacuo. (1) Resulted in yellowish ‘rown con entrate having a threshold odor of approximately 6 billion. b Absorption on activated carbon followed by elution of material with chloroform C Effect of Activated Carbon in Re- moving the Earthy Odo: 1 The odor is practically elimi- nated by 10 ppm carbon. D Effect of Chlorine on Odor 1 Chlorine doea not eliminate the odor but does not Intensify the odor. E Soil perfusion Tests 1 Conducted to determine the extent to which actinomycetes impart odors to a water environ- ment. REFERENCES 1 Fogg, G.E., “The Metabolism of Algae”, John Wiley and Sons, Inc., New Y k, N. Y., 1953. 2 Fox, Leo, ‘Mic oscoptc Organisms in Drinking Water”, Taste and Odor Journal, Vol. 19, No. 10, 1953. 3 Palmer, C. M., and Tarzwe]l , C. M., “Algae of Importance in Water Supplies”, Public Works Magazine, 1955. 4 Whipple, G. C., Fair, G. M., ani Whipole, M. C., “The Microscopy of Drinking Water”, Fourth Edition, John Wiley and Sons, Inc., New York, N.Y., 1948. This out1in was prepared by T. E. Maloney, Former Res arch Biologist, Aquatic Bio1o Activitie8, Research and Development, Cin thnati Water Reearth Laboratory, FWPCA. 30—2 ------- PLANKTON IN OUGOTROPHIC LAKES I INTRODUCTION b Chlorophyll. 1 mg per M 3 or less The term oligotrophic was taken from the c Cells counts, less than 500 per ml Greek words oligos - - small and trophein - - to nourish, meaning poor In nutrients. 2 Zooplankton to phytoplankton volume Lakes with low nutrient levels have low ratio, 19:1. standing crops of plankton. The term is now commonly applied to any water which has a B Quality low productivity, regardless of the reason. I European biologists have found oligotrophic lakes to be dominated by II PHYSICAL AND CHEMICAL CHARACTER- Chiorophyta (usually desmids), ISTICS OF OLIGOTROPHIC LAKES* chrysophyta (such as Dinobryon , and Diatomaceae (Cyclotella and Tabellaria). A Very deep; high volume to surface ratio Eutrophic lakes are dominated by Synedea, Fragilaria, Asterionella, B Thermal stratification common, volume Melosira, blue-green algae, Ceratium, of the hypolimnium large compared to the and Pediastrum. Nygaard devised volume of the epilimnion several phytoplankton quotients based on these relationships C Maximum surface temperature rarely greater than 15°C a Simple quotient D Low concentrations of dissolved minerals Number of species of and organic matter. Chlorococcales if <1, oligotrophlc 1 Phosphorus, less than 1 microgram Desmidiaceae if > 1, eutrophic per liter b Compound index 2 N0 3 -Nitrogen, less than 200 micrograms per liter Myxophyceae+Ch1orococca1e +CentraJ.es+Eu niace.ic DeBmldiaceae E Dissolved oxygen near saturation from surface to bottom if <1, oligotrophic F Water very transparent, Secchi disk readings of 20-40 meters are common if 1-2.5. mesotrophic G Color dark blue, blue-green, or green if >2.5, eutrophic c Diatom quotient Ill PLANKTON Centrales = if 0-0. 2, oligotrophic A Quantity Pennales if 0.2-3.0, eutrophic 1 Standing crop very low a Ash-free weight of plankton, less than 0. 1 mg per liter (compared to 1 mg per liter or more in eutrophic lakes). 31—3 BI.ECO.mic.2. 10.66 ------- Plankton in Oligotrophic Lakes 2 Several lists of trophic Indicators have been publi8hed: Two are listed here Telling. Rawson 1 Swedish Lakes Canadian Lakes Oltgotrophic Tabelleria flocculosa Oligotrophic Asterionella formosa Dactylococcops is M elosira islandica 4 ellipsoldeus Tabellaria fenestrata Tabellaria flocculosa Mesotrophic Kirchneriella lunaris Dinobryon divergens Tetraeadon Fragilarla capucina Pediastrum spp. Stephanodiscus nlagarae Fragilarta crotonenels Staurastrum spp. Atthe zachariasil Melosira granulata Melosira Mesotrophlc Fragtiaria crotonensis Eutrophic Aphanizomenon 8 )• Ceratium hirundinella A naba ena flos - aguae Pe din strum boryanuin Anabaena circinalia Pediastrum di plex Coelosphaerium Pronounced M icrocystis aeruginosa naegelianum Eutrophy Microcystis viridis Anabaena app. Aphanizomenon floe - aquae Microcystis aeruginosa Eutrophic Microcystis flos-aquae 31—2 ------- Plankton in Oligotrophic Lakes Some discrepancies can be seen in the ranking of species in the lists. These may be the result of true differences In the composition of the plankton, or may be only apparent differences which resulted from different sampling methods. Many studies (e. g. those by Hilliard, Olive, and Rawson) have been based on netted samples, which may be highly biased because they contain little of the nannoplankton. Also, it is not uncommon to characterize popu]ationa on the basis of one or two samples collected during the summer months. 3 The dominant plankton in four oligotrophic North American lakes are listed below. The Great Slave Lake and Karluk Lake data are from netted samples taken during the summer, and monthly, re8pecttvely. The Lake Superior and Lake Tahoe data are from grab samples taken twice monthly, and quarterly, respectively. The dominant diatoms are generally similar in the four lakes. Asterionella formosa and Fragilaria crotonensis are common to all. There are also some obvious differences. Melos ira islandica, the dominant diatom in the Great Slave Lake and Lake Superior, is absent from Lake Tahoe and Karluk Lake. It was not found in Crater Lake by Sovereign (1958), in the Mountain lakes of Colorado by Olive (1955) or Brin.ley (1950), and does not occur in WPSS samples in streams west of the Great Lakes. TabeUaria is also absent from Lake Tahoe. It was reported in Colorado lakes by Olive, but was not abundant. Brinley makes no reference to It, and Sovereign indicated that it was rare In Crater Lake samples. It i apparent that the absence of these two diatoms from Lake Tahoe is not related to the lake. Except for the absence of Keratefla cochlearis from Lake Tahoe, the rotl.fer populations are very similar. Data on other segments of the zoo- plankton population are insufficient to permit comparison. 31—3 ------- Hash 5011, Great Slase Lake U5PHS, Lake Superior Hiiltard. Karluk Lake Melosira islardica Aslerlonella formosa Dutobr on do .ergens Cerattum hirunduiella Pedlastrum bor anum Tabellaria fenestrata Cyclatella meneghuuana Fragilarla crotonensis CSpUCUtS Svnectra ulna Eunotta Iunaris Keratella cochlearls KeUlcottia longtsptna Diaptomus tenulcauthtua Limnocalanus macru i-us Senecefla calartosdes Daphnla longispina Bosmuta obtu Irogtrjs Melosira islandica TabeUaria fenestrata Cyclotells kuteingiana M elosira granulata t! elosira amblgua A aterlonella formosa Synedra nana Sc edesmus spp. Arikiatrodesmus spp Dictyosphaerlum app A sterionella formosa TabeUarta flocculosa FragLlaria crotonensis CycloteUa bodanica Cymbefla turgida Dictyosphaerlum app Sphaerocystis app. Staurastrum app KerateUa cochlearis Not reported KeUlcotua 1ongispsna Fragllarta crotonensls Svnedra nana FragtIa rta construens Fragilarta pinnata Nitzschia actcularas A sterloneila Formosa Kelllcottln }onglsplna Daphrna app. Dmptomus t reflt Epischura nevadensis $. PSs. Lake Tahoe Dominant Ph) toplankton Dominant Zoop lankton 0 I- 0 C ‘1 0 V C) F ------- Plankton In Oligotrophic Lakes REFERENCES 9 Rawson, D.S., 1956. Algal indicators of trophic lake types. Limnol. 1 Brinley, F.J. 1950. Plankton population Oceanogr. 1:18-25. of certain lakes and streams in the Rocky Mountain National Park, 10 Rodhe, W., 1948. Environmental Colorado. Ohio 3. Sci. 50:243-250. requirements of fresh-water plankton algae. Synib. Bot. Upsal. 10:1-149. 2 HiliLard, D. K. • 1959. Notes on the phytoplankton of Karluk Lake, Kodiak 11 Ruttner, F. • 1953. Funthmentals of Island, Alaska. Canadian Field- Limnology, 2nd ed., Univ. Toronto Naturalist 43:135-143. Press, Toronto. 3 Jarnefelt, H., 1952. Plankton ala 12 Sovereign, H.E., 1958. The diatoms of Indikator der Trophiegruppen der seen. Crater Lake, Oregon. Trans. Amer. Ann. Acad. Sd. Fennicae A.IV:l-29. Microsc. Soc. 77:96-134. 4 Knudson, B.M., 1955. The distribution of 13 Teiling, E., 1955. Some rnesotrophic Tabeiiaria in the English Lake District. phytoplankton indicators. Proc. lot. Proc. mt. Assoc. Limnol. 12:216-218. Assoc. LImnol. 12:212-215. 5 Nygaard, G., 1949. Hydroblological studies 14 USPHS, 1962. National Water Quality in some ponds and lakes It. The Network, Annual Compilation of Data, quotient hypothesis and some new or PHS Pubi. No. 663. little known phytoplankton organisms. Kig. Danske Vidensk. Seisk. Biol. 15 Welch, P.S., 1952. Limnology, 2nd ed., Skrifter 7:1-293. McGraw Hill Book Co., New York. 8 Olive, J.R.. 1955. Some aspect8 of plankton associations in the high mountains lakes of Colorado. Proc. lot. Assoc. Limnol. 12:425-435. 7 Rawson, D.S., 1953. The standing crop of net plankton in lakes. J. Fish. Res. Bd. Can. 10:224-237. 8 Rawson, D.S., 1956. The net plankton of This outline was prepared by C.!. Weber, Great Slave Lake. 3. Fish. Res. Bd. Chief, Biological Methods Section, Can. 13:53-127. Analytical Quality Control Laboratory, NERC, EPA, Cincinnati, OH 45268. 31—5 ------- THE EFFECTS OF POLLUTION ON LAKES I INTRODUCTION The pollution of lakes inevitably results in a number of undesirable changes in water quality which are directly or Indirectly related to changes in the aquatic community. A Industrial Wastes may contain the following: 1 Sewage 2 Dissolved organics- -synthetics, food processing wastes, etc. 3 Dissolved minerals- -salts, metals (toxic and nontoxic), pigments, acids. etc. 4 Suspended solids--fibers, minerals, degradable and non- degradable organics 5 Petroleum products- -oils, greases 6 Waste heat B The Materials In Domestic Wastes which affect Water Quality are: 1 PathogenIc fecal microorganisms 2 Dissolved nutrients: minerals, vitamins, and other dissolved organic substances 3 Suspended solids (sludge)- -degradable and non-degradable organic materials C Pollution and Eutrophication The discharge of domestic wastes often renders the receiving water unsafe for contact water sports and water supplies. For example, some beaches on the eastern seaboard and in metropolitan regions of the Great Lakes are unfit for swimming because of high coll.form counts. Other effects of domestic pollution Include changes In the abundance and composition of populations of aquatic organisms. 1 As the nutrient level increases, so does the rate of primary production. 2 Shore-line algae and rooted aquatics become more abundant. For example, problems have been experienced with Cladophora and Dichotomosiphon along the shores of Lakes Ontario, Erie, and Michigan. These growths Interfere with swimming, boating, and fishing. and cause odors when the organisms die and decay. 3 The standing crop of phytoplankton increases, resulting in higher counts and greater chlorophyll content. Increases in phytoplarikton abundance may result in taste and odor problems in water supplies, filter clogging, high turbidity, changes in water color, and oxygen depletion In the hypolirnnion. 4 Populations of fish and larger swimming invertebrates increase, based on the increase in basic food production. 5 Changes in dominant species a Diatom communities give way to blue-greens. Toxic blue-greens may pose a problem. b Zooplankton changes include replacement of Bosmina coregoni by B. longirostris . c Trout and whitefish are replaced by perch, bass, and rough fish. d Hypoliinnion becomes anaerobic in summer, bottom sludge buildup results in loss of fish food organisms, accompanied by increase in density of sludgeworms (oligochaeta). II HISTORICAL REVIEW The cultural eutrophication of a number of lakes in Europe and America has been well documented. A Zurichsee, Switzerland WP 1K ic. 4.70 32- 1 ------- The Effects of Pollution on Lakes 1 1896 - sudden increase in Tabellaria B Haliwliersee, Switzerland fenestrata 1 1897 - Oscillataria rubescens not 2 1898 - sudden appearance of Oscillatoria observed up to this time rubescens which displaced Fragilaria capucina 2 1898 - 0. rubescens bloomed, decomposed, formed H 2 S, killing off 3 1905 - Melosira islandica var. helvetica trout and whitefish appeared C Lake Windermere, England (core study) 4 1907 - S phanodiscus hantzschii appeared 1 Little change in diatoms from glacial period until recent times 5 1911 - Bosmina longirostris replaced B. coregoni 2 Then Asterionella appeared, followed by Synedra 6 1920 1924 - 0. rubescens occurred in great 3 About 200 years ago, Asterionella quantities again became abundant 7 1920 - milky-water phenomenon, 4 AsterioneJja abundance ascribed to precipitation of CaCO 3 crystals ( 4 Oii) domestic wastes due to pH increase resulting from photosynthesis D Finnish Lakes 8 Trout and whitefish replaced by perch, Aphanizomenon. Coelosphaerium , bass, and rough fish Anabaena, Microcystis , are the most common indication of eutrophy. TABLE 1 CHANGES IN PHYSIO-CHEMICAL PARAMETERS Zurichsee, Switzerland Parameter Date Value Chlorides 1888 1.3 mg/i 1916 4.9 mg/i Dissolved organics 1888 9.0 mg/i 1914 20.0mg/i Secchi Disk before 1910 16.8M 3.1M 1905- 1910 10.OM 2.1M 1914 - 1928 10.OM 1.4M Dissolved oxygen, at 1910 - 1930 Minimum 100% saturation 100 M, mid-summer 1930 - 1942 9% saturation 32-2 ------- The Effects of Pollution on Lakes E Linsley Pond, Connecticut 1 Species making modern appearance Include Astertonella formosa, Cyclotella glomerata, Melosira itailca, Fragilaria crotonensis, Synecira ulna 2 Asterionella formosa and Melosira Italics were considered by Patrick to indicate high dissolved organics 3 Bosmina coregoni replaced by B. longirostris F Lake Monona, Wisconsin 1 Began receiving treated sewage in 1920, developed blue-green algal blooms. G Lake Washington, Washington 1 1940 - Bosmina longirostris appeared 2 1955 - Oecillatoria rubescens seen for the first time, and constituted 96% of phytoplankton, July 1 H Lake Erie 1 Phytoplankton counts at Cleveland have increased steadily from less than 500 cells/mi in the 1920’s to over 1500 cells/mi in the 1960’a 2 Abundance of burrowing mayflies ( Hexagenia spp.)Ln Western Lake Erie decreased fro n 139/rn 2 in 1930, to less than 1/rn in 1961. Lake Michigan 1 Milky water observed in south end, and in Limnetic region In mid-1950’s and again in 1967. 2 During the period 1965-1967 the Chicago water treatment plant has found it necessary to increase the carbon dosage from 23 lbs/mU gal to 43 lbs/mU gal, and the chlorine dosage from 20 Ibs/mil gal to 25 lbs/mu gal. 3 Phytoplankton counts in the south end now exceed 10, 000/mi during the spring bloom. III FACTORS AFFECTING THE RESPONSE OF LAKES TO POLLUTION rNCLUDE: A Depth-surface area ratio: A large hypolimnion will act as a reservoir to keep nutrients from recirculating in the trophogenic zone during the summer stratification period. Raw8on found an inverse relationship between the standing crop of plankton, benthos, and fish, and the mean depth. B Climate: Low annual water temperatures may restrict the response of the phytoplankton to enrichment. C Natural color or turbidity: Dystrophic (brown-water) lakes may not develop phytoplankton blooms because of the low transparency of the water. IV TROPHIC LEVEL Except in cases where massive algal blooms occur, the trophic status of lakes is often difficult to determine. Core studies are used to determine trends in diatom populations which might indicate changes in nutrient levels over an extended period of time. V CONTROL OF POLLUTION The success of efforts to arrest the eutrophication process, and where desirable, reduce the trophic level of a lake, will depend on a thorough knowledge of the nutrient budget. A Signincant quantities of nutrients may enter a lake from one or more of the following sources: 1 Rainfall 2 Ground water 32—3 ------- The Effects of Pollution on Lakes ‘I A13Li 2 PARAMETERS COMMONLY USED TO DESCRIBE CONDITIONS Oligotrophic Condition 1 Transparency > 10 meters 2 Phosphorus < 1 g/1 3 NO 3 - Nitrogen < 200 g/1 4 Minimum annual near 100% saturation hypolimnetic oxygen concentration 5 Chlorophyll < i mg/rn 3 6 Ash-free weight of seston < 0. 1 mg/i 7 Phytoplankton count < 500/mi 8 Phytopiankton quotients a number of species of Chiorococcales <1 number of species of Desmids b Mlxophycease+Chlorococcales+centra le s+Euglenaceae <1 Desmidaceas c Centrales 0 - 0. 2 Pennales 9 Phytop]ankton species present (Bee outline on plankton in oligotrophic lakes). 3 Watershed runoff c Many methods have been employed to treat the symptoms, reduce the 4 Shoreline domestic and Industrial outfalls eutrophication rate, or completely arrest and even reverse the eutrophication 5 Pleasure craft and commercial vessels process. 6 Waterfowl 1 Use of copper sulfate, sodium arsenite, and organic algicides: It is not 7 Leaves, pollen, and other organic economically feasible to use algicides debris from riparian vegetation in large lakes. B The supply of nutrients from “natural” 2 Addition of carbon black to reduce sources in some cases may be greater transparency. This is likewise than that from cultural sources, and be frequently impractical. sufficient to independently cause a rapia rate of eutrophication regardless of the 3 Harvesting algae by foam fractionation level of efficiency of treatment of domestic or chemical precipitation. and industrial wastes. 32-4 ------- The Effects of Pollution on Lakes 4 Reducing nutrient supply b ’ (a) removal of N and P from effluents, (b) diversion of effluents, and (c) dilution with nutrient-poor water. D Examples of lakes where control has been attempted by reducing the nutrient supply, are: 1 Lake Washington, Seattle The natural water supply for this lake is nutrient poor (Ca -8 mg/i. P <5Mg/I. TDS76 mg/I). Since the turnover time of the water in this lake is only three years, it was expected that diversion of sewage would result in a rapid improvement of water quality. Diversion began in 1963, and improvements were noticeable by 1965 - including an increase in transparency, and a reduction in seston, chlorophyll, and epilimnetic phosphorus. TABLE 3 PHOSPHORUS REDUCTION IN LAKE WASHINGTON Year 1963 1964 1965 Maximum phosphorus in upper 10 meters (Mg/i) 70 66 63 2 Green Lake, Washington The lake has a long history of heavy blooms of blue-green algae. Beginning in 1959, low-nutrient city water was added to the lake, reducing the con- centration of phosphorus by 70% in the inflowing water. By 1966, the lake had been flushed three times. Evidence of improvement l.a water quality was noted in 1965. when Aphanizomenon was replaced by Gleotrichia . 3 Lake Tahoe This lake is still decidedly oligotrophic. To maintain its high level of purity, tertiary treatment incilities were installed in the major sewage treat- ment plant, and construction is now underway to transport all domestic wastes out of the lake basin. REFERENCES 1 Ayers, J.C. and Chandler, D.C . Eds. Studies on the environment and eutrophication of Lake Michigan. Special Report No. 30. Great Lakes Research Division, Institute of Science and Technology. University of Michigan, Ann Arbor. 1967 2 Brezonik, P.L., Morgan, W.H Shannon. E.E., and Putnam, H.D. Eutrophication factors in North Central Florida Lakes. University of Florida Water Res. Center Pub. #5, 101 pp. 1969. 3 Carr, J.F , Hiltunen, J.K Changes in the bottom fauna of Western Lake Erie from 1930 to 1961 Limnol. Oceanogr. 10(4):55l-569. 1965. 4 Frey, David G. Remains of animals in Quatertiary lake and bog sediments and their Interpretation. Schweizerbartsche Ver)agsbuchhandlung. Stuttgart. 1964. 5 Edmondson, W.T., and Anderson, G.C. A rtificial eutrophication of Lake Washington. Limnol. Oceanogr l(1).47—53. 1956. 6 Fruh, E.G. The overall picture of eutrophicatrnn. Paper presented at the Texas Water and Sewage Works Association’s Eutrophication Seminar. College Station, Texas. March 9, 1966. 7 Fruh, E.G , Stewuit, K.M., Lee, G.F., and Rohlich, G.A Measurements of eutrophication and trends. J. W. P.C. F. 38(8): 1237-1258 1966 32—5 ------- The Effects of Pollution on Lakes 8 Hasler, A. D. Eutrophication of lakes by domestic drainage. k co1ogy 28(4):383-395. 1947. 9 Hasler, A. D. Cultural Eutrophication is Reversible. BIoscience 19(5): 425-443. 1969. 10 Herbat, Richard P. Ecological Factors and the Distribution of Cladophora glomerata in the Great Lakes. Amer. Midi. Nat. 82(1):90-98. 1969. 11 National Academy of Sciences. Eutrophication: Causes, Consequence a, Correction. 661 pp. 1969. (Nat. Acad. Sci. ,2101 Constitution Avenue, Washington, DC 20418, 13.50). 12 Neel, Joe Kendall. Reservoir Eutrophication and Dystrophication following Impoundment. Reservoir Fisheries Res. Symp. 322-332. 13 Oglesby, R. T. and Edmondson, W. T. Control of Eutrophication. J.W.P.C.F. 38(9):1452-1460. 1966. 14 Stewart, K.M. and Roh]ich, G.A. Eutrophication - A Review. Publication No. 34, State Water Resources Control Board, The Resources Agency, State of California. 1967. This outline was prepared by C. 1. Weber, Chief, Biological Methods Section, Analytical Quality Control Laboratory, NERC, EPA, Cincinnati, OH 45268. 