AFCEC   TR 77 7 BIOASSAY OF AIR FORCE FC 206 FIRE FIGHTING FOAM Eric H. Wang Civil Engineering Research Facility University of New Mexico Albuquerque, New Mexico 87131 April 1977 Final Report: November 1974   June 1976 Approved for public release; distribution unlimited.

Prepared for AIR FORCE CIVIL ENGINEERING CENTER Air Force Systems Command Tyndall Air Force Base, Florida 32401 ECJ~ iITY CL.~. S~ FI'.; TIOp. j O~ r~ i~ ° ~ .~ W),, f)~. r; r,. r~!'

REPORT DOC'J~ E~ TATIONPAGE ~ LR~ PORT ,. L?. EER I2GC'. T ACc~~ N NO. 1 3. PECI~'~

AFCEC TR   77 7 ~ 4. TITLE ( o~ dSuStiile)

~ BIOASSAYOF AIR FORCE FC 205 FIRE FIGHTING FOAM I i~. TVVE .),~~` O~ T 6 PERIOD : ovEP.~ D Final Technical Report ~. P~ 3~\~ G cr~. REPORT N~ JM3ER .

CERF EE 7 7. AUTHOR( s) 8. CC,~~ 1~. CT O'~ GRA~ iT Y~ JMbER(~)

Harvey Green F29601   76   C 0015 9. PERFORMING ORGANIZATION NAME AND ADDRESS ~`~._ EJ. iE~! T. ~?. 3J~ Z7. TASK Eric H. Wang Civil Engineering Research Facility, ~ C~( UfliT NLMDERS University of New Mexico, Box 25, University T. D. 4.01 Station, Albuquerque, NM 87131 1Z. REPORT tisIE 11. CONTROLLING OFFICE NAME AND ADDRESS Air Force Civil Engineering Center Air Force Systems Command Tyndall Air Force Base, FL 32401 ~ i r. i] 1977 13. NJ~) 3EPO~ PAGES 62   14. MONITORING AGENCY NAME b ADDHESS( If difieren: from CottrolUr~ Office) IS. SECURITY CLASS. ( ot this r~ port~)

Unclassified iSa. DECLASSFICATION/ OOWN3RAOIP4D . SCHEDULE ~_____________________________________________________________ 16. O~ STRIDUTION STATEMENT ( of thfs Report)

Approved for public release; distribution unlimited.

17. D! STRIBUTION STATEMENT ( of the abstract entered in Block 20. If difleren! from Report)

18. SUPPLEMENTARY NOTES Available in DDC 19. KEY WORD$ ( Continue on reverse side if necessary and identify by block number)

Bioassay Eutrophication Ecosystem Acute and Chronic Testing Toxic Lethality Probit Analysis Conspecific 20. ABSTRACT ( Continue on ro" er3e side Ii necessary end Identify by block number)

Tests were conducted to assess the effects of an Air Force fire fighting foam ( FC 206) on an aquatic system. A model ecosystem was constructed to maintain a more natural setting for total ecosystem monitoring. A holding facility was also constructed and used to maintain a variety of test organisms for bioassay experiments. Several flowthrough bioassay systems were used for both invertebrate and vertebrate bioassay testing; data were collected on five different vertebrate species and three invertebrate species.

r~ r~ FORM 1473 EDITION OF 1 NOV 65 IS OBSOLETE IJU 1 JAN 73 UNCLASSIFI ED S~ CURITV CL~ S; IFICATI~ OF THIS PAGE r~ thenDe~ aEnter PREFACE This report documents work performed during the period November 1974 through June 1976 by the University of New Mexico under contract F 2960l 76   C 00l5 with the Air Force Civil Engineering~ Center, Air Force Systems Command, Tyndall Air Force Base, Florida 32401. This research was directed by Dr. David Nyquist as Principal Investigator from November 1974 until February 15, 1976 and by Mr. Harvey Green from February 16, 1976 to June 1976. Dr. Kathryn G. Vogel was responsible for the research contained in the appendix. This program was managed at Kirtland Air Force Base, New Mexico by Maj. Michael G. MacNaughton, Commander AFCEC/ OL   AA and at the Civil Engineering Center by Sgt. Claude Scott.

This report has been reviewed by the Information Office ( UI) and is releasable to the National Technical Information Service ( NTIS). At NTIS it will be available to the general public, including foreign nationals.

This technical report has been reviewed and is approved for publication.

1 ( The reverse of this page is blank.) TABLE OF CONTENTS Section Page I INTRODUCTION 1 II EXPERIMENTAL FACILITIES 3 1. Laboratory 3 2. Hatchery 3 3. Ecosystem Model 6 III TEST PROCEDURES 9 1. Ecosystem Testing 9 2. Bioassay Experiments 9 a. Introduction b. Statistical Analysis Techniques 10 c. Diluter 13 d. Chemical Analysis 14 e. Selection of Test Organisms 23 IV EXPERIt~ 1ENTALRESULTS 24 1. Ecosystem 24 2. Bioassay Tests 25 V CONCLUSIONS AND RECOMMENDATIONS 35 REFERENCES 37 APPENDIX A: ADEOSINE TRIPHOSPHATE ( ATP) ANALYSIS TO ASSESS TOXICITY OF AQUATIC POLLUTANTS 39 111 LIST OF ILLUSTRATIONS 2 Outdoor Hatchery 3 Ecosystem Model 4 Chemical Analysis Rainbow Trout 5 Chemical Analysis Fathead Minnow 6 Chemical Analysis 7 Chemical Analysis 8 Chemical Analysis 9 Tolerance of Test for MBAS of Test Water for for MBAS of Test Water for for MBAS of Test Water for for MBAS of Test Water for for MBAS of Test Water for Species to FC   2O6 Toxicant Catfish Bluegill Flagfish Figure 1 Environmental Testing Laboratory Page 4 5 8 16 18 20 21 22 28 31 32 34 Page 10 Egg Production and Hatchability of P~ imaphaiespromelus and Jordanella floridae 11 Age Versus Lethality to FC 206 for Vertebrate Species 12 Growth Length and Weight Data for Saimo gairdneria and Jordanella floridae LIST OF TABLES Table 1 Chemical Analysis of Dilution Water 15 2 LC 50 Data for Test Species 26 iv SECTION I I NTRODUCT ION 1. BACKGROUND As part of its overall environmental quality program, the Air Force is required to assess the impact of its operations on existing ecosystems. Because of the unique type and locations of many Air Force facilities, the wastes generated by the many varied operations are not common to the civilian sector.

Presently, the acute ( short   term) bioassay is being effectively used to assess the effects of toxic materials on aquatic systems. However, because of the types of wastes discharged by the Air Force, a chronic ( long   term) flowthrough bioassay was deemed necessary.