32—6 ------- APPLICATION OF BIOLOGICAL DATA I ECOLOGICAL DATA HAS TRADITIOMALLY BEEN DiVIDED INTO TWO GENERAL CLASSES: A Qualitative - dealing with the taxonomic composition of communittes B Quantitative - dealing with the populat ion density or rates of processes occurring in the communities Each kind of data has been useful in its own way. II QUA UTATIVE DATA A Certain species have been identified as: 1 Clean water (sensitive) or oligotrophic 2 Facultative, or tolerant 3 Preferring polluted regions (see: Fjerdlnstad 1964, 1965; Gaufin & Tarzwell 1956; Palmer 1963, 1969; Rawson 1956, Teillng 1955) B Using our knowledge about ecological requirements the biologist may compare the species present 1 At different stations in the same river (Gaufin 1958) or lake (Holland 1968) 2 In different rivers or lakes (Robertson and Powers 1987) or changes in the species in a river or/lake over a period of several years. (Carr & Hiltunen 1965, Edmondson & Anderson 1956; Fruh, Stewart, Lee & Rohlich 1966, Hasler 1947). C Until comparatively recent times taxonomic data were not subject to statistical treat- ment. III QUANTITATIVE DATA: Typical Parameters of this type lnclude 2 A Counts - algae/mi; benthos/m fish/net/day B Volume - mm 3 algae/liter C Weight - dry wgt, ash-free wgt. D Chemical content - chlorophyll, carbohydrate; ATP; DNA; etc. E Calories (or caloric equivalents) F Processes - productivity; respiration IV Historically, the chief use of statistics in treating biological data has been In the collection and analysis of samples for these parameters. Recently, many methods have been devised to convert taxonomic data into numerical form to permit: A Better communication between the biologists and other scientific disciplines B Statistical treatment of taxonomic data C In the field of pollution biology these methods Include: 1 Numerical ratings of organisms on the basis of their pollution tolerance (saprobic valency: Zelinka & Sladecek 1964) (pollution index: Palmer 1969) 2 Use of quotients or ratios of species in different taxonomic groups (Nygaard 1949) BI. EN. 35. 12.70 33—1 ------- Application of Biological Data 3 Simple indices of community diversity: a Organisms are placed in taxonomic groups which behave similarly under the same ecological conditions. The number of species in these groups found at “healthy stations is com- pared to that found at “experimental” stations. (Patrick 1950) b A truncated log normal curve is plotted on the basis of the number of individuals per diatom species. (Patrick, Hohn, & Wallace 1954) c Sequential comparison index. (Cairns, Albough, Busey & Chanay 1988). In this technique, similar organisms encountered sequentially are grouped into “runs”. d Ratio of carotenoids to chlorophyll in phytoplankton populations: 0D 4301 0D 665 (Margalef 1968) 0D 4351 0D 670 (Tanaka, et al 1961) e The number of diatom species present at a station is considered Indicative of water quality or pollution level. (Williams 1964) number of species (S ) number of individuals (N) number of species (S) g square root of number of individuals (.1 N) S- 1 log N (Menhinlck 1984) e E n 1 (n - 1 ) (Simpson 1949) N (N - 1) where n 1 number of individuals belonging to the i-th species, N = total number of individuals Information theory: The basic equation used for information theory applications was developed by Margalef (1957). 1 NI I — log N 2NINI...N! a b a where I - information/lndjvjdua l; N • N .. . N are the number of inadivi uals i species a, b, s, and N is their sum. This equation has also been used with: 1) The fatty acid content of algae (Mclntire, Tinsley, and Lowry 1969) 2) Algal productivity (Dickman 1968) 3) Benthic biomasa (Wi]hm 1968) REFERENCES 1 Cairns, J., Jr., Albough, D.W., Busey, F, and Chaney, M.D. The sequential comparison index - a simplified method for non-biologists to estimate relative differences in biological diversity in stream pollution studies. J. Water Poll. Contr. Fed. 40(9):1607—1613. 1968. 2 Carr, J. F. and Hiltunen, J. K. Changes in the bottom fauna of Western Lake Erie from 1930 to 1961. Limnol. Oceanogr. 10(4):551-569. 1965. 3 Dicknian, M. Some indices of diversity. Ecology 49(6):1191-l193. 1968. sd = runs total organisms examined and 33—2 ------- Application of Bio1o tcal Data 4 Edmondson, W.T. and Anderspn, G.C. Artificial Eutrophication of Lake Washington. Limnol. Oceanogr. 1(1):47—53. 1956. 5 Fjerdlngstad, E. Pollution of Streams estimated by benthal phytomicro- organisms. I. A saprobic system based on communities of organisms and ecological factors. InternaUl Rev. Gee. Hydrobiol. 49(1):63-13 1.1964. 6 Fjerdingstad, E. Taxonomy and saprobic valency of benthic phytomicro- organisms. Hydroblol. 50 (4) 475-6O4. 1965. 7 Fruh, E G.. Stewart, K.M., Lee, G.F. and Rohlich, G.A. Measurements of eutrophication and trends. J. Water Poll. Contr. Fed. 38(8):1237—1258 1968. 8 Gaufin, A.R. Effects of Pollution on a midwestern stream. Ohio J. Sd. 58(4):197—208. 1958. 9 Gaufin, A. R. and Tarawell, C. M. Aquatic macroinvertebrate communities as indicators of organic pollution In Lytle Creek. Sew. md. Wastes. 28(7):908— 924. 1858. 10 Hasler, A. D. Eutrophication of lakes by domestic drainage. Ecology 28(4):383- 395. 1947 11 HoUand , R.E. Correlation of Melosira species with trophic conditions in Lake Michigan. Limnol. Oceanogr. 13(3):555—557. 1968. 12 Margalef, R. Information theory In ecology. Gen. Syst. 3 36-71. 1957. 13 Margalef, R. Perspectives In ecological theory. Univ. Chicago Press. 1968. 14 Mclntire, C.D., Tinsley, 1.J. and Lowry, R.R Fatty acids in lotic periphyton: another measure of community structure. J. Phycol. 5:26—32. 1969. 15 Menhinick, E. F. A comparison of some species - Individuals diversity indices applied to samples of field insects. Ecology 45:859. 1964. 16 Nygaard, G. Hydrobiological studies in some ponds and lakes. LI. The quotient hypothesis and some new or little-known phytoplankton organisms. I g. Danske Vidensk. Selsk. Biol. SkriIter 7:1-293. 1949. 17 Patten, B. C. Species diversity In net plankton of Raritan Bay. .J. Mar. Res. 20:57—75. 1962. 18 Palmer, C. M. The effect of pollution on river algae. Ann. New York Acad. Sci. 108:389-395. 1963. 19 Palmer, C. M. A composite ratmg of algae tolerating organic pollution. J Phycol. 5(1):78—82. 1969. 20 Patrick, R., Hohn, M. H. and Wallace, J. H. A new method for determining the pattern of the diatom flora. Not. Natl. Acad. Sci., No. 259. Philadelphia. 1954. 21 Rawson, D.S. Algal indicators of trophic lake types. Limnol. Oceanogr. 1:18—25. 1956. 22 Robertson, S. and Powers, C. F Comparison of the distribution of organic matter in the five Great Lakes. in: J.C. Ayers and D.C. Chandler, eds. Studies on the environment and eutrophication of Lake Michigan. Spec. Rpt. No. 30, Great Lakes Res. Div., Inst. Sd. & Techn., Univ. Michigan, Ann Arbor. 1967. 23 Simpson, E. H. Measurement of diversity. Nature (London) 163:688. 1949. 24 Tanaka, 0. H., Irie, S. Izuka, and Koga, F. The fundamental Investigation on the biological productivity in the Northwest of Kyushu. I. The investigation of plankton. Rec. Oceanogr. W. Japan, Spec. Rpt. No. 5, 1-57. 1961. 33—3 ------- Annileatfon of BioloEical Data 25 Teillng, E. Some mesotrophic phyto- plankton indicators. Proc. Intern. Assoc. L4mnol. 12:212-215. 1855. 26 WtIhm, J. L. Comparison of some diversity indices applied to populations of benthic macroinvertebrates In a stream receiving organic wastes. J. Water Poll. Contr. Fed. 39(1O):1673—1683. 1967. 28 WillIams, L. G. Possible relationships between diatom numbers and water quality Ecology 45(4):810-823. 1964. 29 Zellnka, M. and S]adecek, V. Hydro- biology for water management. State Pubi. Hou8e for Technical Literature, Prague. 122 p. 1964. 27 Wt]hm, J. L.. U8e of biomass units in Shannon’s formula. Ecology 49 153-156. 1968. This outline was prepared by C.!. Weber, Chief, Biological Methods Section, Analytical Quality Control Laboratory, NERCI EPA, Cincinnati, OH 45268. 33—4 ------- THE PROBLEM OF SYNTHETIC ORGANIC WASTES I Sources of organic chemicals in water are varied and of differing compiexity.(1) A Natural pollutants , such as algae, actino- mycetes. etc. contribute to organic pollution. 1 Tastes and odors associated with these materials are probably not merely a result of decomposition, but are closely associated with materials produced during the life cycle of the organisms and plant8. 2 Discharge of nutrients in the form of phosphorus and nitrogen compounds from domestic or other wastes fre- quently stimulate the production of natural pollutants. S Industrial wastes , due to the rate of population Increase and industrial ex- pansion, have made the problem of effective water treatment an acute one in many places. 1 The production of synthetic organic chemical8 has risen steadily over the past years, representing many new and complex products - and of im- portance to us - new and complex wastes. 2 The ideal method of handling lndustriai waste is at Its source. a However, what is often COflBidC red good treatment, still results in materials present in sufficient quantities to affect the taste and odor of water. b Many problems are caused by slug discharges, often accidental. D Miscellaneous sources also contribute to the problem. 1 Wastes from private and commercial boats. 2 ChemIcals applied to the land may be washed into streams. 3 Chemicals applied directly to water. a Evaporation control b Killing off rough fish c Aquatic plant control U Concentrations of organic chemicals in water, even in comparatively minor quantities may cause difficulties. A Wastes may contain from a few mg/i to several hundred mg/i of organic contaminants. B Surface waters may contain from a few ig/1 of organics to several mg/i. 1 Some of the chemicals isolated from water, along with the concentrations which can be detected by odor, are: 2 Concentration Substance Detectable*, ig/l C Domestic wastes in various stages of treatment. 5 Concentrations were determined by taking the median of 4-12 observations. Formaldehyde Picohnes Phenolics Xyle flea Refinery hydrocafbons Petrochemical waste Phenyl ether Chlorinated phenohcB 50, 000 500 - 1,000 250 - 4, 000 300 - 1, 000 25 — 50 15 — 100 13 1 - 100 CH. OTS. 40a. 4.70 34—1 ------- The Problem of Synthetic Organic Wastes UI The damagin effects of organics in water are becoming more apparent. A Taste arid odor in water is usually the first noticed effect from organics. This is a serious public relations and economic problem, it also may be a health problem. B Organic contaminants may interfere with coagulation, damage ion exchangers, and create chlorine and carbc demand. C In the stream they may have adverse effects on aquatic forms that support higher aquatic life, cause off-flavors in fish flesh, or have direct toxic effects on fish. IV The methods of study employed in the collection and identification of organic chemicals in water involve physical and chemical methods and instrumental analysis. A The comparatively small amounts of organic materials may be concentrated by adsorption on activated carbon. 1 This carbon is then extracted with appropriate organic solvents, the solvent extract is taken to dryness, the weighed extract is subjected to solubility group separation, and these individual groups may then be analyzed by various methods. 2 Employing the above method on Ohio River water , the following results were obtained:’ Chemical Group % of Total TOC in p.g/l Relative Odor Contribution Water solubles 20 860 23 Ether and water 22 110 200 insolubles Neutral 14 Amine 4 Weak acid 8 3 575 645 4,670 7 12 Strong acid 6 365 16 Amphoteric 10 5, 000+ Loss 16 -- -- B Chemical separation and analyses may be ccompliahed by means of column chromato- graphy, formation by derivatives, gas chromatography, infrared and ultraviolet spectroscopy, x-ray diffraction. etc. 1 Specific organic chemicals recovered from river and drinking waters by these methods include: synthetic detergents (ABS), phenylether, phenol, DDT, aidrin, o-nltrochlorobenzene, -conedendrin, and xylene. V Some of the types of problems that may be attributed to organic wastes, and more specifically to problems of taste and odor, may be represented by the following examples: 34—2 ------- The Problem of Synthetic Organic Waste8 A By applying the previously mentioned carbon adsorption method, the odor po- tential of organic pollutants and the dilution necessary to reduce this odor potential to a barely perceptible level has been deterrnthed: 4 Industry Source Conc. Required for Detectable Odor i .’g/l Dilution Factor . CHCl Sol. Org. Total Org. CHC1 3 Sol. Org. Total Org. Brewery Chemical Corn Refining Meat Packing Metal Fabrication -Paint Pharmaceutical Refinery Soap 770 28 1, 000 1, 200 890 390 290 84 900 1, 400 32 3, 600 3, 600 1, 600 1, 000 340 510 1,800 14 11,000 1. 4 92 2. 8 69 10 780 640 86 14,000 2. i 140 4. 8 98 32 760 350 B Of all the organic pollutants that can affect the taste and odor of drinking water, phenol has been the most extensively studied. 1 The potential sources of this chemical, both natural arid synthetics have been discussed. 5 ) 2 The course of chlorination of phenol, a common method of treatment in the water plant, has been shown to proceed by a process which starts with the pure compound (in itself relatively tasteless) and proceeds through strong-tasting intermediates to tasteless end products. (6) C The effects of petrochemical wasteB 7 on water quality are becoming increasingly important; It has been predicted that by 1970 the petrochemical production on a tonnage basis may be equal to 41% of all chemicals. 1 The three principal groups of petro- chemicals are the paraffins, the naphthenes, and the aromatics. From these, over 200 basic products are manufactured, having thousands of subordinate uses. 2 Correspondingly, more than 100 identi- fiable compounds have been found in waste streams from petrochemical processes. BIBLIOGRAPHY 1 Middleton, F. M. Taste and Odor Sources and Methods of Measurement. Taste and Odor Control Journal. 26:1. 1960. 34—3 ------- The Problem of Synthetic Organic Wastes 2 Middleton, F. M., Rosen 1 A. A., and Burttschell , R. H. Taste and Odor Research Tools for Water Utilities. Jour. A.W.W.A. 50:21. 1958. 3 Anonymous. Objectionable Organic Con- taminants in Water. San.Eng.Center Actlv. Rep. No. 25, 1855. 4 Sproul, 0. J., and Ryckman, D. W. The Significance of Trace Organics in Water Pollution. PCF 33:1188. 1861. 5 Hoak, R. D. The Causes of Tastes and Odors In Drinking Water. Water and Sewage Works. 104:243. 1957. 6 Burttscheli , B. H., Rosen, A. A., Middleton, F. M., and Ettinger, M. B. Chlorine Derivatives of Phenol Caus- ing Taste and Odor. Jour.A. W. W.A. 51:205. 1959. 7 Gloyna, E. F., and Malina, Jr. • J. F. Petrochemical Wastes Effects on Water. Indus. Water & Wastes, 7: #5. 134. Sept. —Oct. 1962. 8 Baker, R. A. Problems of Tastes and Odors. WPCF. 33:1099. 1961. This outline was prepared by R. L. Booth, Chief, Analyses Unit, Analytical Quality Control Laboratory. NERC, OWP, EPA, cincinnati, OH 45268. 34—4 ------- - I Nil I i A N( I 01’ ‘II M I I ’ [ NG ‘A CTORS ‘10 P01 ‘U LA lION VARIATION INTRODUCTION A All aquatic organism ’, do not react uniformly to the various chemiLal, physical and biologiLal features in their environment. lhroiigh normal evolutionary processes various organisms have become adapted to certain combinations of environmental conditions. The successful develo ment and maintenance of a population or community depend upon harmontous ecological balance b tw en environmental conditions and loleranc’e of the organisms to variations In one or more 0! these Londitlons. 13 A factor whose presenLo 01 absence exerts Nome r(stm aining Influence upon .i popii1 t ion through Incompatibility with species requirements or tolerance is said to be a limiting luLtor . The principle ol limiting factors is one of the major aspects of the environmental control of aquatic organ1 ms (Figure 1). A Liebig’s Law of the Minimum enunciates the first basic concept. In order for an organism to inhabit a particular environ- mt’nt, specified levels of the materials necessary for growth and development (nutrients, respiratory gases, etc. ) must be present. If one of these materials is absent from the environment or present in minimal quantities, a given species will only survive in limited numbers, if at all (Figure 2) II PRINCIPLE OF UMITING FACTORS This principle rests essentiaUy upon two basic concepts. One of these relates organisms to the environmental supply of materials essential br their growth and development. The second pertains to th tolerance which organisms exhibit toward environmental conditions I”igurc 1 ‘I h relationships ol limiting factors to population gm owth and development Figure 2. Relationships of environmental factors and the abundance of organisms. 1 The subsidiary principle of factor interaction states that high concentration or availability of some substance, or the action of some factor in the environ- rnent, may modify utilization of the minimum one. For exampleS .i The uptake of phosphorus by the algae Nitzchia closterlum is influenced by the relative quantities of nitrate and phosphate in the environment, however, nitrate utilization appears to be unaffected by the phosphate (Reid, 1961). b The assimilation of some algae is closely related to temperature The rate of oxygen utilization by fish may he affected by many other sub- stances or factors in the environment. OPTIMUM z 0 z CRITICAL RANGE LbW — MAGNITUDE OF FACTOR — F1IG H x I 0 a I , z 0 a 0 a ‘IJN(IMITID GROWtH DICRIASE IN LWT yiO*u s / — - EQUILIIIIUM wirpi / S INCR eASE IN \ ttliTf bOpis ‘POPULATION DECLINE TIME 131. ECO.2fla.7.6’) 35—1 ------- Signuicance of Limiting Factors’ to Population Variation d Where strontium is abundant, mollusks ,rc able to substitute it, to a partial extent, for calcium in their shells (Oclum, 1959). 2 If a material is present in large amounts, but only a small amount is available for use by the organism, the amount available and not the total amount present deter- mines whether or not the particular material is limiting (calcium in the form of CaCO 3 ). 13 Shelford pointed Out in his Law of Tolerance that there are maximum as well as minimum values of most environmental factors which an he tolerated. Absence or failure of an organism can be controlled by the deficiency or excess of any factor which may approach the limits of tolerance for that organism (Figure 3). MtnIn ,un. IAmft of 1 5.n e of Optfmun Moolmum Ltmft of rolursulon of F,,’toro Toler.ttøn Atm, of Ahun oc. Oreitsit Abwt nc, Decru.in 1 Abient _____J Ab JJ U z a z “a > I- Figure 4. Ftelationship of purely harmful factors and the abundance of organisms. 3 Tolerance to environmental factors varies widely among aquatic organisms. a A species may exhibit a wide range of tolerance toward one factor and a narrow range toward another. Trout, for instance, have a wide range of tolerance for salinity and a narrow range for temperature. Figure 3. Shelford’s Law of Tolerance. I Organisms have an ecological minimum and maximum for each environmental factor with a range in between called the critical range which represents the range of tolerance (Figure 2). The actual range thru which an organism can grow, develop and reproduce normally Is usually much smaller than its total range of tolerance. 2 Purely deleterious factors (heavy metals, pesticides, etc.) have a maximum tolerable value, but no optimum (Figure 4). b All stages in the life history of an organism do not necessarily have the same ranges of tolerance. The period of reproduction is a critical time in the life cycle of most organisms. c The range of tolerance toward one factor may be modified by another factor. The toxicity of most sub- stances increases as the temperature increases. d The range of tolerance toward a given factor may vary geographically within the same species. Organisms that adjust to local conditions are called ecotypes . CONCENTRATION 35—2 ------- Signhlicance of ‘Limiting Factors” _ pula t on Variation e The range of tolerance toward a given factor may vary seasonally. in general organisms tend to be more sensitive to environmental changes in summer than in other seasons. This is primarily due to the higher summer temperatures. 4 A wide range of distribution of a species is usually the result of a wide range of tolerances. Organisms with a wide range of tolerance for all factors are likely to be the most widely distributed, although their growth rate may vary greatly. A one-year old carp, for Instance, may vary in size from less than an ounce to more than a pound depending on the habitat. 5 To express the relative degree of tolerance for a particular environmental factor the prefix (wide) or steno (narrow) is added to a term for that feature (Figure 5). Figure 5. Comparison of relative limits of tolerance of stenothermal and eurythermal organisms. C The law of the minimum as it pertains to factors affecting metabolism, and the law of tolerance as it relates to density and distribution, can be combined to form a broad principle of limiting factors. 1 The abundance, distribution, activity and growth of a population are deter- mined by a combination of factors, any one of which may through scarcity or overabundance be limiting. 2 The artificial introduction of various substances into the environment tends to eliminate limiting minimums for some species and create intolerable maximums for others. 3 The biological productivity of any body of water is the end result of interaction of the organisms present with the surrounding environment. UI VALUE AND USE OF THE PRINCIPLE OF UMLTING FACTORS A The organism-environment relationship Is apt to be 80 complex that not all factors are of equal importance in a given situation; some links of the chain guiding the organism are weaker than others. Understanding the broad principle of limiting factors and the subsidiary principles involved make the task of ferreting out the weak link in a given situation much easier and possibly less time consuming and expensive. 1 If an organism has a wide range of tolerance for a factor which is relatively constant in the environment that factor is not likely to be limiting. The factor cannot be completely eUminated from consideration, however, because of factor interaction. 2 If an organism is known to have narrow limits of tolerance for a factor which is also variable in the environment, that factor merits careful study since it might be limiting. TtMPUATUII 35—3 ------- Significance of “Limiting Factors” to Population Variation B Because of the complexity of the aquatic environment, it is not always easy to isolate the factor in the environment that Is limiting a particular population. Premature conclusions may result from limited observations of a particular situations. Many Important factors may be overlooked unless a sufficiently long period of time Is covered to permit the factors to fluctuate within their ranges of poBsible variation. Much time and money may be wasted on control measures without the real limiting factor ever being dis- covered or the situation being Improved. C Knowledge of the principle of limiting factors may be used to limit the number of parameters that need to be measured or observed for a particular study. Not all of the numerous physical, chemical and biological parameters need to be measured or observed for each study undertaken. The aims of a pollution survey are not to make and observe long lists of possible limiting factors but to discover which factors are significant, how they bring about their effects, the source or sources of the problem, and what control measures should be taken. D Specific factors in the aquatic environment determine rather precisely what kinds of organisms will be present in a particular area. Therefore, organisms present or absent can be used to indicate environ- mental conditions. The diversity of organisms provides a better indication of environmental conditions than does any single species. Strong physlo-chemical limiting factors tend to reduce the diversity within a community; more tolerant species are then able to undergo population growth. REFERENCES 1 Odum, Eugene P. Fundamentals of Ecology, W. B. Saunders Company, Philadelphia. (1959) 2 Reid, George K. Ecology of Inland Waters and Estuaries. Reinhold Publishing Corporation, New York. (1961) This outline was prepared by John E. Matthews, Aquatic Biologist, Robert S. Kerr Water Research Center, Ads. Oklahoma. 35—4 ------- NUTRIENTS: THE BASIS OF PRODUCTIVITY I INTRODUCTION A Nutrients of importance include macro- nutrients: those needed in large quantities, and rnicronutrients: those needed in small amounts. B These nutrients are important because they promote biological reoponses which may interfere with some desired u8e of the water by man. C Other factors (e. g. temperature, light) affect the use of these nutrients and should be considered in an evaluation of the effects of nutrients upon the ecosystem. U Algae, bacteria, fungi and aquatic plants are the forms of life which nutrients affect most directly. A Algae are of Several Types 1 Phytoplankton are small algae suspended in the water and form the basis of pro- ductivity in the aquatic environment. 2 Benthic algae are those forms anchored to substrates of rock and bottom state tials, 3 Periphytic algal are those microscopic forms attached to submersed substrates. B Aquatic plants are of several types. In general they may be referred to as rooted or floating forms. C Heterotrophic bacteria are fungi which respond to organic nutrients Introduced into water. Autotrophic bacteria may re- spond and grow due to inorganic nutrient sources. III BIOLOGICAL LAWS A Liebtg’s “law” of the minimum the essen- tial material avaUable in amounts most closely approaching the critical minimum needed will tend to be the limiting factor. B Shellord’s “law” of tolerance survival of an organism can be controlled by the quantitative or qualitative deficiency or excess with respect to any one of several factors which may approach the limits of tolerance for that organism. C Q 10 “law” with a temperature increase of 10 degrees centigrade metabolic pro- cesses (rates) are approximately doubled. IV The process of photosynthesis Is the fixa- tion of the sun’s energy with the production of organic matter by plants with chlorophyll. A The general reaction is given below: C0 2 +H 2 0 k CH 2 O+O 2 B Chlorophyll contains basically C, 0, H, N and Mg, and in general makes up about 5% of the dry weight of algal cells. V MEASUREMENT OF PHOTOSYNTHESIS A Oxygen production can be used as a measure of photosynthesis because for each mole of CO 2 reduced to organic carbon one mole of free oxygen is liberated. 1 The value cf the molar 0 /C0 ratio has been found experimen 2 tallyato vary within wide limits. B CO 2 Assimilation 1 The CO 2 taken up by algae does not all originate from the dissolved gas. Some algae can use bicarbonate directly as a source of carbon. W.RE.ntr.2e. 10.73 36—I ------- Nutrients: The Basis of Productivity 2 Hence measurement of CO 2 uptake from water is a complicated problem which must consider pH. HCO 3 , and CO 3 concentrations C Fixation of Carbon-14 1 ‘ h use of C 14 as a tracer of C’ 2 in plant metabolism and productivity estimation has been widely used since the early nineteen fifties. 2 In this method a known amount of C 4 is added to the water and after a period of time the proportion of C 14 in the plant cells to C 14 added is found. The amount of carbon assimilated Is then estimated from the following equation. 3 Where K is a constant relating to the slower uptake of C 14 . 4 The total carbon available is determined chemically. D Uptake of Mineral Nutrients 1 The measurement of depletion of nutrients in solution has been tried but found unreliable. E Chlorophyll 1 The quality of chlorophyll present has been found to bear some relation to productivity but not a reliable one. V I Nutrients of significance in the growth and production of algae and plants are discussed below. A Carbon 1 Sources a Gaseous CO 2 b HCO c C0 d Other carbon compounds 2 Effects of the removal of carbon upon the water a Lowered pH b Deposition of CaCO 3 3 The quantity of carbon available is great and it usually is not a limiting factor. B Nitrogen 1 Nitrogen can be taken up by most algae as either ammonium salts or as nitrates. Nitrites can also be used but a high con- centration is usually inhibitory. Some blue green algae can fix atmospheric nitrogen. Certain algae varieties require supplementary amines, growth factors, etc. 2 The quantity of nitrogen in waters has defmitely been shown to limit algal populations. C Phosphorus 1 Phosphate seems to be the only inorganic source of this nutrient. 2 Limiting concentrations of P have been found to range from .01 ppm at a mini- mum and an inhibitory affect if P con- centrations exceed 20 ppm. 3 Optimum concentrations have been found to range from .018 ppm to 15 ppm. 4 Storage of inorganic phosphate by algae has been demonstrated. The extent of this storage may reach 80% of the total phosphorus in algal cells. D Silicon 1 Nutrient ratios in the algal cells of some areas have been found to be Si 23: N16 P1. It can be seen from this ratio that silicon is an important ele- ment in algal growth. activity of pt ytopla nkton activity of C1 4 0 added total carbon (K) assimilated total carbon available 36—2 ------- Nutrients. The Basis of Productivity 2 Silicon is especially important in the population growth of diatoms and may be the limiting growth factor in these populations. E Inorganic micronutrients - Many elements are needed in very small quantities by algal cells. Some of these have a known function in algal metabolism; others do not. I Mg is a cation of major importance in the chlorophyll molecule. 2 Co is known to be necessary for vitamin B 12 . 3 Mn is necessary for 8everal enzyme systems. 4 Mo, V, Zn, and Cu are necessary but these functions are not as well known. F Organic Micronutrients 1 Of 179 algal strains investigated about 40% required vitamin supplementation for optimum growth. Principal growth factors that were not synthesized in sufficient quantity are given as follows along with the percentage of the vitamin deficient strains showing marked pro- ductivity gain after supplementation: a B 12 addition increased growth on 80% of the strains. b Thiamin addition increased growth on 53% of the strains. c Biotin addition increased growth on 10% of the strains. 2 Algae can use and may require many organic compounds depending upon environmental conditions and the ability of the organism to synthesize required building blocks from mineral forms of C.N. & P. This is an area for con- linucd investig.ition with many unappre- ciated or vaguely understood ecological factors. VII PROBLEMS AND BENEFITS RESULTING FROM ALGAE PRODUCTION A Problems to man may result when the total “primary production” by algae leads to an increase in the total organic content of the water that interferes with a desired use. 1 This may consist of a high algal popu- lation that produces a water with high turbidity, taste and odor, or other undesirable effect. High respiratory needs may lead to nocturnal oxygen deficit. 2 Certain algae may cause tastes and odors, clog filters, or otherwise inter- fere with potable water processing. 3 Death of large algal populations may lead to tastes and/or odors through bacterial decomposition. Oxygen deficits may result at any time of day in this process. Deposition of masses of organic sediment or sludge may be considerable. 4 Other problems might be cited. B The primary production of algae can also serve as a supply of food to consumer organisms (animals), resulting in increased production at several (trophic) levels: zo microbes, microinvertebrates, macroinvertebrates, fishes. 1 Earlier notation cited the release of oxygen during utilization of CO 2 during algal photosynthesis. This encourages fungal or bacterial breakdown of pollutants. 2 Photosynthesis occurs in the presence of adequate light and favorable condi- tions. In darkness, the cells continue to respire and may consume more oxygen than they produced because photosynthesis increases the organic load. 36- ------- Nutrients The Basis of Productivity 3 Photo ynthenls tends to occur at the sut fuct where light intensity is greatest. Poor vertical mixing would result in stratification of water supersaturated with oxygen over oxygen deficient water Depending upon conditions, a significant fraction of the oxygen could be lost to the atmosphere. 4 Increased productivity may result in temporary reduction of the free dissolved nutrient level in the water but harvesting at some level is essential to prevent later recycle. VIII CYCLE OF NUTRIENTS A Once nutrients enter a body of water they are cycled through a food chain. B Factors affecting this food chain (e.g. toxicity, removal) will affect the con- centration and distribution of the nu- trients. ACKNOWLEDGEMENT: This outline contains certain material submitted by F. J. Ludzack and H. W. Jackson. 4 Odum, H. T. Primary Production in Flowing Waters. Limnology and Oceanography. 1(2):102-117. April 1956. 5 Ryther. John H. The Measurement of Primary Production. Urnnology and Oceanography. 1(2):72-84. April 1956. 6 Verduin, Jacob. Primary Production In Lakes. Limnology and Oceanography. l(2):85-91. April 1956. 7 Symposium: Factors That Regulate the Wax and Wane of Algal Populations. Inter. Assoc. of Theor. and Appi. Biol. Communications No. 19. 1971. 8 Likens, G. E. Nutrients and Eutrophi- cation. The limiting-nutrient contro- versey. Am. Soc. Limnol. Ocean. Spec. Symp. Vol. 1. 1972. 10 Fitzgerald, G. P. Nutrient Sources For Algae and their Control. Water Pollution Coat. Res. Ser. 16010 EHR 08/71. EPA. 77 p. 1971. H EFERENCES 1 Golterman, H. L. and C]ymo, R. S. Chemical Environment in the Aquatic Habitat (Proc. of an IBP - symposium, Amsterdam and Nieuwerslujs Oct. 1966). 322 pp. (N.y. Noord-HoUandsche Tjltgevers Maatschapplj, Amsterdam. 8.95). 2 Lewln, Ralph A. Physiology and Biochemistry of Algae. Academic Press. 1962 3 Odum, Eugene P. Fundamentals of Ecology. W.B. Saunders Co. 1959. This outline was prepared by Michael E. Bender, Biologist, Formerly with Training Activities, Ohio Basin Region, SEC. 9 Bartoch, A.F. Eutrophication. EPA - R3 - 72 - Role of Phosphorus In Ecol. Res. Ser. 001. 45 p. 1972. ------- ALGAE AND CULTURAL EUTROPHICATION I INTRODUCTION This topic covers a wide spectrum of items often depending upon the individual discussing the sub)ect and the particular situation or objectives that he is trying to “prove”. Since the writer Is not a biologist, these viewpoints are “from the outside-looking in ”. Any impression of bias is Intentional. A Some Definitions are in Order to Clarify Terminology. 1 EutrophIcation - a process or action of becoming eutrophic, an enrichment. To me 1 this Is a dynamic progression characterized by nutrient enrichment. Like many definitions, this one is not precise, stages of eutrophication are classified as aug- , meso-, and eutrophic depending upon Increasing degree. Just how a given body of water may be classified is open to question. It depends upon whether you look at quiet or turbulent water, top or bottom sample 8, season of the year, whether it is a first impression or seasoned judgement. It also depends upon the water use in which you are interested, such as for fishing or waste discharge. The transitional stages are the major problems - it is loud and clear to a trout fisherman encountering carp and scum. 2 Culture Fostering of plant or animal growth. cultivation of living material and products of such cultivation, both fit. Some degree of control Is implied but, the control may have limitations as well as advantages. Human cultural development has fostered human num- bers successfully, but, has promoted rapid degradation of his natural environ- ment 3 Nutrients A component or element essential to sustain life or living organisms. This includes many different materials, some in gross quantities - others in minor quantities. Deficiency of any one essential item make living impossible. Nutrients needed in large quantities include carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur and silica. N and P frequently are loosely considered as “the “nutrients because of certain solubility, con- version and “known” behavior characteristics. 4 Algae A group of nonvascular plants, capable of growth on mineralized nutrients with the aid of chlorophyll and light energy - known as producer organisms, since the food chain is based directly or indirectly upon the organic material produced by algae. B Now that we have “backed into” the title words via definitions, some of the ramifications of eutrophication, nutrient enrichment, and cultural behavior are possible. II NUTRIENTS INTERRELATIONSHIpS A All nutrients are interchangeable in form, solubility, availability, etc. There are no “end” products. We can isolate, cover, convert to gas liquid or solid, oxidize, reduce, complex, dilute, etc. - some tfme, some place, that nutrient may recycle as part of cultural behavior. 1 Water contact is a major factor in recycle dynamics just as water represents two—thirds or more of cell SI. ECO. hum. 3.5 71 37—1 ------- jj .tc wid Cullui al Liitrophicatlon ui’. ss and app.ars to be the medium in wiiwh living foritis started. Waste dispo’,ul intertelationships (Figure 1) stiggi .ts physical interielationships of soil, air and water. The wet apex of tins triangle is the basis for Life. It’s difficult to isolate water from the soil tn atmosphere - water contact means ,i thition of available nutrients. /\ ‘OSAL lNTC LATION5 HPS ATMOSP (RE ST II SMOG \\ ATER$ ii 2 Figure 2 takes us into the biosphere (1) via the soluble element cycle. This refcr .s mainly to phosphorus interchange Phosphorus of geological origin may be solubilized In water, used by plants or animals and returned to water. Natural inovt ment is toward the ocean. Less phosphorus returns by water transport. Phosphorus does not vaporize, hence, atmospheric transport occurs mainly as windblown dust. Man and geological upheaval, partially reverse the flow of phopshorus toward the ocean sink. ATUO P CIE SOLU LE ELE iEUT CVCL 3 The nitrogen cycle starts with ele- mental nitrogen in the atmosphere. It can be converted to combined form by electrical discharge, certain bacteria and algae, some plants and by industrial fixation. Nitrogen gas thus may go directly Into plant form or be fixed before entry. Denitrification occurs mainly via saprophytes. (Figure 3) Industrial fixation is a relatively new contribution to eutrophication. [ I i •’ C1 C GI.E — ii liUflh it ._ — _______— liTt i l li III upullic ,i, i i u I I niuululit ( 1 . 1 1 t h “‘lull. Ii. .Il H I ll Phi, i i luthil uy 11 11 11 tillilTiT! llIl UY ( OSPtEI( 7—2 ------- Algae and Cultural Eutrophication 4 Carbon Convei sion (Figure 4) show most of the carbon in the ioi m of geological carbonate (1) but bicarbonate and CO 2 readl]y are converted to plant cell mass and into other life forms. Note the relatively small fraction of carbon in living mass. - CA DO C flCULATIO I 3IOSPC1E ( Nutrient cycles could go on, but, life depends upon a mixture of essential nutrients under favorable conditions. Too much of any significant item in the wrong place may be consldeied as pollution. Since toxicity is related to chemical concentration, time of exposure and organism sensitivity, too much becomes toxic. If it happens to be too much growth, its a result of eutrophication. ‘How much’ is generally more important than the ‘what’? Both natural and manmade processes lead to biological conversions, to pollution, to eutrophication and to toxicity. Man is the only animal that can concentrate, speed up, invent, or otherwise alter these convu’rsions to make a r’ollossal roe ‘ , S. 1 Life forms have been formulated In terms of e ehienthl or nutrient com- ponents many times. The simplest is C 5 H 8 0 N. A more complex formula is ’ C 10 H 76 0 80 N 20 Ca 6 C1 7 I’ 2 CuF 2 SiMgMn 2 K 2 NaS 21 Zn. This includes 16 elements. More than 30 have been implicated as essential and they still would not “live”, unless they were correct] y assembled. As a nutrient Mnemonic I -I. COPKINS - - Mg(r)- CaFe-MoB does fa,irly well. It also indicates Iod ine-I, Iron-Fe, Molybdenum-Mo, and Boron-B that were not included earlier. 2 The Law of Distribution states that “Any given habitat tends to favor all suitable species - any given species tends to be present in all suitable habitats. ‘ Selection tends to favor the most suitable species at a given place and time. 3 Laebigs Law of the Minimum, states that “The essential material available in amounts most closely approaching the critical minimum will tend to be the limiting growth factor.” 4 Shelfords law recognizes that there will be some low concentration of any nutrient that will not support growth. Some higher concentration will stimulate growth. Each nutrient will have some still higher concentration that will be bacteriostatic or toxic. This has been discussed earlier but was considered in a different manner. III BIOLOGICAL PROGRESSIONS The biological “balance’ appears to be a very transitory condition in cultural behavior. Man favors production. A steady state “balance” does not persist very long unless energy of the system is too low to permit significant growth. A progression of species where each predominent form thrives for a time, then is displaced by another temporarily favored group is usual. ‘Yearly events in the lawn start with chickweed, then dandelion, plantain, crab grass, rag weed, etc , in successive predominence. Occasionally, more desirable grasses may appear on the lawn. Grass is a selected unstable “culture” B Nutrient - Growth Relationships 37—3 ------- A Igae and Cultural Eutrophication 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 dilates, rotifers, etc. Figure 5. S.ct.,l. th,iv. sad finally b.csm. pt.y .1 Ks ciliate., which in •wni sea Is.d is, the roth. ,, sad crustacoani. H Figure 4 shows another progression of bottom dwelling larva. Here the sequence of organi8ms changes after wastewater introduction from aquatic Insects to sludge worms, midges, sow bugs and then to re-establishment of insects. Figure 6. n. p .l.tl.. .i F1.r. 7 is c .mpu.d of a oath.. .1 issal.. I . , individusl • .ci , soak .swltiplying and dying sff a, sins. c..dith.ns swy. $EW1IE UCTEIIA N. PER m l. THE BIOTA 300 200 100 I CIUATE$ .IO.PElmLxl,NI *OTIFER$&ClR$TICEAR$NO.PEImLx25,III — COLFORM (N0 PtR ML) • ;.. .,. . 0 Iii ‘ I I •• 2 1 24 12 0 3 - DAYS 12 24 36 41 SO 72 84 96 108 MILES$WOOEVV*MS THE BIOTA 14 1 I I i 11 II it I I S I I I 37-.4 ------- _Algae and Cultural Eutrophication U Another progression after waste introduction changes the blota from an algal culture to sewage moulds with later return to algal predominence, Figures 5, 6, and 7 are shown separately only because one visual would be unreadable with all possible progressions on it. There are progressions for fungi, protiata, Insect larvae, worms, fish, algae, etc. Each species will perform as it may perform. It it cannot compete successfully, it will be replaced by those that can compete under prevailing conditions at the time. Conditions shift rapidly with rapid growth. IV The interactions of bacteria or fungi and algae (Figure 8) are particularly significant to eutrophication. A The bacteria or the saprophytic group among them tend to work on preformed organic materials - pre-existing organics from dead or less favored organisms. Algal cells produ’e the organics from light energy chlorophyll and mineralized nutrients. This is a happy combination both: The algae release the oxygen for use by tht bacteda while the bacteria release the CO 2 needed by the algae. Since the algae also acquire CO 2 from the atmosphere, from wastewater and from geological sources, it always ends up with more enrichment of nutrients In the water - more enrichment means more growth and growing organisms eventually clump and deposit. The nature of growth shifts from free growth to rooted forms, starting In the shallows. Another progression occurs (Figures 9 and 10). It is this relationship that favors profuse nuisance growth of algae below significant waste discharges. There is a tremendous pool of carbon dioxide available in geological formations and in the air. Transfer to the water Is significant and encourages algal productivity and eventual eutrophication of any body of water, but, this does not occur as rapidly as when the water body is super saturated with CO 2 from bacterial decay of wastewater discharges or benthic deposits from them. • zi.gure ‘s .,,*, . 