2. OBJECTIVES In order to develop sufficient data on the toxicity of Air Force wastes to aquatic organisms, the University of New Mexico Civil Engineering Research Facility ( CERF) was contracted to construct a facility in which to conduct bioassay studies of Air Force wastes and to develop new techniques for assessing their influence on natural aquatic systems. The specific objectives of this research were as follows:

1. To establish a facility to accomplish bioassay and total ecosystem monitoring.

2. To examine the effects of unique Air Force wastes on aquatic systems.

3. To establish an ATP technique to assess the effects of wastes on aquatic systems.

3. SCOPE Toxicity studies were undertaken on a new fluorocarbon fire fighting foam ( FC   206) which is being used by the Air Force. Although not considered a hazard 1 during emergency fire fighting, FC 206 could have adverse environmental effects when used in large volume at a training facility. Continual exposure in a limited area makes specific knowledge of acceptable levels for disposal into natural waters a necessity. Thus, to evaluate the effects that an Air Force waste could have on a total ecosystem, a test model stream was constructed.

2 SECTION II EXPERIMENTAL FACILITIES 1. LABORATORY The main laboratory was housed in a 3000 ft2, concrete block building ( Figure 1). The laboratory was divided by task so that the test animals would be disturbed as little as possible. The test animals were subjected to a 16

hour photoperiod, which was maintained by a timer that was not equipped to simulate dawn/ dusk conditions; however, to prevent spontaneous shock, a small light was left on. Room temperature was maintained at 17 ° Cthroughout the year. One room in the laboratory was maintained for the culturing of tropical fish ( flagfish). This room also housed the refrigerating unit for the culturing of algae.

Acute testing was performed in 20.82 liter test chambers. The cold water species ( rainbow trout, etc.) were placed in a cold   water bath at 15 ° C. The chronic flowthrough experiments were conducted in five banks of six 56.78 liter chambers, each divided in half by a double strength glass panel. The temperature of the water in these chambers was maintained at 15 ° Cby a gas fired air conditioner adapted with a heat exchanger. The water fed to the Mount and Brungs diluters for the warm   water organisms was heated in the feeder tank,

which elevated the temperature to 20 ° C. The tropical fish chambers were further heated by individual 100   watt heaters on each side of the test chambers.

With these individual heaters the temperature in the test chambers could be maintained at any level between 200 and 27 ° C.

2. HATCHERY The outdoor facility was maintained as a typical hatchery ( Figure 2). Only one species of the test vertebrates ( Jordanella floridac) was actually hatched at this facility; the other test animals came from a variety of sources. The facility has six 18,927   liter rearing tanks for vertebrates, six refrigerated ( minnow cool) 473   liter tanks, twelve 1140 liter holding tanks for algae and 3 Storage Wet Chemistry Figure 1. EnvirOnmental Testing LaboratOrY Proportional DiluterS for Bioassay and ~ jomonitOriflg .. ( 71 Solar / Heater~

/ Ecosystem Biomoni tori ng Ii Biomonitoring Test Channel Model Stream Rearing and Holding  Tanks  Laboratory ( See figure 1 for details.)

Figure 2. Outdoor Hatchery invertebrates, and ten 1136 liter circular holding tanks for maintaining invertebrate and vertebrate populations; six 3028 liter holding tanks were available for ecosystem testing. In addition, a 104 meter long model stream with six 7.1   meter   long substreams and holding reservoirs was constructed. Six solar collectors were employed to heat water to facilitate the maintenance of the warm water test organisms during the colder months of the year.

3. ECOSYSTEM MODEL During the past several decades much concern has developed over the continual deterioration of our natural waters. Thus, a total biological ecosystem polluted with FC 206 was examined. The term ecosystem in its broadest sense means the economics of a system. In other words, it encompasses what exactly is occurring within a system at a given time under known conditions. The idea of modeling a total system ( foodweb) is not new, but relatively little work has been accomplished in this field. Since CERF does not have a natural body of water to work with, a model ecosystem was constructed.

Biologically, the significance of modeling a natural environment enhances the data that can be collected in more conventional laboratory testing ( static and flowthrough bioassays). The effects that a foreign substance can have on a total system can be greatly varied by the natural system itself. What occurs in the form of recovery is also helpful in the total assessment of the effect of a toxic substance. If the bottom of the foodweb is destroyed and does not recover quickly, the entire system might be considered dead. The speed of recolonization after the advent of a polluted condition is also important.

Another consideration in assessing effects after introduction of a foreign substance is the nature of the recovery; i. e., does the normal community return or does a new system develop? Combining the data collected from laboratory studies ~ iththat from simulated natural settings can greatly enhance knowledge of the true effects that a particular toxicant will have in a natural setting ( Reference 1).

Reference 1. Modeling the Eutrophication Process, Ann Arbor Science Publishers, Inc., Ann Arbor, Michigan, 1975.

6 To establish the effects that a foreign substance can have on an ecosystem,

total ecosystem monitoring is the best approach. The general objective behind the ecosystem modeling experiment was to evaluate the effects during and after the introduction of a foreign substance to a littoral body of water, since these effects can be reflected in the initial damage done to the total ecosystem and the speed with which the total ecosystem returns to normal.

The main stream of the ecosystem model ( Figure 3) was constructed of 76.2   cm   diameter aluminum culvert cut in half. The unit was 104 meters long with a vertical drop at the headwaters of 1.22 meters to 0.61 meter at the catch basin.

The main channel substrate, composed of clay to stones as large as 5 cm in diameter was varied to best approximate a natural stream bed. The flow rate was established at 127 liters/ mm. The depth of water within the stream varied from 2.5 to 30.5 cm. The depth in each section was maintained by a series of wood weirs. The entire system was fed by domestic water which had been passed through a commercial dechlorinator.

The side channels were 6.1   meter long, 30.5   cm   diameter aluminum culverts cut in half. There was a 5.1   cm drop from the headwaters to the catch reservoir The catch basins at the end of the side channels were 303 liter,

galvanized   steel tanks coated with polypoxy epoxy marine paint to stop leaching from the galvanized metal. 1 The substrate in the side channels was sand and small stones ( 5 cm or less).

The main channel, which was used as a feeder for the side channels during testing and as a source for recolonization after testing, was established to act as a positive control; the actual testing was accomplished in the side channels.

The water entering the side channels was filtered during testing to prevent recolonization or contamination by the main stream. Monitoring of the population numbers within the system without erroneous introductions from the main channel was accomplished in this way. Although there is no effective way to prevent Footnote 1 Routine analysis of the water from this reservoir did not show any changes in water quality after the water entered the system.

7 recolonization from airborne spores, random seeding occurred in both the polluted section and the control section. The apparatus allowed an escape mechanism for the motile organisms. The toxicant was fed into one channel while the other channel was fed only fresh water for a control. Sampling stations were placed along the side channels to monitor the pollutant concentrations. Note: Circled numbers indicate sampling stations.

Figure 3. Ecosystem Model 8 SECTION III TEST PROCEDURES 1. ECOSYSTEM TESTING After preliminary testing of the FC 205 fire fighting foam in the laboratory experiments were conducted on the total ecosystem. The side channel was established 8 weeks prior to testing, and the bluegills which were to be used in these tests were allowed a 30 day period to acclimate themselves. The toxicant was gravity fed into a known volume of water. The concentration at the headwaters was 9320 mg/. e ( volume/ volume) of concentrated FC   2O6. The opposite side channel acted as a control with an equal volume of water flow. It was known in advance that a concentration in excess of 4000 mg/ Z would destroy the fish. The high concentration was established to judge the effects on the standing algae community and on the motile organisms within the test area. The resident fish population was dispersed uniformly throughout the entire system before testing'.

To maintain concentration levels, a complete chemical backup was adminis   tered. The total oxygen content and 1~ J~ S ~ eve~ swere recorded by station.

Sanipies were taken from each of the 12 stations, 3 times each day. Because of the expense and time to process samples, only a random spot check analysis was performed to verify that the ` toxicant levels during the test remained constant.

2. BIOASSAY EXPERIMENTS a. Introduction Bioassay involves the monitoring of effects on living organisms produced by the introduction of a foreign substance to the environment. The techniques for this monitoring are as varied as the effects produced. In this study,

the most commonly accepted procedures for biological monitoring were used acute and chronic test methods.

The acute test is normally done as a screening test for 96 hours or less. Death of the organisms serves as the means of evaluation and provides 9 answers to problems quickly and economically. The chronic test, on the other hand, is normally run over a longer duration of time ( 24 to 2160 hour) and provides monitoring of a wider variety of parameters such as reproduction,

morphological and behavioral changes, etc. This test also provides a closer approximation of what is actually the true effect of a toxicant within an existing aquatic system.

Generally the toxicant level which produces death in 50 percent of the test organisms ( LC 50) is used as a measure of toxicity. A factor of 10 is used in some cases to determine the level of the material which is acceptable for release to the environment; however, this must be determined from other considerations such as the potential harm and the additional cost of reducing the concentration of the material in the waste.

b. Statistical Analysis Techniques Interpretation of the LC 50 is commonly accomplished by the graphic technique described in ~ Reference 2. Saiiilogarithmic paper with the percentage   of survival plotted on the X   axis and the exposure concentration plotted on the Y   axis ° is used. The upper limit is that point at which more than 50 percent and less than 90 percent of the organisms die; the lower limit is that point at which more than 15 percent and less than 40 percent of the organisms die.

The LC 50 is that point at which the line connecting these two points ( high and low limits) intersects the 50 percent line. The LC 50 concentration can then be extrapolated directly from the curve. This graphic expression is attractive since it is quick, economical, and reliable.

Probit analysis, which may also be used to determine the LC 50,

provides a somewhat closer approximation. In a biological assay the potency of a stimulus     physical, chemical, biological, or physiological     is tested by means of the reaction which it produces in living matter. In so doing, a quantal ( all or nothing) response is obtained. In the flowthrough bioassay Reference 2. Standard Methods for the Evaluation of Water and Wastewater, 13th Edition, American Public Health Association, Washington, D. C., 1973.

10 experimental technique, a group of subjects on which a stimulus is applied to the environment, resulting in a response. A response is a measure of the magnitude of the stimulus and, more importantly, the intensity required to produce equal responses.

Probit analysis is one accepted method for handling quantile response data in lieu of the graphic technique described above. Graphic methods are likely to be misleading if the curvature of the dosage response curve appreciably exceeds the range of dosages tested, especially if the distribution of the dosages tested is markedly unsymmetrical about the log of the LC 50. In probit analyses the percent kill is transformed to probit units. The history of this transformation and the statistical technique is outlined in Reference 3.~ Bliss first proposed the name prabit for his modification of Baddum's normal equivalent deviant. The probit of the proportion P is defined as the abscissa which correspond to a probability P in a normal distribution with a mean of 5 and a variance of 1; in mathematical terms, the probit of P is Y where Y 5e 1/ 2 u2du The transformation may be considered a stretching of the percent kill scale to give a probit scale, during which the sigmoid curve becomes straightened The sigmoid curve is the result of plotting percent kill versus concentration thus, the probit straightens, this line. Probits for specific values of P have been prepared by Bliss ( 1935) and reproduced by Fisher and Yates ( 1948)

in their statistical tables for biological, agricultural, and medical research.

After experimental data on the relationship between dose and mortality are obtained, either a graphic or a numerical technique can be used to estimate the probit line. Both these techniques are dependent on the probit transformation The graphic technique is much more rapid and is sufficient for many Reference 3. Bliss, C. I., The Method of Probits  A Correction Science, 79, 409 10.

11 purposes, such as preliminary testing for LC 50 concentration and less   exacting needs. However, for more complex problems, for which an accurate assessment of the precision of estimates is required, the more detailed numerical technique is necessary. In drawing a probit line and judging i~ sagreement with the data,

only the vertical deviations of the points must be considered. The line must be so placed that the difference between the probit values which are plotted and the probits given by the line at the same dosages is as small as possible.

A linear least   squares fit is made to the probit data; extreme probits outside the range of 2.5 to 7.5 ( 1 to 99 percent) carry little weight and should be disregarded. The fitted probit line is therefore a weighted regression line of probit units versus the log of the concentration. Here weighting can be thought of as being related to the reciprocal of the theoretical variance, and the major assumption underlying the applied probit analysis is that the treatments are independent of one another.

In the . probit analysis fiducial limits were set at the 95   percent confidence level. This level was thought to be descriptive of the biological analysis, whereas a fiducial level lower than 85 percent confidence was only descriptive of the experimental design. Therefore, in the analyses only the 85 to 95 percent confidence level is indicative of the experiment and the fiducials shown are a measure of the exactness of the experiment.

Fiducials were calculated when no evidence of significant heterogeneity of the points about the regression line was found. Then, the 95   percent confidence level was computed as a 5 percent probability that the deviation from the true log of LC 50 is the estimate that will be greater than and lower than the true value. These limits will correspond to a concentration and would be equidistant from the estimated LC 50. But, since the limits derived are plotted on a logarithmic scale, they will not be symmetrical when placed on a concentration scale. The difference, however, is trivial for close   fitting data, but with relatively larger standard errors or for more extreme limits, the difference may be considerably greater ( Reference 4).

Reference 4. Finney, 0. J., Probit Analysis, Cambridge University Press, 1962.

12 C. Diluter A modified Mount and Brungs proportional diluter was used to dispense the toxicant. These diluters are composed of glass with tygon tubing for the fittings. The walls of the diluter are made of double strength glass sealed with Dow Corning silicone sealant. The use of glass, limited tygon connections,

and silicone sealant affords a unit of sturdy construction and a unit that is easily disassembled, relatively free from external contamination, and easily cleaned between and during tests. A complete description of the diluter is given by Mount and Brungs in Reference 5.

The modification to the diluter consisted of replacing the flow splitter with a U   tube within the box and a Y connector outside the box. The Yconnector acted as a flow splitter and the U   tube provided a more uniform mix.

The standard flow splitter was found ineffective because heavy bacterial growth,

stimulated by the toxicant, clogged the system.

Proportional diluters have an advantage over serial diluters in that one concentration is diluted to achieve all concentrations; the diluter provides ` five different concentrations PIUS a positive control. Also, this diluter system can provide concominant replicates. The diluters used at this facility were gravity fed ( H20 and toxicant) with domestic water that had been passed through a commercial dechlorinator. This system was employed to preclude experimental losses. The toxicant was also fed into the system on a gravity basis ( dipping bird). The system cycles were set at 3.5 minutes to maintain the dissolved   oxygen and temperature levels.