1.. .. .. dl.ch.,g.. N t. ..uUd. .N.I. .. l*vm 1 ,wNt. This.. ,. .ss.deNd wINt sI..d, dSg.NIIM s .w. I. N t. Urn, curve. Tb. sludge Is dr..mp.-.d gi.d..l$p : as c.adIp4..s c i . ., up, .1g.. gals a f..th.Id .itd .t.ftIply. 37..5 ------- Algae and Cultural Eutrophication L/ ZO ATIO AND AQUATIC PLANTS LIMNETIC DO DEAD BACTERIA (. (I LIGHT MINERALS r o o o@ u ------- A1 ae and Cultural Eutrophication 20m 40m 70m LgiiO AL ZO /\ D AC UATIC PL T D iH LI Nitrogen and phosphorus are essential for growth. They also are prominently considered in eutrophication control, Algal cell mass Is about 50% carbon, l % nitrogen and approximately 1% phosphorus not considering luxury uptake in excess of immediate use. Phosphorus is considered as the most controllable limiting nutrient. It’s control Is corn- plicated by the feedback of P from benthic sediments and surface wash. Phosphorus removal means solids removal. Good clarthcatlon is es8entlal to obtain good removal of P. This also means improved removal of other nutrients- a major ddvantago of the 1’ removal route. I3oth N & P arc easily converted from one form to another, most forms are water soluble. V SUMMARY Cnntrol of eutrophication is not entirely possible. Lakes must eventually fill with benthic sediments, surface wash and vegetation. Natural processes eventually Lause filling. Increased nutrient discharges from added activities grossly increase filling rate. NTS CHA RAPH VIES LAD OP HO RA A We produce more nutrients per capita per day In the United States than In other nations and much more today than 100 years ago. More people in population centers accentuate the problem. B Technology is available to remove most of the nutrients from the water carriage system. 1 This technology will not be used unless water is recognized to be in short supply, 2 It will not be used unless we place a realistic commodity value on the water and are willing to pay for cleanup for reuse purposes. C Removal must be followed by isolation of acceptable gases to the atmosphere acceptable solids into the soil for i euse or storage. Water contact cannot be prevented, hut it must be limited or the enrichment of the water body is hastened. I 10 METERS \\ FERNS MOSSES 37—7 ------- Algae and Cultural Eutrophication I IEFERENCES _______________________________ This outline was prepared by F. J. Ludzack, 1 A collection of articles on the Biosphere Chemist, National Training Center, Sd. Am. 223 (No. 3). pp. 44-208. Water Programs Operations, EPA, Cincinnati, September 1970. OH 45268. 2 Bartch, A.E. and Ingrain, W.M. Stream life and the Pollution Environ- ment. Public Works 90:(No. 7)104- 110. July 1959. 37—8 ------- CONTROL OF PLANKTON IN SURFACE WATERS I PHILOSOPHICAL CONSIDERATIONS A Plankton growths are as natural to aquatic areas as green plants are to land areas and respond to the same stimuli. B Mart i8 currently harnessing plankton forms to accomplish useful work. 1 For generation of oxygen a Stabilization of waste waters in oxidation ponds b Oxygen recovery from CO 2 in space travel 2 For augmentation of food supply a Fish ponds b Nitrogen fixation in rice growing c Harvesting of algae for direct use as food C A growing knowledge of the nutrient re- quirements of plankton organisms will lead to a more enlightened approach to ways and means of controlling their growth when desirable. 11 CLASSICAL METHODS OF CONTROL A Chemical 1 Inorganic a Copper sulfate is used most exten- sively. It is most effective in pre- ventive rather than curative treat- ment. It has long lasting effects in soft waters but is short-lived in hard waters due to precipitation of the Cu as a basic carbonate The pre- cipitated material accumulates m bottom muds and is toxic to certain benthal forms, some of which serve as important fish food. Dosages are normally based on the alkalinity of the water. When alka- linity is < 40 mg/I. the recommended dosage is 0. 3 mg/l of CuSO 4 5H 2 0 in total volume of water. When alkalinity is > 40 mg/l, recommended dosage is 2. 0 mg/l in 8urface foot of water. b Chlorine is preferable to copper sulfate in the control of certain forms of algae. However, it is difficult to apply in most instances and is very short-lived due to photo catalytic decomposition of HC1O — HC1 + 0 2 Organic - Numerous organic compounds have been evaluated, especially in re- lation to control of blue-green algae. “Phygo&’, 2, 3 -dichloronaphthoquinone, has been field tested but is too specific in its action for general application. 111 ECOLOGICAL CONTROL A Theory - Ecological control is based upon the principle of preventing or restricting growth by limiting one or more of the essential requirements. This is an ap- plication of Liebig’s Law of the Minimum. The logical avenues of control are as follows: 1 Elimination of light 2 Limiting nutrient materials B Light - Many cities have solved the prob- lem of plankton growths by the use of covered reservoirs, underground and elevated. Concurrently, they have solved contamination problems created by birds and atmospheric fallout In open reservcxxrs, 131. MIC. con, lOb. 4.70 38-I ------- Control of Plankton in Surface Waters some success has been obtained by limiting light through the use of a film of activa led carbon C Nutrients - Since phytoplankton (algae) serve as the base of the food chain, know- ledge concerning their nutrient require- ments is required for ecological control, when limitation of light is impractical. The nutrient requirements of phytoplankton are as follows ’ I Nature of - The major nutrients are a Carbon dioxide b Nitrogen - ammonia and nitrates (also N2) c Phosphorus - phosphates. Minor nutrients are d Sulfur - sulfates e Potassium f Trace inorganics - magnesium, iroa, etc. g Trace organics-vitamins, amino acids 2 Sources of - See Fig 1 a Atmosphere b Groundwater - springs c Storm water or surface runoff d Waste waters - domestic sewage and industrial wastes. 3 Significance of each major nutrient a Carbon dioxide - Sec Fig 2 Usually present in great abundance. Rapidly replenished from at mosphere and bacterial Uccuinpusition of organ- ic matter No reasonabLe possibility of human control Nature, however, does provide some control through elevated pH levels if carbon dioxide becomes depleted rapidly. b Nitrogen - Like land plants, certain algal forms prefer nitrogen in the form of NH 3 (NH 4 t ) and others prefer it in the form of N0 3 , Both forms often become depleted during the growing season and reach manmum concentrations during the winter season. A level of 0. 30 mg/I of inorganic nitrogen at the time of the spring turnover is considered to be the maximum permissible level All natural surface waters are saturated with nitrogen gas This serves as a source of nitrogen for bacteria and algae capable of fixing it. c Phosphorus - A key element in all plant and animal nutrition. The critical level is considered to be 0 01 mg/i at the time of the spring turnover. Phosphorus is needed to sustain nitrogen fixing forms, D Practice Of 1 Exclusion of hght - Practice we u established l it distribution system reservoirs but impractical on large storage reservoirs, 2 Nutrient limitation a Control of surface run-off quality 1) Agricultural 2) Other b Diversion of sewage plant effluents 1) Madison , Wisconsin 2) Detroit Lakes, Minnesota 3) Pending - State College, Pa. c Tertiary treatment of sewage I) Nitrogen removal - Because of the several forms is very difficult. 18—2 ------- GROUND WATER STORM WASTE WATER WATER (SURFACE (DOM. SEW. RUN .OFF) t IPID.WASTE: OR LAKE RESERVOIR FIG. I SOURCES OF MATERIALS IN SURFACE 0 If ‘1 FERTILIZING OF CONCERN WATERS ------- ATMOSPHERE CO 2 WATER ORGANIC CARBON H 2 0 CO 2 + CO + P420 + HCO 2HC0 HCO 3 +4. +Ca +0W FIG. 2 CARBON DIOXIDE - BICARBONATE - CARBONATE - HYDROXIDE RELATIONSHIPS IN NATURAL WATERS a I : -r PLANTS +H 2 0 ------- Control of Plankton in Surface Waters Also, may be unsuccessful in control unless phosphorus Is con- trolled 1 too, because of nitrogen fixing forms. 2) Phosphorus removal - Phosphorus can be effectively removed by coagulation methods employing lime, alum or ferric salts. It Is expensive and no one has proven its value beyond laboratory experiments. d By Biological Engineering Laboratory studies have shown that effluents essentially free of plant fertilizing elements can be produced by biological treatment of wastes with proper ratios of C to N and P. E Practical Aspects 1 Diversion 2 Nutrient control REFERENCE Muilican, Hugh F. (Cornell Univ.) Management of Aquatic Vascular Plants andAlgae. pp. 464-482. (inEutro- phication: Causes, Consequences, and Correction. Nat. Acad. Sci.) 1969. This outline was prepared by C. N. Sawyer, Director of Research, Metcalf & Edcbr Engineers, Boston, Massachusetts. 3 ExperIences a Madison b Detroit Lakes c State College d Lake Winniequam, N. H. 38—5 ------- CONTROL OF INTERFERENCE ORGANISMS Th4 WATER SUPPLIES I NECESSITY FOR DATA A Information on the number, kinds, and effects of interference organisms in a particular water supply is essential for determining adequate control measures. B Collection of the biological data should be on a regular routine basis. C Interpretation of data requires information on relationship of number and kinds of organisms to the effects produced. D It is generally more satisfactory to an- ticipate and prevent problems due to these organisms than it is to cope with them later. U CONTROL IN RAW WATER SUPPLY A Use of algicides 1 Application of an algicide is to prevent or destroy exces8ive growths of algae which occur as blooms, mats or a high concentration of plankton. 2 Algicide may be applied to control even low concentrations of certain algae such as Synura. 3 Copper sulfate is the only algicide in common use at present. a Application may be by dusting. spraying or dissolving from a porous container over all or part of the water surface, or by continuous feeding of the algicide at the intake of the reservoir or pre-treatment basin. b Effective dosage depends upon the Alkalinity and pH and temperature of the water and the amount and kinds of algae to be controlled. Bartch states that the following arbitrary dosages have been round to be generally effective and safeS M.O. alkalinity > 50 p.p. in 2 p. p. m. in the surface foot of water only (5. 4 pounds per acre). M.O. alkalinity <50 p.p,m. 0.3 p. p. m. in total volume of water (0. 9 pound per acre foot). c Application of copper sulfate should be limited to the minimum effective dosage because of its corrosive properties, and its toxicity to fish and other aquatic animals. 4 Other algicides a Promising types include inorganic salts, organic salts, rosin amines, antibiotics, quinones, substituted hydrocarbons, quaternary ammonium compounds, amide derivatives and phenols. Cuprichloramine which is a combination of copper, chlorine and ammonia, and also chlorine dioxide have shown promise as general aigi- cides. b For domestic water supplies they will have to be not only economically fea- sible but nontoxic to animal life and to green plants other than algae. c Due to higher costs they will prob- ably be used only when adequate plank- ton and algal records are kept, which would permit early localized treat- ment. d Algicides selectively toxic to the particular algae of greatest signifi- cance would be useful. 5 Mechanical removal or spreading out to permit rapid drying may be the sim- plest way of handling massive growths which are detached and washed ashore. 6 Turbidity due to sLit keeps down the plankton population. In shallow reservoirs, fish which stir up the bottom mud will aid in keeping turbidity due to silt high. 7 Provisions for keeping the amounts of nutrients to a minircium may be em- phasized more in the future. 8 For new reservoirs, clearing the site BI.MIC.con.6b.4. 70 39—I ------- Control of Interference Organisms in Water Supplies of vegetation and organic debris before filling will reduce the algal nutrients. Steep rather than gentle elopes will reduce the areas which allow marginal growths to occur. Ill CONTROL IN TREATMENT PLANT A Coagulation and sedimentation 1 When well regulated they often will re- move 90 per cent or more of the plank- ton. 2 With low plankton counts, a coagulant aid may be required. 3 Frequent removal of sludge from the basins, especially during the warm seasons may help to reduce tastes and odors originating from decomposing organic sediment. B Sand filtration I Both slow and rapid sand filters tend to reduce the plankton count of the effluent by 90 per cent or more, when well regu- lated. 2 For rapid filters, accumulated plankton can be removed or reduced by surface scraping and by back washing. C Micro-straining 1 This involves the passing of the water through a finely woven fabric of stain- less steel. AU but the smaller plankton organisms tend to be removed from the water. It is being used in some treat- ment plants in England and elsewhere. D Activated carbon 1 The slightly soluble, organic, taste and odor compounds tend to be readily adsorbed by the activated carbon. It is probably most often applied prior to coagulation, but may be used prior to filtration or in the raw water. E Chlorination 1 Treatment with chlorine is practiced primarily to destroy pathogenic organ- isms. The dosages commonly used are toxic also to many algae and to some of the other groups of aquatic organisms. However, dead as well as living organ- isma are often capable of causing tastes and odors and of clogging filters. F The depth and position of the intake for entrance of raw water into the treatment plant may determine the kinds and amount of plankton which will be drawn into the plant. Plankton algae generally are more concentrated near the surface of the water in lakes and reservoirs. IV CONTROL IN DISTRIBUTION SYSTEM A Maintenance of a chlorine residual con- trols the chlorine sensitive organisms. B Other pesticides such as cuprich.loramine have been used in attempts to control the resistant organisms such as worms, nematodes and copepod eggs. C Flushing of infested portions of the system. especially dead ends may be practiced. D Covering of treated water reservoirs to prevent the entrance of light wifi stop the growth of algae. E Organisms associated with pipe corrosion are probably the most active when the water itself is corrosive. F Mechanical cleaning of the distribution system may be an effective but expensive method of reducing infestations of attached organisms, V SUMMARY A Adequate control is dependent upon ade- quate procedures for detecting and record- ing of organisms. B Control may involve the following: 1 Use of an algicide or pesticide. 39—2 ------- Control of Interference Organisms In Water Supplies 2 Mechanical cleaning of distribution d By reducing the amount of fertilizing lines, settling basins, and filters, nutrients entering the reservoir screens, intake channels and reservoir margins. e By encouraging a balanced develop- ment of the aquatic organisms 3 Modification of coagulation, filtration, chemical treatment or location of raw water Intake. REFERENCE 4 Use of adsorbent, such as activated Mackenthun, Kenneth M. The Practice of carbon, for taste and odor substances. Water Poliution Biology. FWPCA. U. S. Dept. of Interior, Washington, DC. S Modification of reservoir to reduce the 1969. opportunities for massive growths. a By covering treated water reservoirs b By IncreaBing the depth of the water This outline was prepared by C. M. Palmer, Former Aquatic Biologist, Biological Treat- c By eliminating shallow marginal meat Research Activities, Cincinnati Water areas Research Laboratory, FWPCA, SEC. 39—3 ------- CASE PREPARATION AND COURTROOM PROCEDURE I TYPES OF PROCEEDINGS IN WHICH WATER QUAUTY EVIDENCE MAY BE USED A Administrative Proceedings 1 Rule making a Setting up of regulations having general application, e.g. • stream classifications and implementation plan target dates b Factors of safety and absolute prohibitions may be appropriate 2 AdjudicatIons a Determinations by agency having expertise with respect to particular discharge or discharger. e.g., approval of plans and ape s and time schedule of a particular discharger B Court Actions 1 Civil in behalf of state or federal government a Actions to compel action or sus- pension of action - nuisance, health hazard, etc. , - -including court action following federal conference - -hearing procedure b Violations of Water Quality Standards c Violations of Effluent Standards or discharge permits d Tort or contract actions relating to design and/or operation of treatment facilities 2 Criminal (dependent on content of applicable statutes) a Discharge of specific materials b Discharges from specific industries c Littering d Discharges harmful to fish and/or crustaceans e Discharges harmful to specific types of receiving waters f Discharges of poisons NOTE- -In some of these situations doing the act may constitute the violation; in otherH proof of intent or knowledge of effects may also have to be proved. 3 Private actions for damages or to compel action a Alleged harm to plaintiff, e.g., poUution of stream IdUing animals C Procedural Matters 1 See Attached sheet “Administrative and Court Proceedings” on Burden of proof, fact finding, and methods of presentation of evidence. D Classes of Evidence - General Rules 1 Facts - direct a The material was floating from the outfall. 2 Derived values - expert testimony - test results and/or opinion as to effects a The D. 0. was zero; the waterway was polluted; the plant can be built in 6 months. 3 Hearsay a Joe told me W,Q.lc. la. 3.74 40-S ------- Case Preparation and Courtroom Procedure 4 Relevancy 5 Admissibility vs. weight a Even i i admissible, the weight to be given is up to fact finder-- credi- bility. IE Admissibility of Results of Sampling and Testing (Numbers) 1 Sampling a Chain of custody b Tags. etc. c Containers d Place and tune e Retention of samples (Proving that the sample represents what Is at issue in the action (relevancy),that there has been no opportunity for tampering; and availability of portions for analysis by other aide (non-transitory criteria) ). 2 Analysis a Who performed (Can identity of each participant be shown?) b Admission through supervisor - custodian c Scientific acceptance of method. Is there a particular method required to be used by the agency? d Propriety of conduct e Retention of bench cards and other indicia of results. (Your attorney can make arrangements to substitute copies for originals). 3 Tests a Comparison with actual conditions b Mathematical models - how can a computer be cross-examined? F Admissibility of Expert Opinion on Causes and Effects 1 Who has special knowledge - and of what particular areas? 2 Indicators 3 Significance of numerical determin- ations or observations 4 Consistency with own prior publications and testimony 5 Have underlying facts been or need to be proved- -first hand information of this and/or comparable situations. 6 Use of treatises G Conduct on the Witness Stand 1 General a On direct - know what counsel will ask and let him know generally what you will answer, but don’t make it sound rehearsed. b Use layman’s language to extent possible. c Listen to question and answer it to best of your ability. d Speak so that court reporter, judge, Jury, and counsel can hear you. e Speak in language that will be understood; don’t talk down. f Answer only what you are asked - - don’t volunteer; however, answer with precision. g There is nothing wrong with asking to have a question repeated or rephrased. h There is nothing wrong with saying that you consulted with your attorney before you testified, but beware of the question “Did Mr. C tell you what to say?” 40—2 ------- Case Preparation and Courtroom Procedure I There is nothing wrong with thinking out your answer before responding. j You are not expected to know all the answers- -if you do not know, admit it. k Don’t attempt to answer questions outside your area of personal knowledge (hearsay) or beyond your expertise. (Your may be an expert on conducting laboratory tests, but not on epidimeological inferences from results). 1 Don’t try to answer before the judge rules on objection. m Show that you are an impartial dispenser of Information and/or opinion, not a protagonist. n Don’t be afraid to admit what may appear to be damaging. 2 If you are testifying as an expert: a Establish qualifications - - give information relevant to your area of expertise - - educational (in- cluding this course?), work, publications, number of times you have testified previously. a Review and refamiliarize self with materials before you discuss with your attorney. b Be in a position to present all facts known to you simply and concisely: Who, What, When, Where, and Why, How. c Don’t overlook facts and/or test results because you don’t think they’re important. Let attorney decide what he needs. d Use of standard report forms e Ability to recommend additional witnesses with needed specialized knowledge f Ability to aid In cross-examination of other side’s experts and reconcile opinions and/or results g Be candid - sometimes better not to start a lawsuit or accept a settlement than lose in the end. H Non-Verbal Presentation of Evidence 1 Exhibits - including photographs 2 Summaries b Differentiate between physical facts (measurements and observations) and opinion ( derived values) . c Be prepared to discuss theory (in- cluding assumptions) instruments used, techniques(including choice of a particular technique), physical limita ons and errors, inter- ferences. d If experiments were conducted, be able to justify both as to theory and relevancy to this litigation. e If you’re being paid to testify, admit it. 3 ScientifIc personnel as advisers to counsel 3 Buslnes and/or government records a Prepared contemporaneously and in usual course of activities 4 Pre-prepared direct examination a Usually limited to actions before ICC, FPC, and other federal agencies. I Criminal Procedure 1 Privilege Against Self Incrimination (available only to persons) a Warning and suspects b Effect of duty to report spills 40—3 ------- Case Preparation and Courtroom Procedure c Effect of duty to obtain license or This .itline was prepared b r David I. permit and/or furniah operating iedroff , Enforcement Anaiyst, Office of reports Enforcement and General Counsel, Cincinnati Field Investigations Center, d Immunity from prosecution 5555 Ridge Avenue, Cincinnati, OH 45268. 