The calibration of the diluter system was accomplished by using green food coloring. This dye was diluted ( one to one) with tap water. The 50 percent mixture was allowed to flow into the system in the same manner as the toxicant.

The diluter was allowed to cycle for several minutes and then an aliquot was taken from each of the replicate test chambers and analyzed for absorbance on Reference 5. Mount, D. I., and Brungs, W. A., " A Simplified Dosing Apparatus for Fish Toxicology Studies," Water Research, Vol. 1, 1967, p. 21.

13 a l3auch and Lomb Model 20 Spectrophotometer at 510 nm. A straight line color plot was constructed to establish concentration in the test chambers ( Reference 6).

d. Chemical Analysis To properly evaluat° any toxicant, monitoring of the system must be continuous. Routinely in this experimentation, the quality of the water was tested. Throughout the experiments, daily checks were made of the pH,

conductivity temperature, and dissolved oxygen. The temperature and dissolved oxygen were different for each test, but they were never allowed to reach stress levels for any test species. These tests were performed to assure that the quality of the test water did not change. Additionally, after the intror~

Ction of FC 206, the chemical oxygen demand was measured routinely to insure that problems did not arise within the system.

In addition to the routir~ etests, water quality tests were performed to evaluate the quality of the water for aquatic testing. These tests were performed . by the Environmental Chemistry Research Division, AFCEC, located at Kirtland Air Force Base, New Mexico and the Environmental Health Laboratory located at Kelly Air Force Base, Texas. The results of these analyses are given in Table 1; they substantiate that the water quality was in line with the acceptable ranges outlined for bioassay testing.

To evaluate the functioning of the diluters, a means to measure the toxicant concentration had to be determined. It was found that for ease and economy of analysis, the Methylene Blue Active Substance ( MBAS) Method was the best approach ( Reference 2). Further studies showed that the t4BAS was a conservative component of the FC 206 and could be used as a reliable indirect measure of the toxicant concentrations. From the analysis, it was found that the toxicant contained 15,726 mg/!. MBAS. Although t. he MBAS technique gave numbers on the low side of those calculated from the diluters, it did provide a means to monitor the performance of the diluters. Figures 4 through 8 give the results of the MBAS analyses for all the FC 206 toxicant studies.

Reference 6. Water Alnosphere Analysis, 1974 Annual Book of ASTM Standards, Part 23, American Society for Testing and Materials, 1974.

14 Calcium, mgi!.

Magnesium, mg/!.

Sodium, mgi!.

Potassium, mgi!.

Bicarbonate, mg/!.

Carbonate, mg/!.

Sulfate, mg/ L Chloride, mg/!.

Fluoride, mgi!.

Nitrate and Nitrite as Nitrogen, mg/!.

Dissolved Solids at 180 ° C, mg/!.

Calculated ( Sum), mg/!.

Hardness Total, mgi!.

Noncarbonate, . mg/!.

Alkalinity as CaCO3, mgi!.

Carbon Dioxide, mg/!.

Sodium Adsorption Ratio Langlier Ratio at 25 ° C Conductivity, pmhos at 25 ° C Color, Pt Co Units pH Manganese, mg/!.

Iron, mg/!.

Silica, mg/!.

Turbidity, JTU Units Table 1. CHEMICAL ANALYSIS OF DILUTION t~ JATER Characteristic Mean Characteristic Mean ( 31 52 8.3 27 2.9 124 0 51 13.8 0.54 1.64 285 278 165 23 143 18 0.9  0.2 430 0.875 7.48 0.0025 0.14 30 2.0 Chemical Oxygen Demand, mgi!.

Dissolved Solids, mgi!.

Oils and Greases ( Infrared), mg/!.

Surfactants ( LAS), mg/. e.

Phenols, mg/!.

Phosphates, mgi!.

Cadmium, mgi!.

Chromium ( Hexavalent), mg/!.

Chromium ( Total), mg/!.

Copper, mg/!.

Cyanides, mg/!.

Iron, mgi!.

Lead, mg/!.

Silver, mgi!.

Zinc, mg/ c.

Mercury, mg/!.

Total Organic Carbon, mg/!.

Nitrite Nitrogen, mg/!.

Ammonia Nitrogen, mg/!.

Barium, mg/!.

Arsenic, mg/!.

Boron, mgi!.

Nickel , mg/!.

Selenium, mg/!. 5.0 273.0 0.3 1.1 0. cJOl 0.3 0.01 0.01 0.05 0.02 0.01 0.1 0.05 0.01 0.05 0. 001 1.0 0.02 0.2 1.0 0.01 0.5 0.05 0.01 3.0 2.5

2.(

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ci) U 0 C, 11 hNnNnñ lnflnHN : 9k1HflrR9H   Fi TF~ fl  flñ lHhnhhhnflhHHhlnh  Figure 4. Chemical Analysis for MBAS of Test Water for Rainbow Trout 3. C Test Chamber 4 Test Chamber 6 01 E `. 0 c.~ J C i U

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2   1   n Test Chamber 2 H Time Figure 8. Chemical Analysis for MBAS of Test Water for Flagfish e. Selection of Test Organisms Because it was desired to test both warm and cold water organisms,

the experimental animals were obtained from ~ variety of sources. The parent stock of Jordnella fioridae was obtained from the United States Environmental Protection Agency, Duluth, Minnesota. This fish, which is indigenous to Florida was chosen for its ease of breeding and handling under laboratory conditions The fish is unique in that three generations can be observed in one calendar year. This affords the opportunity to observe any morphological changes that might occur in successive generations following exposure to a toxicant.

Fish showing a southern midwest distribution were represented by Ictaluras punctatus ( channel catfish) and Pimaphales prorne7~ us ( fathead minnow).

These test animals originated at the United States National Fish Hatchery located at Uvalde, Texas. Lepornis macrochirus ( bluegill sunfish) came from the National Fish Hatchery located at Dexter, New Mexico. The only cold   water vertebrate species tested was Sairao gairdneria ( rainbow trout). This test group came from the United States National Fish Hatchery located at Mescalero, New Mexico; the eggs for these animals were supplied to the hatchery from a commercial hatchery in Northern California.

The test animals were examined to insure that no disease was introduced into the facility. These animals were given a 2   to 4   week period to acclimate themselves to the new water conditions before testing occurred.

The invertebrate animals were selected on the same basis as the vertebrates They were as follows: Chironomus, midge ( larval stage); Daphnia magna, water flea ( first instar and adult); and Gastropoda, snail. These invertebrates were reared at the CERF facility.

23 SECTION IV EXPERIMENTAL RESULTS 1. ECOSYSTEM Within 1 hour after ecosystem testing began, the population of fish showed signs of behavioral searching patterns. The animals in the test channel migrated into the catch basin; i. e., the population detected a foreign substance and was reacting by looking for escape routes from the affected area. As is characteristic of many toxic substances, this toxicant seemed to disorient some of the test animals. The animals that chose the wrong route of escape fell victim to the toxicant in less than 6 hours; the remaining animals were able to maintain themselves. After 12 hours of exposure, the catch basin contained 4660 mg/!. of FC 206; from previous tests this amount was shown to be a lethal dosage for this test animal. Because many fish frequented this area after the first day of testing, it was felt that some damage had already been done to the biological mechanism for escape that caused these animals to remain in the toxic reservoir. However, because they were able to remove themselves from the toxicant for part of the time, these animals survived. This indicates that in nature if there are means of escape ( avoidance of the toxicant), total elimination of the species will probably not occur. Throughout the 21   day test, 56 percent of the total fish population was destroyed. Some dead animals were found in the control channel at station 11; many of these animals probably died because they remained too long in the polluted reservoir. As stated earlier,

this toxicant seems to act much like a sedative and this could account for some of the deaths in the reservoir. Residual effects were shown in this experiment in that after stopping the administration of the toxicant on the eighth day,

16 percent of the population still expired.

Probably more apparent in this study were the effects of the toxicant on the invertebrate population of the polluted channel. Within 48 hours the polluted channel of the stream was devoid of life, except for an occassional snail.

The snails that remained alive did so by actually crawling up out of the water from time to time. Within 96 hours after the test began, the snail population had moved to the control channel.

24. Because of the nonavailability oF baseline workups of species diversity and population levels, it was impossible to speculate on recovery rates. It can be noted that within 72 hours the toxic test channel was devoid of life while the control channel was teaming with life. In general , the control side was a true representation of a typical seasonal community with no apparent ill effects due to testing.

As the toxic channel began to recover, an intense bacterial bloom occurred this indicated that residual eutrophic effects were occurring. Within 96 hours after stopping the administration of the toxicant, chemical analysis showed no MBAS present. The bacterial bloom occurred 6 weeks after testing was stopped. The control side experienced an algae bloom devoid of bacteria.

The recovery of the polluted side was hampered by the presence of the bacteria and seasonal cold weather. However, the polluted side compl'etely recovered in the spring with no ill effects. The bacterial community was replaced by a normal seasonal algal community.

2. BIOASSAY TESTS To best evaluate the FC   206 toxicant, its effects need to be looked at,

not by genus or family, but by individual groups and their habitat orientation.

There appeared to be a direct correlation between the toxicity expressed by warm water, low oxygen   level habitat organisms and cold water, and high

oxygendemanding organisms. The rainbow trout was the most sensitive of the fish tested ( Table 2). The sensitivity of trout to foreign substances has been shown in a variety of other studies. The more tolerant organisms, as stated before, appear to be those that do not need high levels of oxygen to survive.

Figure 9 shows graphically the tolerance to FC 2O6 expressed by all the organisms tested.

To account for the toxicity, the mode of action produced by the toxicant should be known. FC   2O6 appears to affect the transfer of oxygen across the gill membranes. Before death the vertebrate organisms go through the classical stages exhibited when low dissolved oxygen levels are present; they become alarmed easily. Sudden changes in light intensity caused the fish to become excited to the point of bringing on premature death. The racing motion 25 Table 2. LC 5O DATA FOR TEST SPECIES Species Age Test Period LC 5O ( 95% Confidence Level)

Fathead Minnow Channel Catfish Flagfish Bluegill Rainbow Trout Young ( 1 month) 96 hr 24 hr Young ( 3 months) 72 hr Adult 96 hr 36 hr Young ( 6 weeks) 96 hr Young ( 2 weeks) 96 hr Eggs 96 hr Adult 96 hr Eggs 144 hr 144 hr Adult ( 6 months) 72 hr 2527.0 2527.5 2550.2 3378.0 2113.0 1198.0 2113.0 97.0 1635.0 20.0 18.0 1901.6 33.59 77.60 79.82 43.33 37.61 42.43 53.07 37.61 43.77 63.73 51.90 58.97 959.10 1141.0 1666.2 1702.5 1161.9 1700.0 950.0 2 month start fry 2 month start fry 2 month start fry 4 month start fry 4 month start fry 4 month start fry 4 month start fry 4 month start fry 4 month start fry 1 month start fry 1 month start fry 1 month start fry 1 month 1 month 2 months 3 months 3 months 2 months 2 months 29 days 29 days 29 days 76 days 76 days 76 days 76 days 76 days 76 days 28 days 28 days 28 days 96 hr 96 hr 48 hr 48 hr 96 hr 48 hr 96 hr 26 Table 2. LC 50 DATA FOR TEST SPECIES ( Concluded)

Sp2cies Age [ Test Period ( 95% Confidence Level)

1 month 96 hr 1 month 96 hr 1 month soft 96 hr 1 month soft 96 hr 1 ` month soft 96 hr 1 month soft 96 hr 1 month soft 144 hr 1 month soft 144 hr Instars 96 hr Instars 96 hr Instars 96 hr Instars 288 ` hr Instars 288 hr Instars 48 hr Instars 48 hr Instars 48 96 48 48 48 hr hr hr hr hr Instars 96 hr Instars 48 96 48 hr hr hr 6 weeks 90 days Fry 9~ Tdays Adult 90 days Adult 90 days Adult 90 days Eggs 96 hr Adult 96 days Mayfly Chironomid Mayf1 y Daphnia Chi ronomjd Daphnia Rainbow Trout Flagfish Combined Fathead Flagfish Combined Flagfish Flagfisha Bluegill 814.64 963.44 1008.1 1023.7 1008.1 889.55 780.27 680.77 69.16 46.00 49.92 198.87 149.61 228.14 153.79 245.96 1922.4 4189.1 4189.1 3973.0 413.0 840.0 4626.0 12,955.0 30.65 303.0 198.0 292.0 298.0 145.0 2921.0 ~ With aeration.

27 7003 4000

3000

2000

1000 ~` 3~ 4~

13 Figure 9. Tolerance of Test Species to FC 2O6 Toxicant 2 C,

`. O e C)

o~ .,

0) U C,

C, Legend 1. Fathead ( Adult) 2. Fathead ( Fry) 3. Channel Catfish ( Fry)

Do  4. Flagfis. h ( Adult) 5. Flagfish ( Young) 6. Flagfish ( Eggs  No Aeration). 7. Flagfish ( Eggs   Aeration) 8. Bluegill ( Adult) 9. Rainbow Trout ( Fry) 10. Rainbow Trout ( Fry  Soft Water)

5000   11. Mayfly ( Larva  First Instar) 12. Water Flea ( First Instar). 13. Chirnomid ( Larva  First Instar)

8 6~ nfl. r, 11 n 28 produced by external disturbances speeded up the opercular movements and eventually caused the death of the organisms. Displays of escape behavior were common; the fish actually jumped from the water. Thus all of the test chambers were covered during this experiment to eliminate the biasing of the data by animals jumping from the test chambers. There appeared to be hemorrhaging of the temporal region of the head. Before the stages just described, the animals underwent other physiological changes. There was a darkening of the skin; the animals became lethargic; and feeding was sporadic the fish merely striking the food and not actually eating it. In many cases the cause of death was probably starvation. This behavior had occurred in other studies in which animals were subjected to MBAS, LAS, and ABS contamination ( Reference 7). It should be added that at no time during the test or when these symptoms were recorded was the oxygen level in the test chambers to a point at which the animals should have been stressed. If gill   associated anoxia was the mode, the trout's sensitivity may be explained because the organisms most tolerant to the FC   206 toxicant were also those most tolerant to the low oxygen levels.