2 Double Jeopardy 3 Unreasonable search and seizure Deacripto s : Courtroom Procedure, Law Enforcement, Legal Aspects, Sampling, a Available to persona and Water Analysis. Water Pollution corporations Control, Water Quality Standards 4 Procedures and need for arreBt and search warrants - -possible cause Administrative & Court Proceedings, and cerpts from Revised Draft of Proposed Rules of Evidence for the United States Courts can be found on the following pages. 40—4 ------- ADMINiSTRATIVE & COURT PROCEEDrNGS Court or Agency Fact Finder Burden of Proof Comments State Pollution Control Agency As per statute - Hearing may be conducted by hearing Agency usually weight of examiner, agency member, or full evidence, agency. Appeal may be on facts and Rule making-adjudi- law or law alone, depending on statute. 0 cation Federal Water PoLlution Control Act Conference Head of agency Reports acceptable. Hearing Hearing Board Specific testimony. o 0 C Court Judge Uses prior material, and may take additional testimony. Court ‘1 Civil Case - - - for money only Judge or jury Weight of evidence ‘1 - In lunction 0 preliminary or Judge Must show immediate Must also show likelihood of success at temporary harm or danger, final hearing - bond required for non- government plaintiff. permanent Judge Usually clear and convincing. “Balance Equities” - administrative Judge - whether Sometimes have complete new trial. appeal “arbitrary and capricious” or sub- stantial evidence. C ” Criminal case Jury unless waived. Beyond reasonable Proof of intent may be required. includes penalties doubt. ------- Case Preparation and Courtroom Procedure Excerpts from Revised Draft of Proposed RULES OF EVIDENCE FOR THE UN 1TED STATES COURTS GENERAL PROCEDURES Rule 102. PURPOSE AND CONSTRUCTION These rules shall be conBtrued to secure fairness In administration, elimination of unjustifiable expense and delay, and promotion of growth and development of the law of evidence to the end that the truth may be ascertained and proceedings justly determined. Rule 101. PREUMINARY QUESTIONS (a) Questions of Adnttssthility Generally. Preliminary questions concerning the qualification of a person to be a witness, the existence of a privilege, or the admissibility of evidence shall be determined by the j idge, subject to the pro- visions of subdivision (b). In mald.ng hia determination he is not bound by the rules of evidence except those with respect to privileges. (b) Relevancy Conditioned on Fact. When the relevancy of evidence depends upon the fu].filhxnent of a condition of fact, the judge shall admit it upon, or subject to, the introduction of evidence sufficient to support a finding of the fuiflhlnient of the condition. Rule 815. EXCLUSION OF WITNESSES At the request of a party the judge shall order witnesses excluded so that they cannot hear the testimony of other witnesses, and he may make the order of his own motion. This rule does not authorize exclusion of (1) a party who Is a natural person, or (2) an officer or employee of a party which Is not a natural person designated as Its representative by its attorney, or (3) a person whose presence is shown by a party to be essential to the presentation of his cause. Rule 611. MODE AND ORDER OF INTERROGATION AND PRESENTATION (a) Control by Judge. The Judge may exercise reasonable control over the mode and order of interrogating witnesses and presenting evidence so as to (1) make the interrogation and presentation effective for the ascertainment of the truth, (2) avoId needless consumption of time, and (3) protect witnesses from harassment or undue embarrassment. (b) Scope of Cross-Examination. A witness may be cross-examined on any matter relevant to any ls9ue In the case, including credibility. In the interests of justice. the judge may limit cross-examination with respect to matters not testified to on direct examination. 40-8 ------- Case Preparation and Courtroom Procedure Rule 613. PRIOR STATEMENTS OF WITNESSES (a) Examining Witness Concerning Prior Statement. In examining a witness concerning a prior statement made by him, whether written or not 1 the state- ment need not be shown or its contents disclosed to him at that time, but on request the same shall be shown or disclosed to opposing coun e1. JUDICIAL NOTICE Rule 201. JUDICIAL NOTICE OF ADJUDICATIVE FACTS (b) Kinds of Facts. A judicially noticed fact must be one not sublect to reasonable dispute In that it Is either (I) generally known within the territorial jurisdiction of the trial court or (2) capable of accurate and ready determination by resort to sources whose accuracy cannot reasonably be questioned. (g) Instructing Jury. The judge shall instruct the jury to accept as established any facts judicially noticed. RELEVANCE Rule 401. DEFINITION OF “RELEVANT EVIDENCE” “Relevant evidence” means evidence having any tendency to make the existence of any fact that is of consequence to the determination of the action more probable or lees probable than it would be without the evidence. Rule 402. RELEVANT EVIDENCE GENERALLY ADMISSIBLE, iRRELEVANT EVIDENCE INADMISSIBLE All relevant evidence is admissible, except as otherwise provided by these rules, by other rules adopted by the Supreme Court, by Act of Congress. or by the Constitution of the United States. Evidence which is not relevant is not admissible. COMPETENCY OF WITNESSES Rule 601. GENERAL RULE OF COMPETENCY Every person is competent to be a witness except as otherwise provided in these rules. 40-7 ------- Case Preparation and Courtroom Procedure Rule 602. LACK OF PERSONAL KNOWLEDGE A witness may not testify to a matter unless evidence is introduced sufficient to support a finding that he has personal knowledge of the matter. Evidence to prove personal knowledge may, but need not, consist of the testimony of the witness himself. This rule is subject to the provisions of Rule 703, relatIng to opinion testimony by expert witnesses. EXPERT TESTIMONY Rule 702. TESTIMONY BY EXPERTS If scientific, technical, or other specialized knowledge will assist the trier of fact to understand the evidence or to determine a fact in issue, a witness qualified as an expert by knowledge, skill, “xperience, training, or education, may testify thereto in the form of an opinion or otherwise. Rule 703. BASES OF OPINION TESTIMONY BY EXPERTS The facts or data In the particular case upon which an expert bases an opinion or inference may be those perceived by or made known to him at or before the hearing. If of a type reasonably relied upon by experts in the particular field in forming opinions or inferences upon the subject, the facts or data need not be admissible in evidence. Rule 705. DISCLOSURE OF FACTS OR DATA UNDERLYING EXPERT OPINION The expert may testify in terms of opinion or inference and give his reasons therefore without prior disclosure of the underlying facts or data, unless the judge requires otherwise. The expert may in any event be required to disclose the underlying facts or data on cross-examination. Rule 706. COURT APPOINTED EXPERTS (a) Appointment. The judge may on his own motion or on the motion of any party enter an order to show cause why expert witnesses should not be appointed, and may request the parties to submit nominations. The judge may appoint any expert witnesses agreed upon by the parties, and may appoint witnesses of bin own selection. An expert witness shall not be appointed by the judge unless he consents to act. A witness so appointed shall be informed of his duti.es by the judge in writing, a copy of which haU be filed with the clerk, or at a conference In which the parties shall have Opportunity to participate. A witness so appointed 8hail advise the parties of his findings, if any, his deposition may be taken by any party, and he may be called to testify by the judge or any party. I-Ic shall be subject to cross-examination by each party, Including a party calling him as a witness. 40-8 ------- Case Preparation and Courtroom Procedure flEA USA Y Rule 801. DEFINITIONS The following definitions apply under this Article (a) Statement. A “statement Is (1) an oral or written assertion or (2) nonverbal conduct of a person, if it is intended by him as an asserticn. (b) Declarant. A “deelarant” is a person who makes a statement. (c) Hearsay. “Hearsay” is a statement, other than one made by the declarant while testifying at the trial or hearing, offered in evidence to prove the truth of the matter asserted. Rule 802. HEARSAY RULE Hearsay is not admissible except as provided by these rules or by other rules adopted by the Supreme Court or by Act of Congress. Rule 803. HEARSAY EXCEPTIONS: AVAILABILITY OF DECLARANT IMMATERIAL The following are not excluded by the hearsay rule, even though the declarant is available as a withess: (5) Recorded Recollection. A memorandum or record concerning a matter about which a witness once had knowledge but now has insuffici nt recollection to enable him to testify fully and accurately, shown to have been made when the matter was fresh in his memory and to reflect that knowledge correctly. If admitted, the memorandum or record may be read into evidence but may not itself be received as an exhibit unless offered by an adverse party. (6) Records of Regularly Conducted Activity. A memorandum, report, record, or data compilation, in any form, of acts, events 4 conditions, opinions, or diagnoses, made at or near the time by, or from information transmitted by. a person with knowledge, all in the course of a regularly conducted activity, as shown by the testimony of the custodian or other qualified witness, unless the sources of informatioi or other circumstances indicate lack of trustworthiness. (18) Learned Treatises. To the extent called to the attention of an expert withess upon cross-examination or relied upon by him in direct examination, statements contained in published treatises, periodicals, or pamphlets on a subject of history, medicine, or other science or art, established as a reliable authority by the testimony or admission of the witness or by other expert testimony or by judicial notice. If admitted, the statements may be read into evidence but may not be received as exhibits. 40—9 ------- Case Preparation and Couitroom Procedure IDENTIIICATION OF PERSONS AND SAMPLES Rule 901. It EQIJIR EM ENT OF AUTHENTICATION OR IDENTIFICATION (a) General Provision. The requirement of authentication or identification as a condition precedent to admissibility is satisfied by evidence sufficient to SUpport a finding that the matter In question is what its proponent claims. (b) Illustrations. By way of illustration only, and not by way of limitation, the following are examples of authentication or identification conforming with the requirements of this rule: (1) Testimony of Witness with Knowledge . Testimony that a matter is what it is claimed to be. (3) Comparison by Trier or Expert Witness . Comparison by the trier of fact or by expert witnesses with specimens which have been authenticated. (9) Process or System . Evidence describing a rocess or system used to produce a result and showIng that the process or system produces accurate result. ADMISSIBILITY AND PROOF OF SPECIAL MATTERS Rule 406. HABIT; ROUTINE PRACTICE (a) Admissibility. Evidence of the habit of a person or of the routine practice of an organization, whether corroborated or not and regardless of the presence of eye- witnesses, 18 relevant to prove that the conduct of the person or organization on a particular occasion was in conformity with the habit or routine practice. (b) Method of Proof. Habit or routine practice may be proved by testimony In the form of an opinion or by specific instances of conduct sufficient In number to warrant a finding that the habit existed or that the practice was routine. Rule 612 WRITING USED TO REFRESH MEMORY If a witness uses a writing to refresh hie memory, either before or while testifying, an adverse party is entitled to have it produced at the hearing, to inspect it. to cross-examine the witness thereon, and to introduce in evidence those portions whith relate to the testimony of the witness. Rule 1006. SUMMARIES The contents of voluminous writings, recordings, or photographs which cannot conveniently be examined In court may be presented In the form of a chart, summary, or calculation. The originals, or duplicates, shall be made available for examination or copying, or both, by other parties at a reasonable time and place. The judge may order that they be produced in court. 40—10 ------- KEY TO SELECTED GROUPS OF FRESHWATER ANIMALS The following key Is intended to provide an introduction to some of the more common freshwater animals Technical language Is kept to a mlnlmwn in using this key. start with the first couplet (la. ib), and select the alternative that seems most reasonable. If you selected HiaIl you have identified the animal as a member of the groups Phythm PROTOZOA If you selected “Ib’, proceed to the couplet indicated. Continue thiq process until the selected statement is terminated with the name of a group If you wish more information about the group, consult references (See reference l1 t ) B !. AQ.21b. 5.71 41—1 ------- i’ y to Selec’tcd Gioiips of Freshwater Ammals l.i ‘l lie hoily of the organis n comprising a single microscopic independent cell, or many similar and indepen- dently functioning cells associated in a colony with little orno differ- enre between the cells i e with- out fern ing tissues, or body com- prised of masses of rnultinucleate protoplasm Mostly microscopic, single celled animals. Phylum PROTOZOA it) The body of the organism com- prised of many cells of different kinds, i.e., forming tissues. May be microscopic’ or macro- scopic. 2a Body or colony usually forming irregular masses or layers some- times cylindrical, goblet 8haped, vase shaped, or tree like. Size range from barely visible to large. 2h Body or colony shows some type 4 of definite symmetry. ‘ Ia Colony surface rough or bristly in appearance under microscope or hand lens. Grey, green, or brown. Sponges. Phylum PORIFERA (Fig. 1) ‘lb Colony surface relatively smooth. General texture of mass gelatinous, transparent. Clumps of minute individual organisms variously distributed. Moss animals, bryozoans. Phylum BRYOZOA (Fig. 2) 4a Microscopic. Action of two ciliated (fringed) lobes at an- terior (front) end in life often gi er appearance of wheels. Body often segmented, accordian- like. Free swimming or attached. Rotifers or wheel animalcules, Phylum I’ROCIIELMIN’l’)IES (liotifera) (Fig. 3) 4h Larger, worinlike, or having 5 strong skeleton or shell. 5a Skeleton or shell present. Skel- 15 eton may be external or internal. 5b Body soft aridfor wormlike. Skin may range from soft to parchment-like. 6a Three or more pairs of well 19 formed jointed legs present. Phylum ARTHROPODA (Fig. 4) 6b Legs or appendages, if present, 7 limited to pairs of bumps or hooks. Lobes or tenacles. if present, soft and fleshy, not jointed. 7a Body strongly depressed or 8 flattened in cross section. 3 7b Body oval, round, or shaped like 10 an inverted “U” in cross section. 8a Parasitic inside bodies of higher animals. Extremely long and flat, divided into sections like a Roman girdle. Life histor) may involve an intermediate host. Tape worms. Class CESTODA (Fig. 5) 8b Body a single unit. Mouth and 9 digestive system present, but no anus. 9a External or internal parasite of higher animals. Sucking discs present for attachment. Life his- tory may involve two or more in- termediate hosts or stages. Flukes. Class TREMATODA 9b Free living. Entire body covered with locomotive cilia. Eye areas in head often appear ‘crossed’. Free living flatworms. Class TURBELLARIA (Fig. 6) lOa Long, slender, with snake-like motion in life. Covered with glis- tening cuticle. Para8itlc or free- living. Microscopic to six feet in length. Round worms. Phylum NEMATHELMINTHES (Fig. 7) lOb Divided into sections or segments 11 2 #1 1 — ------- Key to Selected Groups of Freshwater Animil’ ., 10,’ tJnc( Inentcd head blunt one 18 i two retractile tentacles. Flat pointed, tail. liii Head a more or less well-formed, hard, capsule with jaws, eyes, and antennae. Class INSECTA order DIPTERA (Figs. RA, 8C) lib Head structure soft, except 12 jaws (if present). Fig. 8E.) 12a }k ’acl conical or rounded, lateral appendages not conspicuous or numerous. 12h Ilpad somewhat broad and blunt. Retractile jaw’; usually present. Soft fleshy lobes or tentacles, often somewhat flattened, may be present in the head region. Tad usually narrow. Lateral lobes or fleshy appendages on each segment unless there is a large sucker disc at rear end. Phylum ANNELJDA (Fig. 9) 13a Minute dark colored retractile jaws present, body tapering somewhat at both ends, pairs or rings of bumps or “legs” often present, even near tail. Class INSECTA Order DIPTERA 13b No jaws, sides of body generally parallel e’ccept at ends. ThLcken- ed area or ring usually present if not ill the way back on body. Clumps of minute bristles on most segments Earthworms, sludge- worms. Order OUGOCHA ETA l4a Scgmentb with bristles and/or fleshy lobes or other extensions. Tuhc ’ builders, borers, or burrowers. Often reddish or greenish in color, Brackish or fresh water. Nereid worms. Order POLYCIIAETA (Fig. 9A) 14b Sucker disc at each end, the large one posterior. External blood- sucking parasites on higher anirnal . often found unattached to hoqi. Leaches. Class HIRUDINEA (Fig 9B) 15a Skeleton internal, of true bone. 40 (Vertebrates) 15b Body covered with an external 16 skeleton or shell. (Figs. 10, 13, 17, 18, 24. 25, 28) 13 16a External skeleton jointed, shell 19 covers legs and other appendages, often leathery in nature. Phylum ARTHROPODA 16b External shell entire, not jointed. 17 unless composed of two clam- like halves. (Figs. 10, 11, 12) 17a Half inch or less in length. Two leathery, clam-like shells. Soft parts inside include delicate jointed appendages. Phyllopodc or branchiopods. Class CRUSTACEA, Subcbssei, BRANCHIOPODA (Fig. 12) and OSTRACODA (Fig. 11) 17b Soft parts covered with thin skin, mucous produced, no jointea I c Phylum MOLLUSCA 14 iSa Shell single, may be a spiral cone. Snails. Class GASTROPODA (Fig 13) 18b Shell double, two halves, hinged at one point. Mussels, clams. Class BIVALVIA (Fig. 10) 19a Three pairs of regular walking legs, or their rudiments. Wings present in all adults and rudiments in some larvae, Class INSECTA (Figs. 22, 24D 25, 26, 28, 29) 19b More than three pairs of legs 20 apparently present. 20a Body elongated, head broad anu flat ‘4 (Fig. 8) 29 41—3 ------- Ke) to Selected Gr p of Freshwater Animals with strong jaw8. Appendages follow- ing first three paiis of legs are round- dcii tapering filaments. Up to 3 inches long. Dobson fly and fish fly larvae. Class INSECTA Order MECAI.OPTERA (Fig. 14) 20b Four or more pairs of legs. 21 2 1d Four pairs of legs. Body rounded, bulbous, head minute. Often brown or red. Water mites. Phylum ARTHROPODA, Cla8s ARACHNIDA, Order ACARI (Fig. 15) 21h Five or more pairs of walking or swimming legs; gills, two pairs of antennae. Crustaceans. Phylum ARTHROPODA, Class CRUSTACEA 22a Ten or more pairs of flattened, leaflike swimming and respiratory appendages. Many species swim constantly in life, some swim upside down. Fairy shrimps, phyllopods. or branchipods. Subclass BRANCHIOPODA (Fig. 16) 22b Less than ten pairs of swimming 23 or respiratory appendages. 23a Body and legs inclosed in bi- valved (2 halves) shell which may or may not completely hide them. 2 lb Body and legs not enclosed in bivalve shell. May be large or minute. (FIgs. 17, 18, 19) 24a One pair of branched antennae enlarged for locomotion, extend outside of shell (carapace). Single eye usually visible. “Water fleas” Subclass CLADOCERA (Fig. 12) 24h Locomotion accomplished by 25 body legs, not by antennae. 25a Appendages leaflike, flattened, more than ten pairs. Subclass B} ANCHIOPODA (See 22 a) 25b Ammal less than 3 mm, in length. Appendages more or less slender and jointed, often used for walking. Shells opaque. Ostracods. (Fig. 11) Subclass OSTRACODA 26a Body a series of six or more 27 similar segments, differing mainly in size. 26b Front part of body enlarged into a somewhat separate body unit (cephalothorax) often covered with a single piece of shell (cara- pace). Back part (abdomen) may be relatively small, even folded underneath front part. (Fig. 19b) 27a Body compressed laterally i.e., organism is tall and thin. Scuds. amphipods. Subclass AMPHIPODA (Fig. 17) 27b Body compressed dorsoventrally, i.e., organism low and broad. Flat gills contained in chamber beneath tail. Sowbugs. Subclass ISOPODA (Fig. 18) 28a Abdomen extending straight out behind, ending in two small pro- 24 jections. One or two large masses of eggs are often attached to female. Locomotion by means of two enlarged, uribranched antennae, the only large 26 appendages on the body. Copepods. Subclass COPEPODA (Fig. 19) 28b Abdomen extending out behind ending in an expanded “flipper ’ or swim- ming paddle. Crayfish or craw fish. Eyes on movable stalks. Size range usually from one to six inches. Subclass DECAPODA 29a Two pairs of functional wings, one pair may be more or less har- dened as protection for the other pair. Adult insects which normally live on or En the water. (rigs. 25, 28) 22 28 41—4 ------- Key to Selected Groups of Freshwater Animals 29b No functional wings, though p.ids in which wings are develop- inj tony h visible. Some may r ‘o’mhle adult insects very closely, others may differ ex- treincly from adults. 