Additional work was performed to evaluate the eff. ects produced when trout fry were exposed to excessively high levels of FC 206 for from 1 to 6 minutes.

At a concentration of 30,000 mg/. C, trout fry went under water and gave the appearance of sedated animals within 30 seconds after the introduction of the toxicant. Upon removal of the test fish, recovery occurred in 3 to 5 minutes.

When left in this concentration for more than 4 minutes, the entire population was lost. When exposed to 60,000 mg/ L., the animals survived for only 2 minutes before substantial losses were incurred. However, no permanent damage to the exposed animals was evident. These test animals were observed for several days;

no losses or behavioral differences between the control and the test groups were noted. In the sublethal work, this was not the case. After extended exposure to low levels of toxicant, the sick animals died when placed in fresh, clean water. This shows that gradual irreversible damage is done by extended perio! is of exposure to low levels of FC 206. This experiment showed that in a natural situation the animals have only a few seconds to properly evaluate the situation and escape before the toxicant takes effect.

Reference 7. Howes, D., Newson, C., and Tovell, P. W. H., " Effects of Water Hardness on the Toxicity of Nonionic Detergents to Fish," The Journal of the International Association of Water Pollution Research, Vol. 9, January 1975.

29 A 90 day study was conducted to evaluate the effects of the FC 206 toxicant on the spawning of selected test organisms. It was known from previous work that a concentration in excess of 145 mg/~ e. would be fatal to development and thus care was taken to protect the adults from these high concentrations.

However, after 1 month no eggs were produced at any concentration over the control It appears that the eggs initially produced in the lowest concentration were eggs that were already prepared for spawning. These test fish were randomly selected from the breeder tanks. This test was conducted in accordance with the methods outlined by Mr. Don Allison of the Environmental Protection Agency, National Water Quality Laboratory ( Reference 8). The data indicate that at concentrations as low as 77 mg/. tL egg production did not occur after the first 30 days of the study. Since it cannot be clearly stated that FC 206 prevents spawning ( Figure 10) further investigation is warranted.

In an additional study, fathead minnow adults were encouraged to spawn.

Although this study was conducted to obtain a 90 day LC 50, spawning did occur in~ the control group and at a concentration of 52.49 mg/ L However, at the next higher concentration ( 147 mgf~.) spawning did not occur. It appears that the potential for the cessation of spawning exists when fish are subjected to sublethal dosages of FC   206 for extended periods. This theory is supported by the testing of flagfish eggs. Eggs allowed to hatch without aeration or agitation showed much lower tolerance to FC 206 than those hatched in aerated or agitated waters; agitated and aerated eggs showed a five fold better hatchability The development of a surface film appeared to interfere with oxygen uptake and thus the eggs were killed. There was also a time delay in the hatching of the eggs after the introduction of FC 206. This may indicate low dissolved oxygen availability to the eggs.

It was also found that those invertebrates normally found in low

oxygenlevel water survived the best during testing. The mayfly larva showed a very low tolerance to FC 206 and required high oxygen levels for survival; whereas the water flea was more tolerant to low oxygen levels and to FC   206.

Reference 8. Goode and Beane, Recommended Bioassay Procedures for Jordanella fiorid. ae Chronic Testing, United States Environmental Research Laboratory, Duluth, Minnesota.

30 ~ 20I Concentration of FC 206, mgfC Figure 10. Egg Production and Hatchability of Pimaphales promelus and Jordanella floridae 0 50 147. 0 77~ 90 31 3000 2500

~ 2000 ° l500 C. 0'

4',

~. 100o   .4,, C: 0),

0   Figure 11. Age Versus Lethality to FC 206 for Vertebrate Species The differences between the LC 50s among different tests of conspecific is well within the range of acceptability ( Table 2). Although there is a lack of sufficient data on soft water, it appears that there is no substantial change in the lethality of the FC   206 between treatments of soft and hard water.

Although the data are inconclusive to speculate on the effects on the same species at. different chronological periods of development, it appears that there is no substantial difference in the toxicity of FC   206 at different developmental stages in fish ( Figure 11).

Normal respiration of Daphnia is accomplished by the passage of oxygen across the body membrane. This could account for the high tolerance in that it would be more unlikely that the total body system would be engulfed in a film than it would for a surface film to develop on the gills of a fish.

Although tests were performed on channel catfish and bluegill fry for a 90

day period, an LC 50 was not determined. The concentrations used in these tests were not sufficient to cause substantial kills. There was a difference in the Juvenile ~ Adult~

/ /

I I 32 rate of growth shown in th2 bluegill tests; however, in the catfish experiment,

there was no concrete evidence of any growth changes at the levels tested. The levels established for catfish were from 18 to 100 mg/ c; the levels for bluegill were from 30 to 200 mg/ L The nongrowth differentiation shown by the catfish could reflect again that a species tolerant to low oxygen levels would not be as seriously affected in its feeding patterns as would an organism dependent on a high oxygen level. Therefore, if the animal is not being affected in its feeding or normal functions it would stand to reason that its growth rate would not change. In all the experiments conducted, except those on the channel catfish, the organism's normal growth patterns were disturbed. It is not known at this time if the somatogenic level of secretion was affected.

However, it is not felt that this is the case, since the channel catfish did not show growth changes. Examples of these data are given in Figure 12. As can be seen in the graphic representation for trout, these organisms appeared to show a crossover between 19 and 16 mg/. e; this probably occurred because at levels below 20 mg/ Se., no effects were being felt by the test animals. The apparent crossover at 37 mg/. P~ is brought about by having too few points to reflect a true distribution. Here the graph is skewed because of one abnormally large, hardy animal in the test chamber.

The data for the flagfish were a good representative collection which reflected a true growth pattern from juvenile to adult.  The atrophy established by the exposed animals was evident. These two tests were chosen to reflect growth rate differences between a hardy, tolerant species and a very nontolerant species. The data presented in this figure represent combined data from the replicates of these tests.

33 Concentration of FC 206, mg/~ C.

Figure 12. Growth Length and Weight Data for Sa7. mo gairdneria and Jordanelia floridae 0.88 Jordanella floridae U Length 0 Weight cr, ~

. C ~ 0~ c~ C ~ 16 14

12   10   8

6

4~.

2

0 0.8 4.0 0.7 3.5 0.6 3.0 0. E  2. E 0.4 2.0 0.3 1.5 0.2 1.0 0.1 0.5 Bairno qairdncria o Length ~ Weight ` 1 ` 1 ` 4 /

` 5 0.0 16 19 29: 37 0.0 0 100 200 250 350 500 34 SECTION V CONCLUSIONS AND REC0~ MENDATIONS 1. CONCLUSIONS 1. FC   206 has devastating effects at concentrations as low as 50 mgJ. C to some invertebrate and vertebrate species. 2. The family Salmo is the most susceptible of the fish tested to the FC   205 toxicant.

3. Spawning is probably affected by FC 206 even at low levels ( 100 mg/. C~

or less).

4. FC 205 seriously affects the growth and development of vertebrate organisms.

5. FC 206 may retard the hatching of eggs.

6. FC   205 at low levels probably prevents the formation and disposition of eggs by vertebrate species.

7. FC   206 does not appear to cause genetic morphological changes nor does it appear to produce infertility in the F1 or F2 generations of offsprings of fish subjected to low levels.

8. Fish are capable of detecting low levels of FC 206 and will, if given the opportunity, escape from the area of contamination.

9. The use of model stream systems to evaluate toxicants is not only feasible but gives data that are more representative of what is actually happening in nature.

10. Algae populations are destroyed by FC 206 at levels of less than 9420 mg/ L 11. FC   206 appears to increase the eutrophication process of an aquatic system.

2. RECOMMENDATIONS 1. From the results of these tests, it is recommended that the toxicant be used for training purposes only when proper precautions to prevent water contamination have been taken.

35 2. If feasible, the foam should not be used during known seasons of local spawning. Training schedules should be set up in accordance with local fish spawning conditions. 3. Even though FC   206 is biodegradable, it should be diluted as much as possible before it is allowed to enter a water supply or domestic treatment plant.

4. Additional follow   up studies should be performed to ascertain the mode of action produced by this toxicant.

5. Additional local land u~ eand water   use evaluations should be made prior to extensive training uses of this product.

6. Additional work should be accomplished on the effects of this toxi   cant on terrestrial organisms.

36 REFERENCES 1. Modeling  the Eutrophication Process, Ann Arbor Science Publishers, Inc., Ann Arbor, Michigan, 1975.

2. Standard Methods for the Evaluation of Water and Wastewatcr, 13th Edition,

American Public Health Association, Washington, D. C., 1973.

3. Bliss, C. I.., The Method of Probits  A Correction Science, 79, 409 10.

4. Finney, D. J., Probit Analysis, Cambridge University Press, 1962.

5. Mount, 0. I., and Brungs, W. A., " A Simplified Dosing Apparatus for Fish Toxicology Studies," Water Research, Vol. 1, 1967, p. 21.

6. Water Atmosphere Analysis, 1974 Annual Book of ASTM Standards, Part 23, American Society for Testing and t~ 1aterials, 1974.

7. Howes, D., Newson, C., and Tovell, P. W. H., " Effects of Water Hardness on the Toxicity of Nonionic Detergents to Fish," The Journal of the International Association of Water Pollution Research, Vol. 9, January 1975.

8. Goode and Beane, Recorivnended Bioassay Procedures for Jordanella floridae Chronic Testing, United States Environmental Research Laboratory, Duluth, Minnesota.

37 ( The reverse of this page is blank.) APPENDIX A ADEOSINE TRIPHOSPHATE ( Alp) ANALYSIS TO ASSESS TOXICITY OF AQUATIC POLLUTANTS by Kathryn G. Vogel, Ph. D.

39 TABLE OF CONTENTS Section Page I INTRODUCTION 41 II TEST METHODS 44 1. Extraction of ATP 44 2. Determination of ATP 44 3. Determination of Chlorophyll a 46 4. Determination of Dry Weight 47 III EXPERIMENTAL RESULTS AND ANALYSES 48 1. Algae Treated with Sodium Azide 48 2. Daphnia Treated with FC 206 48 3. Fathead Minnows Treated with FC 206 50 4. Pollution Monitoring Methodology 50 5. Sidestream Pollution 54 IV CONCLUSIONS 60 REFERENCES 61 LIST OF ILLUSTRATIONS Figure Page A l Growth and ATP Content of Unicellular Green Algae ( Chiorella vuigaris) in Pure Culture 42 A   2 Standard Curve for JRB ATP Photometer with duPont Luminescence Reagents 45 A 3 Azide Poisoning of Chiorella vulgaris 49 LIST OF TABLES Table Page A i AlP Content for Various Organisms 41 A 2 Twenty Four Hour Effect of FC 206 on ATP Content of Daphnia magna 49 A 3 Seventy Two Hour Effect of FC 206 on ATP Content of Whole Fathead Minnows 51 A 4 Assay of Periphyton Community from One Outdoor Tank 53 A 5 Assay of Sidestream Transects Before and After Pollution with FC   206 55 A   6 Assay of Periphyton Community Attached to Plexiglass Slides in Sidestreams During Administration of and Recovery from Pollution with FC 2O6 57 40 SECTION I INTRODUCTION 1. BACKGROUND The basic assumption for the development of an adeosine triphosphate ( ATP)

analysis methodology is that every living organism contains an approximately constant amount of ATP per unit of dry weight. A measure of ATP can therefore be translated into units of biorr. ass ( Reference 1). An equally important assumption is that only living organisms contain ATP. These assumptions have been tested and verified by several workers ( References 2, 3). In our laboratory ATP and dry weight for several different organisms were determined ( Table A i); these numbers were always in the range of 0.1 to'l. O pg ATP/ mg dry weight.

It was also found that increasing ATP concentration is directly proportional to increasing cell number in a pure algal culture ( Figure A i). Several experiments such as killing adult Daphni~ by freezing and then thawing them for various periods of time before determining the AlP content showed that the ATP content of an organism drops rapidly to a negligible amount after death.

Table A i. ATP CONTENT FOR VARIOUS ORGANISMS .

Organism ATP Cont pg/ mg dry ent, weight Filamentous Green Algae ( Ulothrix) 0.1 Snail Egg Case 0.1 Daphnia 0.3  0.8 Whole Young Flagfish ( Jordanella) 0.6 Whole Young Fathead Minnows ( Pirneph~ 7, es) Mixed Periphyton Community 0.5

0.06  0.8 0.2 References 1. Brezonik, P. L., Bowne, F. X., and Fox, J. L., " Application of ATP to Plankton Biomass and Bioassay Studies," Water Research, 9, 1975, pp. 115   162.

2. Hoim Hansen, 0., " ATP Levels in Algal Cells as Influenced by Environmental Conditions," Plant Cell Physiol., ii, 1970, pp. 689   700. 3. Weber, Cornelius I., " Recent Developments in the measurement of the Response of Plankton and Periphyton to Changes in Their Environment," Bioassay Techniques and Environmental C~. e. mistry, Ann Arbor Science Publishers, 1973, pp. 119   138.

41 I

`   I L)

` 4 0 S. C) . c~ E 7   6

5   4.

3   2   Gwth     ATP Content 0'~~ C)   0.16   0.14   0.12 o   0.10 I I   I   / I   0.08   0.06   0 04   0.02   0.00 32; , / /

0' E I   Ui 41 lj 8 12 16 20  24 1 28 Time, day&

Note: Chiorella vuigaris was grown at 18 ° Cin 100 m e of phosphatebuffered Beijerinck's medium ( pH 6.5) plus trace elements in cotton stoppered 300 m. e Erhlenmeyer flasks with shaking at 75 oscillations per minute. Cell number was determined with a haemocytometer. ATP was extracted by centrifuging 40 m. C of culture and immersing the cell pellet in 5 mL of boiling 0.0211 Tris, pH 7.4. The ATP content of the buffer was then determined.