30a External pads or cases in which 35 wings develop clearly vieible.(Figs. 2426, 27) 30b More or less wormlike, or at 31 least no external evidence of wing development. 31a No jointed legs present. Other structures such as hooks, sucker dii cs, breathing tubes may be present. Larvae of flies, midges. etc. 31b Three pairs of jointed thoracic 32 legs, head capsule well formed. 32a Minute (2-4mm) living on the water 8urface film. Tail a strong organ that can be hooked into a “catch” beneath the thorax, When released animal jumps into the air. No wings arc ever grown. Adult spring- tails. Order COLLEMBOLA (Fig. 20) 32b Larger (usually over 5 mm) 33 wormlike, living beneath the surface. 33a Live in cases or webs in water. Cases or webs have a silk foundation to which tiny sticks, stones, and/or bits of debris are attached. Abdominal segments often with minute gill filaments. Generally cylindric In shape. (‘addisfly larvae. Order TBJCHOPTERA (Fig. 21) 33b Free living, build no cases. 34 34a Somewhat flattened in cross ‘ction and massive in appear- .lfl( e, Each abdominal segment with rather stout, tapering, lateral filaments about as long as body is wide. Aiderflies, fishflies, and dobsonflies, Order MEGALOPTERA (Fig. 22, 14) 34b Generally rounded In cross section, Lateral filaments if present tend to be long and thin. A few forms extremely flattened, like a suction cup. Beetle larvae. Order COLEOPTERA (Fig. 23) 35a Two or three filaments or other 37 structures extending out from end of abdomen. 35b Abdomen ending abruptly, unless 36 terminal segment itself is extended as single structure.(Figs. 24A, 24C) 36a Mouth parts adopted for chewing. Front of face covered by extensible folded mouthparts often called a “mask”. Head broad, eyes widely spaced. Nymphs of dragonflies or darning needles. Order ODONATA (Figs.24A, 24C, 24E) 36b Mouthparts for piercing and sucking. Legs often adapted for water lo- comotion, Body forms various. Water hugs, water scorpions, water boatmen, hackswimmers, electric light bugs, water striders, water measurers, etc. Order HEMIPTERA (Fig. 2 37a Tail extensions (caudal filaments) two. Stonefly larvae. Order PLECOPTERA (Fig. 26) 37b Tail extensions three, at times greatly reduced in sue. 38a Tail extensions long and slender. Rows of hairs may give extensions a feather-like appearance. Mayfly larvae. Order EPHEMEROPTEflA (Fig. 27) 38b Tail extensions flat, elongated plates. Head broad with widely spaced eyes, abdomen relatively long and slender. Damseifly nymths. Order ODONATA (Fig. 24D1 30 Order DIPTERA (Fig. 8) 41—5 ------- l’.ei to Selpeted Groups of Freshwater Animals ‘a l.xt ’riia1 wings or wing co ers form a hard protective dome over the Inner wings folded beneath, and over the abdomen I teetles. Order COLEOPTERA (Fig 28) 39b flxternal wings leathery at base, Mernbranareoua at tip. Wings sometimes very short Mouth- parts for p1 rcing and sucking l3odv form various. True bugs Order HEMIPTERA (Fig. 25) 40a Appendage present in pairs (fins. legs, wings) 40h No paired appendages. Mouth a round suction disc 41a Body long and slender Several holes along side of head Lampreys. Sub Phylum VERTEBRATA, (‘lass C YCLOSTOMATA lib Rndy plump, oval Tail extendin out abruptly. Larvae of frogs an toads. Legs appear one at a time during metamorphosis to adult form. Tadpoles. Class AMPHIBIA 42a Paired appendages are legs 42b Paired appendages are fins, gills covered by a flap (operculum) True fishes. Class PISCES 43a Digits with claws, nails, .or hoofs 43b Skin naked \in claws or digits Frogs, toads, and salamanders Class AMPUH3IA 42 44a Warm blooded 41 44b Cold b1i oded. Body covered with horny scales or plates Class REPTILLA 451 Body covered with feathers Birds Class AyES 45b Body covered with hair Mammals Class MAMMALLA 41 44 4 41—6 ------- Key to Selected Gro ps of Freshwatcr Animals 1EFERENCES - Invertebrates I Eddy, Samuel and Hodson, A.C. Taxonornic Keys to the Common Animals of the North Central States. Burgess Pub. Company. Minneapolis. 162 p. 1961. 2 Edmondson, W. T. (ed.) and Ward and \Vhipple’a Freshwater Biology. John Uley & Sons, New York. pp. 1-1248. 1959. 3 Jahn, T. L. and Jahn, F. F. How to Know the Protozoa. Wm. C. Brown Compai , Dubuque, Iowa. pp. 1-234. 1949. 4 Kiots, Elsie B. The New Field Book of Freshwater Life. G.P. Putnam’s Sons. 398 pp. 1966. 5 Kudo, R. Protozoology. Otarles C. Thomas, Publisher, Springfield, Illinois. pp. 1-778. 1950. 6 Palmer, E. Lawrence. Fieldbook of Natural History. Whittlesey House, McGraw-Hill Book Company, Inc. New York. 1949. 7 Pennak, R.W. Freshwater Invertebrates of the United States. The Ronald Press Company, New York. pp. 1-769. 1953. 8 Pimentel, Richard A. Invertebrate Identification Manual. Reinhold Publishing Corp. 151 pp. 1967. 9 Pratt, U. ‘.. A Manual of the Common Invertebrate Animals Exclusive of Insects. The Blaikston Company, Philadelphia, Pa. pp. 1-854. 1951. REFERENCES - Fishes 1 American Fisheries Society. A List of Common and Scientific Nami-’s of Fishcs from the United States and Canada. Special Publication No. 2, Am. Fish Soc. Executive Secretary AFS. Washington Bid. Suite 1040, 15th & New York Avenue, N. V I. Washington, DC 20005. (Price$4.00 paper, $7.00 cloth). 1970. 2 Bailey. Reeve M. A Revised List of the Fishes of Iowa with Keys for identification, IN Iowa Fish and Fishing. State of Iowa, Super, of Printing. 1956. (Excellent color pictures). 3 Eddy, Samuel. How to Know the Freshwater Fishes. ‘Nm. C. Brown Company. Dubuque, Iowa. 1957. 4 Hubbs, C.L. and Lagler, K.F. Fishes of the Great Lakes Region. Bull. Cranbrook Inst. Science, Bloomfield Hills, MichIgan. 1949. 5 Lagler, K,F. Freshwater Fishery Biology. Wm. C. Brown Company, Dubuque, Iowa. 1952. 6 Trautman, M. B. The Fishes of Ohio. Ohio State University Press, Columbus. 1957. (An outstanding example of a State study). Descriptors: Aquatic Life, Systematics. 41-7 ------- Key to Selected Group. of Freshwater Animals 3W RoWer, KrstsUa Up to • 3 mm, IC. Retlf.r Philodiss Upto .4 mm. 4C. Joimed leg Oetr.cod 5. Tapeworm beads TunIa . Upto 25ydm. long 7. Nematodes. Free Uvthg forms commoni) ’ up to 1 nun., occasionafly more. i At \ ‘ \ 1. 5pongLlla spiculs. Upto .2 mm. long. 3*. Retlfer, arthra Upto .3 mm. 2B. Bryotosi mass. Up to several feet diam. 2A. Brycios PlumMsll* . isdividuala ep to 3 mm. hdertwtasd ma.e . maybe very satonelve. 4*. Joi ed leg Caddiaf ly Jo et.d log Crayfish SB. Turb.U ri.. f Up to 1.6 cm. aria M..ost a .1 IV £ m. 41’8 ------- K.y to Selected Groups of Freshwater Animals SB. Diptera, Mosquito pupa, Upto5mm. SA. Dipitra. Mosquito larvae Up to 15 mm. long. 8C. Diptera, larvsie. Up to 2 cm. 12B. Branchiopod, Bosmina. Up to 2mm. chironomid BE. Diptera. crane fly pupa. Upto2.Sczn. BA. Annelid. • 1 gj td worm. up to 1$ meter SD. Dipters. Rattailed maggot Up to 25mm. without tub.. - 1 ) bA. PeI.cyopod. Alasmidonta Side view, up to 18 cm. long. SB AnneUd. l..ch up to 20 cm. 108. Alasmidonta , end view. 12A. BranchiopOd, Daphnia . Up to 4mm. hA. Ostracod, Cypericu . Side view, up to 7 mm. 111. Cypericua , end view. 41 9 ------- Key to Selected Group f Freshwater Anim 1s 13. Gastropod, Caapelom* Up to 3 Inches. 14. segaloptera, Siali . Alderfly larvae Up to 25 am. 16. Fairy Shrimp, Eubranchipuu Up to $cm. clopoid copepod Female Up to 25 am. 15. Water mite, up to 3 mm. 18. leopod, Asellus Up to 25 am. 20. Collembola, Podura 1OA. Up to 2 am. Tong cope—, l9B. Female Up to 3 mm. 41.10 ------- hey to Selected Groups 01 Freshwater Anirnai,’, I ‘ (\ t J 21A. 21B. 2 1C. I 21. Trichoptera, larval cases, mostly 1-2 cm. 2 • Odonata, tail of daaeelfly nymph (side view) uborder Zygoptera (248, D) 241). Odonata, damseifly nymph (top view) 22. Megaloptera ,alderfly Up to 2 cm. Odonata, front view of dragonfly nymph showing “mask” partially extended Suborder Anisoptera (24A, E, C) 24C. Odonata, tail of dragonfly nymph (top view) 21D. 21E. 23A. Beetle larvae, Dytieidae, Usually about 2 23B. Beetle larv , Hydrophilidse cm. Usually *boUt J. cm. 24A. Odonata, dragonfly nymph up to 3 or 4 cm 41 ’11 ------- Key to Selected Groups of Freshwater Animals 37.Eph.meroptera. M — tip to 3cm . ISA. Dipt.ra. Crsi fly. Up to *1 cm. 25B. Hemiptera, Water Scorpion About 4 cm. 25A. H.mlptirs. Water Boatman About 1 cm. tJpto 5cm. 3M. Co1sopt.r ,, Water Ioavsse r bs.tle, Up to 4 cm. 31B. Colsoptera Dytiseid beetle Usuallyupto4 cm. 39B. Dlpt.rs, Mosquito UptoIO mm. 41—12 ------- 11 KEY TO ALGAE OF IMPORTANCE IN WATER POLLUTION I Plant atube.thread. strand, ribbon, or membrane, frequently visible to the unaided eye 2 1’ Plants of microscopic cells which are isolated or in irregular, spherical, or microscopic clueter5, cells not grouped into threads . . . 123 2 (I) Plant a tube, strand, ribbon, thread, or mernhrsne composed nf cells . 3 Plant a branching tube with continuous protoplasm, not divided into cclls . 120 3 (2) Plant a tube, strand, ribbon, thread, or a mat of threads . . ... . 4 3’ . Plant a membrane of cells one cell thick (and 2 or more cells wide) 116 4 (3) Cells in isolated or clustered threads or ribbons which are only one cell thick or wide 5 44 Celia in a tube, strand, or thread aLL (or a part) of which is more than one cell thick or wide . ... 108 5 (41 Heterocysts present . 6 5’ Heterocysts absent , , . 23 6 (5) Thread. gradually narrowed to a point at one end . . 7 6’ Threads same width throughout. . . . . 12 7 (61 Threads as radii, In a gelatinous bead or mass . . . 8 7’ Threads not in a gelatinous bead or mass . ... 11 8 (7) Spore (akinete) present, adjacent to the terminal heterocyst ( Gloeotrichia ) 9 8’ No spore (akinste) present ( Rivularia ) .. . ... - . . .. 10 9 (8) GelatInous colony a smooth bead. . ... . .. . Cioeotrichia echinulata 9’ Gelatinous colony irregular .. . , Oloeotrlchia natans 10 (8’) Cslls near the narrow end as long as wide . Rivularia 10’ Cells near the narrow end twice as long as wide .. .. Rivularia haematites 11 (7’) Cell, adjacent to heterocyst wider than heterocyst . . . Calothrlx braunii Il ’ Cells adjacent to heterocyst narrower than hetsrocyst . . Calothrix p4rietina 12 (6’) Branching present . , , 13 12’ BranchIng absent . .. . 14 13 (12) Branches in pairs . , Scytonema tolypothricotdee 13’ Branches arising singly . , . . . .. . . Tolypothrix tenuie 14 (12’) Heterocyst terminal only ( Cyclindrospermum) . . 15 14’ Hstrocyste intercalary within the filament) . 16 15 (14) Heterocyst round . . . . . Cy1indrosi rmum musci cola 15’ Heterocyst elongate . , . ... Cylindrospermum stagnale 16 (14’) Thread. encased in a gelatinous bead or mess . . . .. . 17 16’ Threads not encased In a definite gelatinous mass . . is I i (16) Heterocysts and vegetative cells rounded Nostoc pruniforme I ?’ Heterocysts and vegetative cells oblong . . . . . Nostoc csrneum 18 (16’) Heterocyste and vegetative cells shorter than the thread width Nodularia gpumIgena 18’ Heterocysts and vegetative cells not shorter than the thread width, . . . . . .19 19 (18’) Heterocyets rounded ( Anabaena ) 20 19’ Heterocysts clindric. . .. . Aphanleomenon flos-aquae 20 (19) Cells elongate, depressed in the rniodle. heterocyste rare. Anabaena conetricta 20’ Cells rounded, heterocyets common . ... . 21 21 (20’) Heterocyste with lateral extensions. . . Anabaena planctonica 21’ Ileterocyste without lateral extensions .22 B1.MIC ,c la ,8b ,8 ,5 9 42—1 ------- 22 (21’) 22’ 23 (S’) 23’ 24 (23) 24 25 (23) 25’ 26 (25’) 26’ 27 (26’) 27 28 (27) 28’ 29 (28) 29’ 30 (29’) 30’ 31(30) 3’, 32 (28’) 32’ 33 (32) 33’ 34 (32’) 34’ 35 (27’) 35’ 36 (35) 36’ 37 (36’) 37, 38 (37) 38’ 39 (35’) 39’ 40 (39’) 40’ 41(40) 41’ 42 (41) 42’ 43 (40’) 43’ 44 (43) 44’ 25 49 285 26 Microcoleus subtorulosue 27 • . Lyngbya ocracea 30 Lyngbya lagerheimii 31 Lyngbya digueti Lyngbya ver.icolor 33 34 Phormidium uncinstum Phormidjum autumnale Phormidium inundatum • . . Phormjd [ um retail 36 39 • O ,cillatorja ornata 37 • . 38 O.cillatoria limosa Oscjljatorja curviceps Oscillatoria princep. Oacjll&torja rube,cer 40 41 43 Osciliatorla putrida • . . . 42 Oscillatoria lauterbornit Oscillatoria chlorina 44 • . 48 Oscillatoria pseudogeminata 45 rhreads 4-8ii wide . Anabaena fioe- Thread. 8- )4, wld. ’ • . Anabaena circina3is Branching absent . . 24 Branching (including “false” branching) present . . 84 Cell pigments distributed throughout the protoplasm Cell pigments limited to plaetid. Thread, short and formed as an even spiral • .. Threads very long and not forming an even spiral Several parallel thread. of cells in one common aheath ____________ ____________ One thread per sheath if present Sheath or gelatinous matrix present . . • . . . . .28 No sheath nor gelatinous matrix apparent ( Oscillatoria ) . . . 35 Sheath dIstinct, no gelatinou, matrix between threads ( Lyngbya ) . 29 Sheath indistinct or absent, threads Interwoven with gelatinous matrix between ( Phormidium ) . 32 Cells rounded , Cells short cyltndric Threads in part forming spirals l’hrends straight or bent but nut in spirals Maximum cell length 3 5g , sheath thin Maximum cell length 6 5. . sheath thick Ends of some threads with$ rounded swollen “cap” cell Ends of all threads without a “cap” cell . End of thread (with “cap”) abruptly bent End of thread (with “cap”) .traight .. . Thread. 3-5, in width . Thread. 5-12, in width Cell. very short, generally less than 1/3 the thread diameter Cells generally 1/2 as long to longer than the thread diameter Cross walls constricted . . Cross wall, not constricted . .. . End . of thread, if mature, curved Ends of thread straight . Thread. 10-14, thick , . lhreads 16-60, thick Threads appearing red to purplish Threads yellow-green to b)ue-green Thread, yellow-green • , Threads blue-green . . . • . Cells 4-7 times a. long as the •t read diameter Cell, less than 4 times as long a, the thread diameter rrominent granules (“pseudovacuc le.”) in center of each cell No prominent granules In center of cells Cells 1/2-2 times as long a. the thread diameter Cell. 2-3 time. as long as the thread diameter Cell walls between ccli, thick and transparent Cell walls thin appearing a. a dark line 42—2 ------- 45 (44’) End, of thread straight . . . Oscillatoria agardhii 45’ Ends of mature thread. curved . . . . . . . .46 46 (45’) Prominent granule. present eepeclsliy at both ende of each cell . . Oacillatoria tenuia 46’ Cell, without prominent granule. .. . . 47 47 (46’) Cross wall. conatricted . . . . .. Oscillatorla chalybea 47’ C rose wall, not conetrictad Oscillatoria formoaa 48 (43’) End of thread long tapering . . . Oscillatoria eplendida 48 Fed of thread not tapering . . . . . .. . . . . Oscillatoria amphibia 49 (24’) Cell, separate from one another and enclosed in a tube ( Cyrnbella ) . 251’ 49’ Cell, attached to one another as a thread or ribbon 50 50 (49’) Ccli. separating readily Into disco or short cylinder.. their circular face showing radial marking. . . . . 233 50’ Cells either not separating readily, or if eo. no circular end wall with radial marking. 51 51(50’) Cells in a ribbon, attached elde by side or by their corners . 52 SI’ Cell. In a thread, attached end to end . . . 56 52 (51) Numerou, regularly spaced marking. in the cell wall . 53 52’ Numerous markings in the cell wall absent ( Scenedesmus ) . . . 128 53 (52) Wall marking, of two types, one coarse, one fine .. . . 185 53’ Wall marking. all fine ( Fragilaria ) . . 54 54 (53’) Cell, attached at middle portion only . Fragilaria crotonensls 54’ Cells attached along entire length .. .,,, , .55 55 (54’) Cell length 25-l00 t Fragilaria capucina 55’ Cell length ‘7-ZSsi ... . . .. Fragilarla conetruens 56 (51’) Plastid in the form of a sj ral band ( Spirogyra ) . . . . .. . . 57 56’ Plastid not a spiral band .‘ . 61 57 (56) One plastid per cell . . 58 5?’ Two or more plsstids per cell 60 58 (57) Threads i 8 -Zbp wide Spirogyra communie 58’ Threads Z8-50 wide 59 59 (58’) Threads 28 - 4 0p wide . , .. . Spirogyra variana 59’ Thread. 40-SOp wide . . . . . . Spirogyra porticalis 60 (57’) Thread. 30-45 . wide. 3.4 plastid. per ,etl . . . Spirogyra fluviatilie 60’ Threads 50-80, wide. 5-8 pla.tid. per cell . . Spirogyra majuscula 61(56’) Plaetid. two per cell , . . . . . . . . 62 61’ Pla.tids either one or more than two per cell . 66 62 (61) Cells with knob . or granules on the wall . . 63 62’ Cell, with a smooth outer wall , . . , 64 63(62) Each cell with two central knob, on the wall Deemtdium grevillit 63’ Each cell with a ring of granule. near one end Hyalotheca mucoaa 64(62’l Cells denne green, each pla,tid reaching to the wall ygnema sterile 64’ Cells light green. plastids not completely filling the cell 65 65 (64’) Width of thread U’ 32p. maxlrnun, cell length 6Op Zygnema inaigne 65’ WIdth of thread .30 -36, ,, maximum “all length IZOp . Zygnerna pectinatum 66 (61’) Plastid a wide ribbon. psesing through the cell axle ( Mougeotia ) . 67 66’ Plastid or plastids close to the cell wail (parietal) 69 42-3 . IJ,I C’ t’ ’’’ 2003W 3hSk7o .e ( C r I k. Orc ’qc 97 D ------- 1 ,7 ((.1,) Thread, with occasional ‘knee-joint” bend. .. . . . Mougeotia genuflexa 1,7’ lhrende straight . . 68 ( H (67) Thread. l’)-Z 4 wide. pyrenoids 4-16 per cell. . . Mougeotia sphaerocarpa 68’ Thread. 2O -34 , wide. pyrenoid. 4-10 per cell . Mougeotia acalaris 69 (66’) Occasional cell. with one to .everal transverse wall lines near one end ( Oedogonium ) 70 69’ Occasional terminal transverse wall line. not present. .. . .. 73 70 (69) Thread diameter less than 24g 71 70’ Thread diameter 25j,i or more . 72 71(70) I’hread diameter 9 -l4 (* . . . Oedogonium .uecicum 7) Thread diameter l4-23g . Oedogonium bo.cii 72 (70) Dwarf mile plant. attached to normal thread, when reproducing Oedogonium idioandro .porum 72’ No dwarf male plant, produced . .. Oedogonium gre.nde 73 (69’) Cells with one plastid which has a smooth surface , 74 73’ Cells with several plastids or with one nodular piastid . . . . . . 78 74 (73) C cli . with rounded ends . . Stichococcus bacillari . 74’ Cells with flat end. (Ulothrtx) 75 75 (74’) Thread. lOg or lee, in diameter . . . 76 75’ ‘1 breads more than lOp in diameter . . . 77 76 (75) Thread. 5- 6 p in diameter . . . . . Ulothrix variabjije 76’ Thread. h.lOp in diameter . tJiothri.x tenerrima 77 (75’) rhread. ll-l?p in diameter . Ulothrtx aeguali . 77’ Threads 20-60 in diameter , , ‘ . ‘ . Ulothrix Zonata 78 (73’) Iodine test for starch positive; one nodular plastid per cell . .79 78’ Iodine test for starch negative, .everal plastids per cell . . . . 80 79 (78) Thread when broken, forming “H” ehape segments Microapora amoena 79’ Thread when fragrr(ente4 separating irregularly or between cells ( Rhizocionium) . 100 HO (78’) Side walls of cell. straight. net bulging A pa t?rn of fine lines or dote present in the wall but often indistinct ( Melosire ) 81 HO’ Side wall, of cells slightly bulging Pattern of wall markings not present ( Tribonema) , 83 HI (80) Spine-like tceth at margin of end walls .. ‘ . . 82 81’ No spine-like teeth present , . . Melosira variant 82 (81) Wall with (the granule., arranged obliquely .. . . . Melosirs crenulata H. ’ Wall with coarse granules, arranged parallel to side. . Meloeira ranulata 83 (80’) Plastid. 2-4 per cell . . . . . Tribonema minue Hi’ Plastide more than 4 per cell . , . . ‘ .. Tribonema oombyclnum 84 (23’) Plastids present, branching “true” . 85 144’ Plastide absent, branching “false” Plectonema tomaqiniana 145 (84) Branches reconnected, forrnIn , net . . Hydrodictyon reticulaturn 85’ Branches not forming a distinct net , . . . . . . 86 86 (85’) Fach cell in a conical sheath open at the broad end tDinobryon ) .. . 87 M I” N , conical sheath sroun ,I each cell . 90 8 ? (86) l3ranchee diverging, often almost at a right angle . Dinobryon divergena 87’ ranche. compac.. often almost parallel . ‘ 88 88 (87’) Narrow end ol sheath sharp pointed . , , , 8q 8$’ Narrow end of i heath blunt pointed . , , Dinobryon .ertuiaria 42—4 ------- 89 (88) Narrow end drawn out into a stalk . .. . . . Dinobryon atipitaturn$9’ Narrow end diverging at the base . .. . . . . . - Dinobryon sociale 90 (86’) Short branchee on the main thread in whorls of 4 or more (Nitella) . . . 91 90’ Branching commonly single or in paire . . .92 91 (90) Short branches on the main thread rebranehed once . Ntte llaflexi lis 91’ Short branches on the main thread rebranched two to four times Nitella gracilis 92 190’) Terminal cell each with a colorless spine having an abruptly swollen base ( Bulbochafle l 93 92’ No terminal spines with abruptly swollen bases . . 94 93 Vegetative cells Z0-48p long . .. Bulbochaete rnirabilis 93’ Vegetative cc Its 4\$-€Sp tong .... . . . . Bulbochaete insign)j 94 (92’) Cell, red, brown, or violet . . Audouinella violacea 94’ Cells green . . . . 95 95 (94) Threads enclosed in a gelatinous head or mass . . . . 96 95’ Threads not surrounded by a gelatinous mass . . . . 99 96 (95) Abrupt change in width from main thread to branches ( Draparnaldia ) 97 91 , ’ Gradual change in width from main thread to branches ( Chaetophora ) 9 1 1 97 (96) 3 ranches (from the main thread) with a central, main axis . . Draparnaldia plumosa 97’ Rrsnchrs diverging and with no central main axis Draparnaldia glomerats yR (96’) End cells long-pointed, with colorless tips . Chaetophora attenuata 98’ End cells abruptly pointed, mostly without long colorless tips Chaatopltora elegans 99 (95’) Light and dense dark cells intermingled In the thread . . .. Pithophora oedogogonia 99’ Most of the calls essentially alike in density . . . . . . . . 100 100 (99’) Branches few in number, and short, colorless Rhiaoclonium hierogiyphicum 100’ Branches numerous and green . .. ... 101 1011100’) ‘Terminal attenuation gradual, involving two or more cells ( Stigeoclonium ) .. 102 101’ Terminal attenuation absent or abrupt. involving only one cell ( Cladoohora ) 104 102 (10)) Branches frequently In pairs 103 102 ’ Branches mostly single Stigeoclonium stagnatile 103 (102) Cells in main thread 1-2 times as long as wide Stigeoclonium lubricum 103 ’ Cells in main thread 2-3 times as long as wide Stigeocloniom tenue 104 (101’) Branching often appearing forked, or in threes .....,...... Cladophora aegagropila 104’ Branches distinctly lateral 105 lO S 1104’) Branches forming acute angle with main thread, thus forming clusters.Cladophora glomerats 105’ Branches Forming wide angles with the main thread . . . ,. 106 106 (105’) Threads crooked and bent Ciadophorafracta l O b ’ lhreads straIght . . . . . . . 107 107 (106’) Branches few, seldom rebranching . Cladophorn insignis 107’ Branches numerous, often rebranching Cladophora crispata 108 (4’) Plant or tubc with a tight surface layer of cells and with regularly spaced awellings (nodes) . . Lemanen annulata 108’ Plant not a tube that has both a tight layer of surface cells and nodes - . 109 109 (lO S’ ) Cells spherical end loosely arranged In a gelatinous matrix Tetraspora geiatinosa 109’ Cells not as loosely arranged spheres. . , 110 110 (109’) Plants branch .. . . I D 1 10’ Plants not branched . . .. . . . . Schizomeris leibleinil I II (110) Clustered branching . . . . . . . . . . - 112 Ill’ Branches single , , . 115 42—5 ------- 112 (III) Threads embedded in gelatinous matrix ( Batrachosperm ) . 113 HZ’ No gelantlnou. matrix (Char.) . . 114 113 (lIZ) Nodal masses of branches touching one another Batrachospermum vagum 113’ Nodal masses of branches separated by a narrow space . Batrachospermum moniliforme 114 (112’) Short branches with 2 naked cell, at the tip Citarn globularje 114’ Short branches with 3-4 naked cells at the tip . Chara vulgaris 115 (Ill’) Heterocy.te present. plastids absent . Stigonema minutum 115’ Heterocyets absent. pla.tid . present Compsopogon coeruleus 116 (3’) Red eye spot and two flagella present for each cell 125 116 No eye spots nor flagella present . . . 117 II? (116’) Round to oval cells, held together by a flat gelatinous matrix ( A menellurn ) .. . 131 117’ Cell, not round and not enclosed in a gelatinou, matrix . . 118 118 (117’) Cell, regularly arranged to an unattached disc. Number of cells 2, 4. 8, 16, 32. 64. or 128 . . . . . . 133’ 118’ Cells numerous, membrane attached on one surface 119 119 (118’) Long hairs extending from upper surface of cells . . . Chaetopeltis megalocystis 119’ No hairs extending from cell surfaces Hildenbrandia rivularle 120 (2’) Constriction at the base of every branch Dichotomosiphon tuberosus 120’ No constrictions present in the tube ( Vaucheria) . . , . .. . . . . 121 121 (120’) Egg sac attached directly, without a stalk, to the main vegetative tube Yaucheria sessilis 121’ Egg sac attached to an abrupt, short, side branch 122 122 (121’) One egg sac per branch . Vaucheria terrestris 122’ Two or more egg sacs per branch Vaucheria geminata 123 (1’) Cells in colonies generally of a definite form or arrangement . 124 123’ Cells isolated, in pairs or in loose, irregular aggregate. . 173 124 (123) Cells with many transverse rows of markings on the wall .. . . . 185 124’ Cell. without transverse rows of marking. 125 125 (124’) Cells arranged as a layer one cell thick 126 125’ Cell cluster more than one cell thick and not a flat plate . . . .. . . 137 126 (1251 Red eye spot and two flagella present for each cell Gonium pectorale 126’ No red eye spots nor flagella present . 127 127 (1Z( , ’) Cells elongate, united side by side in 1 or 2 rows ( Scenedesmus ) 128 127’ Cells about as long a. wide .. . . . . . . . . 131 128 (127) Middle cells without spines but with pointed ends Scenedesmus dimorphus 128’ Middle cells with rounded ends . .. . .129 129 (128”) Terminal cells with spines .,, , . , 130 129’ Terminal cells without spines . Scenedesmu. bijuRa 130 (129) Terminal cells with two spines each . , . . .. Scenedosmus guadrtcauda 130’ Terminal cells ith three re ” more spines each Scenedesmue abundans 131 (117) Cells In regular rows, immersed in colorless matrix ( Agmenellum Quadrjduplicatum ) 132 131’ Cells not immersed In colorless matrix . . . . , . . . 133 II? (Ill) Cell diameter I 3 to L Agmenellum guadriduplicatum , tenuissima type 13t’ Cell diampter 3 -Sp . Agmenellum guadriduplicatum. glauca type 133 1131’) Coils without spines, projections, or Incision.. .. . . .. . Crucigenia guadrata 133’ Cell, with spine., projections, or incision. . . . . 134 42—6 ------- 134 (133’) Call. round.d . . Micractintum pusillum 134’ Cell, angular (PedIsetru, , ,) . , 135 135 (134) Nu,narou. spaces between elIs . . . Pediastrum duplex 1 1 5 ’ Cells fitted tightly together . 136 l3( (135’) Cell incisions deep and narrow. , . . Pediastrum tetres 136’ Cell Incisions shallow and wide Pediastrum boryanum 137 (125’) Celle sharp-pointed at both ends, often arcuate 138 137’ Cells not sharp-pointed at both ends; not arcuate. . . 141 138 (137) Cells embedded in a gelatinou. matrix . Ktrchneriella lunaria 138’ Cells not embedded in a gelatinous matrix ... 139 139 (138’) Cells all arcuate, arranged back to back . Selenastrum g aci1e 139 Cells straight or bent in various ways, loosely arranged or twisted together ( Ankistrode.mus ) 140 140 (139’) Cells bent . . . . . . Ankistrodesmus falcatue 140’ Cells straight Ankistrodeemus falcatus var acicularts 141 (137’) Flagclla present, eye spots often present 142 141’ No flagella nor eye spots present . . 152 142 (141) Each ‘cl i in a conical eheath open at the wide end ( Dinobryon ) . 86 142’ IndIvidual cell, not In conical sheaths . , , 143 141 (142’) Fach cell with l-Z long .iralght rods extending , . . Chryeoephaerella longispina 143’ No long straight rods extending from the call. 144 144 (143’) Cc 11cc touching one another Ins dense colony 145 144’ Cell, embedded separately In a colorle, naatr 149 145 (144) Cell, arranged radially, facing outward .. 146 145’ Cells all facing in one direction 147 146 (145) Plastid. brown, eye spot absent . . . Synura uvelia l4f ’ Plastid. green, eye spot present in each cell . . . . . Pandorina morum 147 (145’) Eaich cell with 4 flagella . Spondylomorum guaternarium 147’ 1a, h cell with 2 flagella ( Pyrobotrys ) . . . 148 148 1147’) Eye spo t In the wider (anterior) end of the cell Pyrobotry8 etellata 148’ Eye spot In the narrower (posterior) end of the cell . Pyrobotrys precut , 149 (144’) Plastid. brown . Uroglenopsus americana 149’ Pla.tlds green 150 150 (149’) Cells 16. 32, or 64 per colony . Eudorina elegans I SO’ Cells more than tOO per colony . . . 151 151 (1501 Colony spherical each cell with an eye spot. . Volvox aurcus 151’ Colony tubular or irregular no eye spots ( Tetraspora ) ioq 152 (141’) Flongate cells, attached together at one end, arranged radially ( Actinastrum ) 153 152’ Cells not elongate, often spherical.. . . 154 153 (152) Cells cylindric . . . . . . . Actinastrum gracullumum 153’ Cells listInctly bulging . Acttnastrum hantzschui 154 (1521 PlasI ide present . . ‘ 355 154’ Plaetids absent, pigment throughout each protoplast 168 155 (154) Colonies, including the outer matrix, orange to red-brown Botryococcus braunui It S’ Matrix If any, not bright colored, cell pla.tids green 156 42—7 ------- 156 (155) Colonies round to oval . . .. . . . . . . . . . 160 56’ ColonIes not round, oftrn Irregular In form . . . 157 l’;7 (156’) StraIght (flat) wall, between adjacent celia ( Phytoconis ) 278 IS ?’ Wall. b t wean neighboring cell. rounded . . . 158 58 (157’) Cells arranged a. a surface layer in a large gelatinou, tube ( Tetra.pora) . .. 109 158 Colony not a tube, cell, in irregular pattern .. . 159 159 (158) Large cell, more than twice the diameter of the small cell. ( Chlorococcum ) . . . 280’ 159’ Large cell, not more than twice the diameter of the email cell. ( PalmelIa ) 281 160 (156) Cell, touching one another, tightly grouped. . ... . . Coeia.truin microporum 160’ Cells loosely grouped . . . . 161 161 (160’) Colorless threads extend from center of colony to cells. . . 162 161’ No colorless threads attached to cell, in colony . . . 164 162 (161) Cell, rounded or straight, oval ( D1ctyosphaer um ) . . . ‘ . 163 162’ Cell, elongate, some cello curved Dimorphococcus lunatus 163 (1621 Cell. rounded. . Dictyo.phaerium pulchellum l6 ’ Cells straight, oval Dictyosphaerium ehrenbergianum 164 (161’) Cell, rounded 165 164’ Cells oval . Oocysti, borgei 169 (164) One plastid per cell . . 166 165’ Two to four plastids per cell . . . Cloeococcu, achroeteri 161 (165) Outer matrix divided into layers ( Cloeocyetla ) . . . 167 16(.’ Outer matrix homogeneous Sphaerocy tjs achroeteri 167 (166) Colonies angular , . . . Gloeocy,tis planctonica 167’ Colonies rounded Gloeocy.tj. giga . 168 (154’) Cell, equidistant from center of colony ( Gompho.phaeria ) 169 168’ Cells irregularly distributed in the colony . .. . . 172 169 (168) Cell, with pseudovacuoles . .. . ... , Gompho.paeria wichurar 169’ Cells without pseudovacuoles . . . 170 170 (169’) Cells -4 in diameter ( Comphosphaeria lacustris ) . . . 171 170’ Cells ovate . . . . . . Compboephaeria aponina 171 (170) Cells .phorical . . . .. Oomphosphaeria lacuetri,, kuetaingianum type Ill’ Cells 4-15 in diameter , . . . Gomphosphaerta lacustri,, collin.li type 172 (168’) Cells ovid, divl .Ion plane perpendicular to long axis ( Coccochlorip ) . . . . . 286 172’ Cells rounded, or division plane perpendIcular to short axis ( Aztacystis ) 286’ 173 (123’) Calls with an abrupt median transver.e groove or incision . , 174 173’ Cells wIthout an abrupt tranever.e median groove or incision . 184 174 (173) Cell, brown, flagella present (armored flagellates) , . 175 174 C dl. green, no flagella (deimids) . . .. 178 175 (174) Cell with 3 or more long horns Ceratium hirundinella 175’ Cell without more than 2 horn, 176 176 (175’) Cell wall of very thin smooth plates Clenodinium palustre 176’ Cell wall of very thick rough plates ( Peridinium ) 177 177 (176’) Ends of cell pointed . . . Peridinjum wiscon,inense 177’ End, of cell rounded . . . . Peridinfum cinctum 178 (174’) Margin of cell with sharp pointed . deeply cut lobes or long .pikeo . .. , 179 178’ Lob.., if prelent, with rounded end, . . .. . 182 42—8 ------- 119 (178) Median incision narrow, linear . , MJcras erjas truncita 179’ Median Incision wide. “V’ or ‘U’ shaped ( Staurastrum ) 180 180 (179) Margin of cell with long spikes.. Staurastrurn paradoxum 180’ Ms rgtn of cell without long spikes 181 181 (180”) Ends of lobes with short spines Staurastrum polymorphum 181’ Ends of lobes without spine. Staurastrum punctulttum 182 (178’) Length of cell about double the width Euastrum oblongum 182’ Length of cell one to one ..nd one.half times the width ( Cosrnarium ) 183 183 (182’) Median incision narrow linear Cosmariurn botrytis 183’ Median tnciston wide, “U” shaped Co.marium pprtianurn 184 (173’) Cells triangular Tetraedron muticum 184’ Cell, not triangular 185 185 (124) Cells with one end distinctly different from the other 186 Cell, with beth ends essentially alike 225 186 (185) Numerou. transverse (not spiral) regularly spaced wall markings present (diatoms) . 187 186’ No transverse regularly spaced wall marking. 193 187 (186) Cell, curved (bent) In girdle view Rholcosphenia curvata 187’ Cells not curved In girdle view 188 188 (187’) Cell, with both fine and coarse transverse linea Merjdton circulare 188’ Cells with transverse line, all alike in thickness 189 189 (188’) Cells essentially linear to rectangular, one terminal swelling larger than the other ( Asterloneila ) 190 189’ Cells wedgs-.haped; margins sometimes wavy ( Goinphonema) . . .. 191 190 (189) Larger terminal swelling 1-1/2 to 2 tIme. wider than the other Asterionella formosa 190’ Larger terminal swelling less than 1 -1/2 time, wider than the other. . Aa erionella g c llima 191 (189’) Narrow end enlarged in valve view Gomphonema geminatum 19!’ Narrow end not enlarged in valve view 192 192 (191’) Tip of broad end about as wide as tip of narrow end in valve view . Gomphonema parvulum 192’ Tip of hroad end much wider than tip of narrow end in valve view Comphonema olivaceurn 193 (186’) Spine present at each end of cell Schrooderia setigera 193’ No spine on both ends of cell 194 194 (193’) Pigments in one or more plastids 195 194’ No plastid. pigments throughout the protoplast Entophysalis lemaniae 195 (194) Cells in a conical sheath ( Dinobryon ) 86 195’ Cells not In a conical sheath 196 196 (195’) Cell covered with scales sod long spines M*ilo,nena. caudata 196’ CelIa not covered with scale, and long spines 197 197 (196’) Protoplasts separated by a space from a rigid sheath (lorica) . 198 197’ No loose sheath around the cells 202 198 (197) Cells Compressed (flattened) .. Phacotus lenticularis 198’ Cells not compressed 199 199 (198’) Lorica opaque, yellow to reddish or brown Trachelomonag crebea 199’ Lorica transparent, colorless to brownish ( Chrysococcus) . . . 200 200 (199’) Outer membrane (lorica) oval Chrysococcu, ovalis 200’ Outer membrane (lorica) rounded 201 42—9 ------- 101 (200’) Lorica thickened around opening . . . . . . Chrysococcue rufeecena 20)’ L.orica not thickened around opening .... . Chrysococcu. major 202 1)97’) Front end flattened diagonally . . . 203 202’ Front i ’nd not flattened diagonally . 206 203 (202) Plnetid. bright blue-green ( Chroomonse ) 204 203’ Plastids brown, red, olive-green. or yellowish 205 204 (203) Cell pointed at one end Chroomona. nordetetii 204 Cell not pointed It one end Chroomonas setoniensia 205 (203) Gullet present, furrow absent Cr’yptomona. eroas 205’ Furrow present, gullet absent Rhodomonas lacustris 206 (202’) Plastids yellow-brown Chrotnulina ro.snoffj 206’ Plastid. not yellow-brown, generally green 207 207 (206’) One plastid per cell 208 207’ Two to aeveral plastid. per cell . . 211 208 (207) Cells tapering at each end . Chiorogoniwn euchiorum 208’ Cells rounded to oval 209 209 (208’) Two flagella per cell ( Chismydomonas ) . .210 209 Four flagella per cell Caterta multifilia 210 (209) Pyrenoid angular: eye spot in front third of cell Chlamydoinonas reinhardi 210’ Pyrenoid circular, eye spot in middle third of cell Chlamydomonas globosa 211 (207’) Two pla.tids per cell . 211’ Several plastids per cell 212 212 (211’) Cell compressed (flattened) ( caal 213 212’ Cell not compressed 214 213 (212) Posterior spine short. bcnt Phacu. pleuronectea 213’ Posterior spine long, straight Phacue longicauda 214 (212) Cell margin rigid . 215 214’ Cell margin flexible ( Eunlena ) . 217 215(214) Cell margin with spiral ridges Phacus pyrum 2 )5’ Cell margin wIthout ridge., but may have spiral lines ( Lepocinclis) . . . . 216 216 (215’) Posterior end with an abrupt, spine-like tip . . Lepocinclieovtun 216’ Posterior end rounded Lepocindli . texta 217 (2)4’) Green plastids hidden by a red pigment in the cell Euglens eangulnea 217’ No red pigment except for the eye spot . . . . . 218 218 (2)7’) Pla.tid. at least 1/4 the length of the cell . . .219 218’ Plastide discoid or at least shorter than 1/4 the length of the cell.. . . 220 219 (2)8) Plastids two per cell . . Euglena 219’ PlistSdn several per cell, often extending radiately from the center Euglena viridis 220 (2)8’) Posterior end extendLng a. an abrupt colorless spine . . . 221 220’ Posterior end rounded or at least with no colorles, spine. .. . 222 221 (220) Spiral morkinga very prominent and granular Euglens apirogyra 22)’ Spiral markings (airly prominent, not granular Euglena oxyuria . 4Z (220’) Small, length 35-Sip . . . Euglena gracilis /2 1’ Medium to large, length 69,. or more . . 223 123 (222’) Medium in sue, length 69-2001. . . . . 224 / 2 1’ Large in alze, length Z50-Z90i . . . Euglena ehrenbergii 42—10 ------- //4 (223) lastidi, with irregular edge, flagellum 2 times as long as cell. . Euglena polymorpha 224 Plastids with smooth edge, flagellum about 1/2 the length of the cell . . Euglena deses 2Z’ (lH’’ ( Cr 1 1, distinctly bent (arcuate), with a spine or narrowing to a point at both ends 226 zzc’ Cells not arcuate ‘ 230 ?26 ( 22 g .) Vacuole with particles showing Brownian movement at each end of cell Cells not in clu.ters ( Closterium ) . . . .. . . . 227 226’ No terminal vacuole. Cells may be in cluster, or colonies . 228 227 (226) Cell wide, width 30-7O . . . Closterium moniliferum 227’ Cell long and narrow width up to 5 p Clo.terium aciculare 228 (226’) Cell with a narrow abrupt spine at each blunt end . Ophiocytium capitatum 228 No blunt ended cell, with abrupt terminal spines . . . . 229 229 (228’) Sharp pointed ends as separate colorless spines . . 193 229’ Sharp pointed ends as part of the green protoptast 137 230 (225) One long spine at each end of cell . . . . . . . . 231 230’ No long terminal spines . . . . . . . . 232 231 (230) Cell gradually narrowed to the spine . , 13 231’ Cell abruptly narrowed to the spine . . . . . Rhizosolenia gracili . 232 A regular pattern of fine lines or dots in the wall (diatoms) . . 233 232’ No regular pattern of fine lines or dot. in the wall... . . . 276 233 (50. Cell, circular in one (valve) view, short rectangular or square in other (girdle) view 234 232) 233’ Cells not circular in one view 240 234 (233) Valve surface with an Inner and outer (marginal) pattern of striae ( Cyclotella) . . . 235 234’ Valve surface with one continuou, pattern of .trtae ( Stephanodiecue ) . . . 238 235 (234) Cell, small, 4-lOg in diameter. . . . . . . . Cycletella glornerata 235’ Cells medium to large. 10-80 in diameter . , . . . . . . 236 236 (235’) Outer hall of valve with two types of lines, one long, one short . , 237 236’ Outer half of valve with radial lines all alike . Cyclotella meneghtniana 237 (236) Outer valve tone constituting more than lIZ the diameter . Cyclotella bodanica 237’ Outer valve Lone constituting more than 1/2 the diameter. Cyclotella compta 238 (234’) C cl i 4- 2 5p in diameter . 239 238’ Cell 2 S- 65 g in diameter. Stephanodiscu, niagaree 239 (238) Cell with two transverse band,, In girdle view Stephanodiscus binderanus 239’ Cell without two transverse bands, in girdle view . Stephanodiscue hantc,chii 240 (233’) Cell, flat, oval ( Cocconeis ) . . ‘ 241 240’ Cell. neither flat nor oval , , 242 241 (240) Wall markings (.triae) (8.20 in lO , . .. . Cocconeis pediculue 241’ Wail markings (striCcl 23-25 In I0i . . Coccorteis placentula 242 (240’) Cell sigmoid in one view. . ‘ 243 242’ Cell not sigmoid in either round or point ended (valve) or square ended (girdle) surface view 244 243 (242) Cell .igrnoid in valve surface view . . Gyrosigma attenuatum 43’ Cell slgmoid in square ended (girdle) surface view Nitzechia acicularis 244 (242’) Ccli longitudinally uneymniet rical in at leaet one view 245 244’ Cell longitudinally Ayninietrii .il 254 24c (244) Cell well will, both fine and coarse transverse line, (striae and costae) 246 245’ Cell well with fine transverse lines (striac) only 247 42—li ------- /4/, (245) Valve Ia e about as widc at , , , ,d ,Ile as git die face . . . Epithernia turgida ‘4/i ’ Valve face if?. or c ,. as whir at middle as girdle face Rhopalodia 247 245) inc of pores dod raphe located at edge of valve lace . . . . . . 248 /4 ! ISaphe out at ,‘xtren ,e edge of valvi face 250 4411 (247) l(aphe of r .u h valve atjncent to the tame girdle outface . Henteichla amphioxys 24R’ Raph. ( each valve adjacent to different girdle surfaces ( Nitasehia ) . 249 24’) (248) Cell 20-65p long N&tzschia 249’ Cell 70- 18G 0 long Niti.chia linearia 150 (247) Cell longitudinally unsymmetrical in valve view . . 251 250 Cell longitudinally unsymmetrIcal in gIrdle view . . Achnanthes microcephala 251 (250) l5aphc bent toward one side at the middle . .. . Amphora ovalie 251’ Raphe a smooth curve throughout ( Cymbella ) . . . . . . . 252 252 (251’) Cell only slightly unsymmetricaL Cymbella ce,ati (246) Cell distinctly unsymmetrical . 253 253 (252) Striation. distinctly cross lined, width l0-30 s . . Cymbella prostrata 253’ StriatIon. indistinctly cross line . t , width S-lZ a . . Cymbella ventricosa 154 (244’) Longitudinal line (rapho) and prominent marginal markings near both edges of valve 255 ?54 ’ No marginal longitudinal line (raphel nor keel, raphe or pseudoraphe median 257 Z ’ c (254) Margin of girdle face wav . . Cymatopleura solea 255’ Margin of girille face straight ( Surirella ) . . . . . 256 256 (4S5 ( Cell width S.23g . . . . . . . . . . . . Surirella Ovata 256’ Cell width 40-601 . . Surirella splendida 457 (254) Gridle face genrTally In view and with two or more prominent longitudinal linee In valve view, swollen central oval portion bounded by a line ( Tabeliaria ) . 258 257’ Girdle lace with less than two prominent longitudinal lines. In valve view, whole central portion not bounded by a line , . . . .. 259 258 (2”7) Girdle face lee, than /4 as wide aa long . . . . Tabellaria fenestrata 258’ GIrdle face m.,re than 1/2 as wide as long . . Tabellaria flocculosa 259 (257’) Valve (are with both coarse and fine transverse lines Diatoma vulgare 259’ Valve face with transverse lines, if vIsIble alike in thickness . 260 260 1259’) Valvi face naviculoid true raphe present . . 261 2h0’ Valve face linear to linear-lanceolate. true raphe absent . 270 261 (260) Valve lace with wide transverse line. (co,tae( ( Pinnularia ) 262 Valve face with thin transverse lines (striar) , 263 i ( ,4 (21,1) C.ell 5 - 6 ii broad . . . . . . . Pinnularia subcapitata Cell 34-50 i broad . . . Pinnularia nobilis 4 / 121,1’) Transverse lines (strlar) ab ,cnt a ross transverse axis of valve face Staurpneis phoenicenteron I ransverse lines (ilriae) present acros, transverse axis of valve face. 264 264 (2611 fl&phe strictly median ( Navicula ) 265 4 / , 5’ Raph located nlightly I.. one side 252 .‘65 (/64) Fnda of valve face abruptly narrowed to a beak . . Navicula exigun var ‘(.5 Ends of valve face gratially oar rowed . 266 ,/ 6 (/1.5’) Moat of striation, air,, tly tranavi rec . . . Navicula gracili , 266’ Most of striation. ratliai (oblique) . . 267 42—12 ------- 267 (266) Striac distinctly cum 1 ,o,cd of dot (pu ctae) Navicula lonceolata 267 Striac essentially a. continuous lines . . 268 268 (267) Central clear area on valve face rectangular Navlcula graciloicles 268 Cent rd clear area on valve face oval 269 269 (268’) C.ll length 29 -40p. ends slightly capitate. Navicula cryptocephala Ciii length 3O l20li, ends not capitate . Naviculo radlosa 270 (260’) Knob at one end larger than at tb othcr ( Astertonella ) 1 ) 19 270 Terminal knobs if present equal in st e ( Synedra ) 271 271 (270’) Clear space (pseudonodule) in central area Synedra pulchella 271’ No p.eudonoduie in central area. 272 272 (271) Sides parallel In valve view, each end with an enlarged nodule Synedra capitata 27.: SIdes converging to the ends in valvi view 271 273 (272) V4IVC linear ti lanceolate-linear, R-ll striac per IO )J. Synedra ulna 273 Valve narrowly linear-lanceolate, (2-lB otrtae per lOp .274 ?74 Valve 5-61J wide . Synedra acue 274’ Valve 2.4k Wi(le . . . . . . 275 275 (274) Cells up ti , (iS time, as long as wide. entral area absent to small oval Synedra acus var radian . 275 ’ Cells 90-14(1 times as long as wide, central area rectangular Synedra acuavar augustissima 276 (232’) Green to brown pigment in one or more plastid 277 276’ No plastid., blue end green pigments throughout protopleot 284 277 (276) Cello long and narrow or flat 233 277’ Celle rounded 278 278 (277) Straight, flat wall between adjacent cells in colonies . - Phytoconis botryoides 278 Rounded wall between adjacent cells in colonies 279 279 (278’) Cell eIther with 2 opposite wall knob, or colony of 2-4 cells surrounded by distinct mem- brane or bo’h . 164 279’ Cdl without 2 wall knobs, colony not of 2-4 cello surrounded by rltotince membrane 280 280 (279’) Cells essentIally similar in sice within the colony 28) 280’ Cello of very dIfferent sires within the colony Chlorococcum ‘ umicola 2)11 (159’) Cello , ‘,nhe ,Ided in art extensive gelatinous matrix Palm.’lla mucooa 2141’ Cells tth lIttle or no gelatinous matrix around them Chlorella ) 2 ) 12 282 (281’) C. 110 rounded 281 282’ Cells rIlIpsoid 1 to ,,vold Chiorella ellipsoidea 283 (282) Cell 5 -lOp in diameter. pyrenoid indistinct Chlorella vulgaris 483’ Ci II 3 -5 in diameter, pyrcnoid distini t Chlorella pyrenoidosa 2)14 (276’) Cell a spiral rod . . 285 284’ ( elI not a opiral rod . 286 ?85 (25) Thread aeptate (with crosewallo) Arthrospira Jennerl 2115’ Thread non-aeptate (without eroaswallo) Spirulina nordotedtit 286 (172) Cello dividing in a plane at right . .ngles to the long axis , Cocc’ochiorjs stagnina (284’) 286’ (172’) Cello op. ri al or dividing in a plane parallel to the long axis ( Anacystis ) 287 2117 (2111) Cell containing pseudovacuoles Anacystis cyanca 287’ Cell not containing pseudovacuoles 288 42—13 ------- 88 (287’) Cell 2-6k in diameter, heath often colored montana 288 Cr11 6 -SOp in diameter. sheath colorless . .. . . . 289 289 (288’) C cl i 6 -IZ a in diameter. cell. in colonieo Cr. mostly spherical . . . Anacystis thermalis 289’ Cell lZ-5O In diameter, cells in colonies ar, often svgular.. . , . Anacy.ti. dimidiata 42—14 ------- FOREWORD The following work is more easily defined in terms of what it is not, than what it is: it is not a ukeyhl in the usual biological sense of the word, nor is it a glossary of a dictionary. It is rather a device for determining what general kind of organism or group is designated by some unrecognized name, be it common or scientific. if one has access to one or more of the references cited, he can find the same name, and learn much more about It; but not everyone has all of these books, and the Information is often couched in highly technical terms. The nonbiologist would be well advised to read Part I before attemp- ting to use the Finder. The experienced biologist on the other hand may proceed directly to the index and quickly be referred to the larger group to which his unknown organism belongs. No professional systematist will find himself completely at home. In an effort to present a relatively simple concept of relationships couched in standard terms for all groups or organisms, some violence was done to certain highly sophisticated systems of classification. It is hoped, however, that the layman will find accuracy sufficient for his needs, and the specialist will be referred to technical literature where he can satisfy his needs for greater detail. While every effort has been made to ensure accuracy, it is inevitable that errors have crept in. Please call them to our attention. Grateful appreciation is extended to Michael E. Bender and Charles L. Brown, Jr., both former Biologists with the Water Pollution Training A ctlvitles, for their valuable contributions and encouragement. H.W. Jackson Chief Biologist National Training Center 13-I ------- CLASSIFICATION - FINDER for NAMES OF AQUATIC ORGANISMS iii WATER SUPPLIES AND POLLUTED WATERS Part I. The System of Classification INTRODUCFION A Every type of living creature has a favorite place to live. There are few major groups that are either exclusively terres- trial or aquatic. The following remarks will therefore apply in large measure to both, but pri- mary attention will be directed to aquatic types. B One of the first questions usu- ally posed about an organism is: “What is it?”, usually meaning “What is it’s name?”. The nam- ing or classification of bio- logical organisms is a science in itself (taxonomy). Some of the principles involved need to be understood by anyone working with organisms however. 1 Names are the “key number”, “code designation”, or “file references” which we must have to find Information about an unknown organism. 2 Why are they so long and why must they be in Latin and Greek? File references in large systems have to be long in order to designate the many divisions and subdivi- sions. There are over a million and a half items (or species) included in the system of biological nomen- clature (very few libraries have a million books). 3 The system of biological no- menclature is regulated by international congresses. a It is based on a system of groups and super groups, of which the foundation (which actually exists in nature) is the species. Everything else has been devised by man and is sub- ject to change and revision as man’s knowledge and understanding increase. b The basic categories em- ployed are as follows: (1) Similar grouped ______ (genus) (2) Similar genera are grouped into families (3) Similar familes are grouped into orders (4) Similar orders are grouped into classes (5) Similar grouped _____ (phylum) (6) Similar phyla are grouped Into kingdoms 4 The scientific name of an or- ganism Is its genus name plus its species name. This is ana- logous to our system of sur- names (family names) and given names (Christian names). a The generic (genus) name is always capitalized and the species name written with a small letter. They should also be underlined or printed in italics when used in a technical sense. For example: Homo sapiens - modern man Homo neanderthalis - neanderthal man Esox niger — Chain pickerel Esox lucius - northern pike Esox masquinongy - muskellunge b Common names do not exist for most of the smaller and less familiar organisms. For example, if we wish to refer to members of the species are into genera classes are into phyla BI. AQ. 24. 5.71 43—1 ------- RELATIONSHIPS 8LIWE N L1VIN OR( ItN1SM5 LOWER PROTISTA (ORMONERA) ASCOMYcETE8 265 HIGHER PHYCOMYCETES 281 4% BLUE GREEN ALGAE 7 PH O’ T OTR OPIC BACTERIA 252 CREMOTROPIC BACTERIA 252 NOTE: NUMERALS REFER ‘TO PARAGRAPHS IN PARTS 2 AND 3. • B. COOKE AND H. W. JACKSON, AFTER WHITTAI R SI.ECO.pl.2b.4.66 ACITNOMYcETES 253 SPIROCHAETES 255 MYXOBACFERIA 254 PARASIT IC BACTERIA 251 AND VIRUSES 3APROBIC BACTERIA 251 PLANTS 5 ORGANIC MATERIAL PRODUCED, USUALLY BY PHOTOSYNTHESIS ENERGY FLOWS FROM LEFT TO MGWr, GENERAL EVOLUTIONARY SEQUENCE IS UPWARD ANIMALS 81 ORGANIC MATERIAL INGESTED OR CONSUMED DIGESTED INTERNALLY ENERGY STORED ENERGY RELEASED ENERGY RELEASED - FUNGI 250 ORGANIC RATER IAL REDUCED BY EXTEACELLULAR DIGESTION AND IN- TRACELLULAR HE rA— BOLISM TO MINERAL CONDITION FLOWERING PLANTS AND GYM 14OSPERMS 76 CLUB MOSSES, FERNS 76 LIVERWORTS, MOSSES 73 ALGAE 12 BASIDIOMYcE’TEs 266 MAMMALS 243 BIRDS 242 REPTILES 241 AMPHIBIANS 240 FISHES 195 PROCHORDATES 191 STARPI5H GROUPS 185 ARACMEIDS 167 INSECTS 154 CRUSTACEANS 129 SEGMENTED WORMS 121 MOLLUSCS 172 MOSS ANIMALS 120,181 WHEEL ANIMALS 116 ROUNDIOEMS 113 YLATWORMS 108 SPONGES 99 JELLYFISH - CORAL GROUP 103 DIATOMS 38 PIGMENTED FLAGELLATES : ..DEVELOPMENT)f MuLrjcL [ ,.uL4R.oRcOENoC Ift ORG NISMS. - HICJH R PRQTISTA PROTOZOA 82 AMOEBOID PROTOZOA 86 FLAGELLATED PROTOZOA 85 FLAGELLATES 85 CILLIATED PROTOZOA 92 SPOROZOA 98 IA 97 ( si’)!’i; 1 .f-I - DEVELOPMLN1OF -4 UC1E4RMFMI3R N LOWER PRYCOMYCETES 261 43—2 ------- Classification - Finder genus Anabaena (an alga), we muet simply use the generic name, and: Anabaena planctonica, Anabsena constricta, and Anabaena flos—aguae are three distinct species which have different signi— ficances to water treatment plant operations. 5 A complete list of the various categories to which an organism belongs is known as its “classi- fication”. For example, the classification of a type of frog spittle, a common fila— mentous alga, and a crayfish or crawdad are shown side by side below. Their scientific names are Spirogyra crassa and Cambarus sciotensis . a Examples of the classifica- tion of an animal and a plant: b These seven basic levels of organization are often not enough for the complete de- signation of one species among thousands; however, and so additional echelons of tenii’i are provided by grouping the various cate- gories into “Super. ..“ groups and subdividing them Into “sub.. .“ groups as: Superorder, Order, Suborder, etc. Still other category names such ns “tribe”, “di- vision”, “va iety” , “race”, “section”, etc. are used on occasion. c Additional accuracy is gained by citing the name of the authority who first described a species (and the date) m- mediately following the spe- cies name. Authors are also often cited for genera or other groups. d A more complete classification of the above crayfish is as follows: Kingdom Animalia Phylum Arthropoda Class Crustacea Subclass Malacobtraca Order Decapoda Section Nephropsidea Family Astacidae Subfamily Cambarinae Genus Cambarus Species sciotensis Rhoades 1944 e It should be emphasized that since all categories above the species level are essen- tially human concepts, there is often divergence of opin- ion in regard to how certain organisms should be grouped. Changes result as knowledge grows. f The most appropriate or cor- rect name for a given species is also sometimes disputed, and so species names too are changed. The species itself, as an entity in nature, how- ever, is relatively timeless and so does not change to man’s eye. II T GENERAL RELATIONSHIPS OF LIVING ORGANISMS A Living organisms (as contrasted to fossil types) have long been group- ed into two kingdoms: Plant King- doms and Animal Kingdoms. Modern developments however have made this (Frog Spittle) (Crayfish) P]antae Kingdom Anizrialla Chiorophyta Phylum A rthropoda Chlorophyceae C]ass Crustacea Zygnernatales Order Decapoda Zygnemataceae Family Palaemonidae Spirogyra Genus Cambarus crassa Species sciotenais 43—3 ------- Classification - Finder Simple pattern technically unten- able. It has become evident that there are as great and fundamental differences between certain other groups and these (two), as there are between the traditional “plant” and “animal”. The accompanying chart consequently Shows the Fungi as a third kingdom. B The three groups are essentially defined as follows on the basis of their nutritional mechanisms: 1 Plantae: Photosynthetic; synthesizing their own organic substance from inorganic min- erals. EcologicaLly known as PRODUCERS . 2 Animalia: ingest and digest solid particles of organic food material. Ecologically known as CONSUMERS . 3 Fungi: extracellular digestion (enzymes secreted externally). Food material then taken in through cell membrane where it is metabolized and reduced to the mineral condition. Ecolo- gically known as REDU RS . C Each of these groups includes simple, single celled representa- tives, persisting at lower levels on the evolutionary stems of the higher organisms. 1 These groups span the gaps be- tween the higher kingdoms with a multitude of transitional forms. They are collectively called PROTISTA . 2 Within the protista, two prin- ciple sub-groups can be defined on the basis of relative com- plexity of structure: a The bacteria and blue algae, lacking a nuclear membrane, may be considered as the lower protista or MONERA . b The single celled algae and protozoa having a nuclear membrane, are best referred to simply as the higher protlsta. 43—4 ------- Classification - Finder Part II. Biological Classification I NTRODUCT ION What 18 it? Policies Procedures PLAHY KINGDOM “Algae” defined PHYLUM CYANOPHYTA - blue-green algae CLASS Myxophyceae Order Chroococcale Order Hormogonales Suborder Hetercystineae C PHYLUM CHLOROPHYTA - green algae CLASS Chlorophyceae Order Volvocales Order Ulotrichales Order Cbaetophorales Order Chlorococcales Order Siphonales Order Zygnematales Order Tetrasporalee Order Ulvalee Order Schizogoniales Order Oedogonialea Order Cladophorales CLASS Charophyceae Order Charales D PHYLUM CHRYSOPHYTA - yellow- green algae or yellow—brown algae CLASS Xanthophyceae Order Rhizochloridales Order Heterocapsales Order Heterococcales CLASS Chrysopbyceae — yellow-green algae Order Chrysomondalas Order Rhizochrysidales Order Chrysosphaerales Order Chrysocapsales Order Chrysotrichales CLASS Bacillariophyceae - Diatoms Order Pennales - pennate diatoms Order Centrales — centric di atoms E PHYLUM EUGLENOPHYTA - eugle - noid algae F PHYLUM PYRRHOPHY”I’A - yellow brown algae CLASS Desmokontae Order Desmononadales CLASS Dinophyceae — dinoflagellates Order Gymnodiniales Order Peridiniales Order Dinocapsales Order Chioromonadales CLASS Cryptopbyceae G PHYLUM CHLOROMONADOPHYTA H PHYLUM RHODOPHYTA - red algae CLASS Rhodophyceae Order Rangiales Order Nemallonales Order Gelidiales I A B C I I A B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 131. AQ. 24 5.71 43-5 ------- Classification — Finder 57 58 59 60 61 62 63 64 65 86 67 88 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 a r “U 97 98 99 100 101 102 103 104 105 106 107 108 110 111 112 Order Cryptonemiales Order Gigartinales Order Rhodymenjales Order Ceraniales PHYLUM PHAEOPH’YTA - brown algae CLASS Phaeophyceae Order Ectocarpales Order Sphacelarjales Order Tilopterjda].ee Order Chordjales Order smarestjalas Order Punctarialea Order Dictyosiphonales Order Laminarjales Order Fucalee Order Dictyotales J PHYLUM BRYOPHYTA CLASS Hepaticae — 1iver orts CLASS Musci - mosses K VASCULAR PLANT GROUP Emergent vegetation Rooted plants — floating leaves Submerged vegetation Free floating plants III ANIMAL KINGDOM A PHYLUM PROTOZOA - protozoa CLASS Mastigophora Subclass Phytomastigina Subclass zoomastigiria CLASS Sarcodina - amoebojd protozoa Order Amoebina Order Foraminifera Order Radiolarja Order Heliozoa Order Mycetozoa (Myxomycetes) CLASS Ciliophora - ciliates Order Holotricha Order Spirotricha Order Peritricha Order Chonotrjcha CLASS Suctorja — suctorja CLASS Sporozoa B PHYLUM PORIFERA - sponges CLASS Calciepongea CLASS Hyalospongea CLASS Demospongea C PHYLUM COELENTERATA CLASS Hydrozoa - hydroide CLASS Scyphozoa — Jellyfish CLASS Actinozoa (Anthozoa) — corals D PHYLUM CTENOPHORA - comb Jellies H PHYLUM PLATYHELMINTHES - flatworms CLASS Turbellarja — turbella— 109 ians CLASS Trematoda - fluke CLASS Cestoidea — tapeworme F PHYLUM NEMERTEA - proboscis worms G PHYLUM NEMATODA - threadworme,1l3 roundworms 4 ‘ —6 ------- Classification — Finder H PHYLUM NEMATOMORPHA - 114 Horsehair worms I PHYLUM ACANTHOCEPHALA - thorny 115 headed worms J PHYLUM RaFIFERA - rotifer, 118 wheel animalculee K PHYLUM GASTROTRICHA - gastro- 117 triche L PHYLUM M PHYLUM N PHYLUM 0 PHYLUM worms CLASS Polychaeta — polychaet 122 worms CLASS Oligochaeta — briltie 123 worms CLASS CLASS CLASS CLASS worms P PHYLUM ARTHROPODA - jointed 128 legged animals CLASS Crustacea — crustaceans 129 Subclass Branchiopoda 130 Order Anostraca — fairy 131 shrimps Order Notostraca — tadpole 132 shrimps Order Conchostraca - clam 133 shrimps Order Cladocera — water fleasl34 Subclass Ostracoda — seed 135 shrimps, ostracodes Subclass Copepoda - copepods 136 Subclass Branchiura — fish 137 lice Subclass Cirripedia — 138 barnacles Subclass Malacostraca Order Leptostraca Order Hoplocardia (Stomatopoda) — mantis Order Syncarida Order Peracarida Suborder Mysidacea Suborder Cumacea Suborder Tanaidacea Suborder leopoda — sowbugs pilibugs Suborder Amphipoda — scuds 148 Order Eucarida 149 Suborder Euphaueiacea — 150 kr ill Suborder Decapoda — shrimp,l51 lobster, crab Macrurous group (4 tribes) 152 shrimps, prawns, lobsters, c ny fish Brachyurous group 153 (2 trIbes) — crabs and hermit crabs CLASS Insecta — the insects 154 155 KINORHYNCHIA PRIAPULIDA ENDOPROCTA AIINELIDA - segmented 118 119 120 121 139 140 141 shrimps 142 143 144 145 146 ,147 Hirudinea — leeohe Archiannelida Echiuroidea Sipunculoidea - peanut 124 125 126 127 Orders represented by imma- ture stages only. Order Plecoptera — stone— 156 flies Order Ephemeroptera - 157 may files Order Odonata - dragon and 158 damself lies Order Megaloptera — alder- 159 flies, dobsonfiles, fisbflies Order Neuroptera — spongilla—l60 flies 43-7 ------- Classification — Finder Order Trichoptera — caddjs— 161 flies Order Lepidoptera — aquatic 162 caterpillars Order Diptera - two winged 163 flies Orders including aquatic 164 adults Order Coleoptera - beetles 165 Order Hemiptera — true bugs 166 CLASS Arachnoidea — spiders, 167 Scorpions, mites Order Xiphosoura - horse— 168 shoe or king crabs Order Hydracarina — aquatic 189 mites Order Pantopoda (Pycnogonida)_ 170 Pycnogonids Order Tardigrada 171 Q PHYLUM MOLLUSCA 172 CLASS Amphineura - chitons 173 CLASS Gasteropoda — snails 174 Order Prosobranchiata 175 Order Opisthobranchiata ],76 Order Pulmonata — air breath— 177 log snails CLASS Scaphopoda - tusk 178 she l ls CLASS Bivalvia 179 (Pelecypoda) CLASS Cephalopoda - squid, 180 Octipua, nautilus H PHYLUM SRYOZOA (Ectoprocta) — 181 Moss animals S PHYLUM BRACHIOPODA - lamp 182 she 1 is T PHYLUM CHABTOGNATHA - arrow 183 worms U PHYLUM PHORONIDEA - tufted worms V PHYLUM ECHINODER ATA - echinoderms CLASS Asteroidea — starfishes CLASS Ophiuroidea — brittle stars CLASS Echinoidea - sea urchins CLASS Holothuroidea — sea cucumbers CLASS Crinoidea — sea lilies W PHYLUM CHORDATA - chordates Subphylum Henichordata — Acorn worms Subphylum Urochordata — tunicates, sea squirts Subphylum Cephalochordata - lancelets Subphylum Vertebrata ( Craniata) — vertebrates CLASS Agnatha — jawlesa 196 fishes Order Myxinifornes — 197 hagf ishes Order Petromyzontjformes — 198 lampreys CLASS Chrondrichthys — 199 cartilage fishes Order Squaliformes — sharks 200 Order Rajiformes — skates, 201 rays Order Chimaerifo es — 202 chimaeras CLASS Osteichthys (Pisces) — 203 bony fishes Order Acipenseriformes — 204 Sturgeons Order Polyodontidae — 205 paddle fishes 184 185 186 187 188 189 190 191 192 193 194 195 43—8 ------- Classification — Finder Order Semionoteformes - ears 206 Order Amliformes - bowfisa 207 Order Clupeiformee — soft 208 rayed fishes Family Clupeidae — herrings 209 Family Salmonidae — trouts, 210 salmon Family Esocidae — pikes, 211 pickerels Order MyctopbifOrmes — 212 lizard fishes Order Cypriniformea — 213 Family Cyprinidae - minnowS, 214 carps Family Catoetomidae - euckers2l5 Family Ictaluridae — fresh— 216 water catfishes Order Anguiliiformes — eel— 217 like fishes Order Notacanthiformeb - 218 spiny eels Order Beloniformes — needle— 219 fishes, flying fishes Order Cyprinodontiformes - 220 killifishes, livebearers Order Gadiformes — coda and 221 hake 8 Order Gasterosteiformea — 222 atickelbacks Order Lampridiformea — Opahe, 223 ribbon fishes Order Beryciformes — beard— 224 fishes Order Percopsiformes — trout 225 and pirate perches Order Zeiformes - dory 228 Order Perciformes — spiny— 227 rayed fishes Family Serranidae — sea 228 basses Family Centrarchidae - 229 sunfishes, freshwater basses Family Percidae — perch 230 Family Sciaenidae — drum 231 Family Cottidse — sculpins 232 Family Magilidae — mullets 233 Order Pleuronectiformes — 234 flounders Order Echeneiformes — remoras235 Order Gobiesociformes — 236 clingflshes Order Tetraodontiform — 237 spikefishes Order Batrachoidiformes — 238 toadfishes Order Lophiiformes — 239 goose fishes LA8S Amphibia — frogs, toade,240 salamandera CLASS R.eptilia — turtles, 241 snakes, lizards CLASS Ayes - birds 242 CLASS Mammalia — whales, 243 seals, walrusses IT FUNGUS KINGDOM 250 A Bacteria 251 Eubacteria 252 Actinomycetes 253 Myxobacteria 254 Spirochaetes 255 Other bacterial types 256 B FUNGI 260 “Phycomycete” group 261 43—9 ------- ClasRification - Finder CLASS Chytridionycetes 262 CLASS Oornycetes 263 CLASS Zygomyce tee 264 CLASS Ascoinycetes 265 CLASS Basidlomycetes 266 CLASS Fungi Imperfectj 267 43—10 -------