Figure A l.  Growth and ATP Content of Unicellular Green Algae ( ChZ. orella vulgaris) in Pure Culture 42 Both dry weight and total organic carbon determinations combine measurement of living and dead material. Because ATP determinations measure only living tissue, the ATP method is useful in distinguishing living from dead

organisms. It would, of course, be a foolish exercise to use the method on macroscopicaily visible organisms, for there are much easier and cheaper ways of telling whether a fish is dead or alive. When one is dealing with microorganisms algae, or mixed communities of organisms that cannot be separated for individual inspection, however, a test that can generate a number that ~ n   dicates living bioniass is no longer trivial. ATP measurements can thus be used both to quantify the living organisms of a mixed periphyton community and to assess changes in this community caused by pollution.

2. OBJECTIVE The AlP project was undertaken for two reasons: ( 1) to determine whether measurement of the AlP content of periphyton samples would give useful information for assessing the toxicity of certain Air Force pollutants, and ( 2) to develop a methodology that would enable relatively untrained personnel to monitor the pollution of aquatic ecosystems.

43 SECTION II TEST METHODS 1. EXTRACTION OF ATP The living material from which ATP was to be extracted was immersed quickly and totally into boiling 002M Tris buffer, pH 7.75 for 5 minutes. The supernatant was then analyzed for ATP content or frozen for analysis up to 3 months later. Periphyton samples were concentrated by centrifugation.

In practice it is convenient to keep the buffer solution boiling in an electric frypan filled with hot sand. The amount of material added should not be enough to cool the buffer. If a large sample is being extracted, the amount of buffer should be increased. Some water will evaporate from the buffer, but this is not critical as long as volume corrections are made at the time of the AlP assay. If the Iris is not hotter than 90 ° C, or if the material is not brought immediately to boiling, the amount of ATP extracted will be significantly reduced. The extraction procedure, therefore, is very important to the assurance of reproducibility and accuracy.

2. DETERMINATION OF ATP A JRB, Inc., ATP photometer and duPont reagents were used. One buffer tablet and one vial of enzyme were dissolved in 3 m~ of distilled water. 2 0.1 m. e of the hydrated enzyme was dispensed into small reaction cuvettes provided by duPont. The solution was allowed to stand for 30 minutes before it was assayed.

During this period, the ATP photometer was turned on and warmed up. ( For this study the instrument was usually operated at an HV Adjust of 6.0 V and a Zero of 4.3 V.) The samples were then thawed and brought up to a known volume. A glass scintillation vial with a hole bored in its screwcap was used as a cuvette holder. The sample was injected directly into the enzyme mixture with a l0 p~ Hamilton syringe. The injection step was critical; variations in the Footnote   2The 3 m~ volume is recommended by duPont. If more water is used the sensitivity of the assay is less, but more assays are possible.

44 injection technique will produce great variations in the results. In this case, reproducibility was achieved by placing the needle tip against the bottorn of the reaction cuvette and dispensing the entire sample with one smooth and fairly rapid motion so that air bubbles  would not be introduced with the sample. As the injection was made, the foot lever that activates a delayed count cycle of the photometer was simultaneously pressed. During the next 15 seconds, the pipette was set down, the vial was smoothly placed in the counting chamber, the light tight lid was placed over the chamber and pushed and rotated slightly until it clicked, and the lever that exposes the photocell to the reaction was pulled. The instrument then measured emitted light for 60 seconds and displayed the counts on a digital panel. At the end of this counting period the yellow " Reset SC" light came on. The final display was recorded. The photocell lever was then pushed in and the display panel was reset to zero. The cuvette holder was then removed from the holder and a new cuvette with enzyme was placed in the holder.

The ATP standards that were routinely used were 0.002, 0.005, 0.01, and 0.015 pg AlP in 10 p.~ of distilled water ( Figure A 2). If the cpm of a sample 2i 2 cH 0ci   ~ ~ t 0.0OO~ 0.004 0.008 0.012: 0.016 AlP, pg Note: ATP standard ( Sigma Chemical Co.) was added in 10   p C volume to 0.1 rme of duPont luminescence enzyme reagent. After a 15 second delay, emitted light was read by the photometer for 60 seconds and displayed as counts per minute. Instrument settings: HV = 6.0 V, Zero = 4.3 V.

Figure A 2. Standard Curve for JRB ATP Photometer with duPont Luminescence Reagents o~~ o I I 45 exceeds 300,000, the " Reset HV" light will come on and all counting will stop.

That sample must then be diluted before the assay can be made.

After completing the test, the instrument was turned off and the photochamber was sealed to prevent damage to the photocell. Cuvettes may be rinsed by hand or discarded, depending on the economics of the situation. All glassware should be thoroughly cleaned because ATPase is a ubiquitous enzyme.

To determine the amount of AlP in a sample, the cpm was located on the standard graph produced during the same run, and the amount of AlP in the 10 p C.

sample was read from this. The amount of ATP in the total sample was calculated as follows: ( AlP/ lU p~ f.) x ( p. C/ sarnpie) = ( ATP/ sample). For example, if there is 0.01 pg of AlP in 10 p C of a. 5 m~ Csample, there is 0.01/ 10 x 5000 =

5 pg ATP in the entire sample.

A useful discussion and description of the theoretical and practical aspects of this methodology may be found in Reference 4.

3. DETERMINATION OF CHLOROPHYLL a Algal or periphyton samples were concentrated by centrifugation and then added to 90 percent V/ V acetone/ water. In routine assays, the periphyton pellets were added to 5 tnt of acetone in a standard test tube. This acetone/ algal solution was covered with parafilm and placed in the refrigerator for at least 24 hours. Extraction was assured if the solid material at the bottom of the tube was white and the acetone was green. The chlorophyll of some algae cannot be extracted by this gentle procedure ( Scenedesmus was unacceptable for laboratory testing for this reason), but most can be, and all the periphyton samples were treated in this manner.

The acetone   chlorophyll solution was clarified by low   speed centrifugation if necessary. The optical density of the clear acetone solution was then read Reference 4. JRB ATP   Photometer Instruction Manual, JRB Associates, Inc., La Jolla, California, August 1972.

46 at 665 mp before and after the solution was acidified with one drop of concentrated HC1. The amount of chlorophyll a was determined by the following formula ( Reference 5):

where Chlorophyll a( mg/ m2) 26.73( 665b  665a) x V A 665b = 0D665 before acidification 665a = OD665 after acidification V = volume extracted ( liters)

A = area of periphyton substrate harvested ( meters2)

For pure chlorophyll a, the OD ratio 665b" 665a = 1.7. Pheophytin, the degraded chlorophyll present in dead material, shows no change in optical density after acidification ( OD b/ a ratio = 1).

Small porcelain crucibles were cleaned and dried in the oven. Their tare ` weight was determined to the 4th decimal place on a Mettler balance. The sample was poured into the crucible and then dried at 85 ° Ffor 48 hours. The dry sample and the crucible were weighed together, and the ` dry weight of the sample was determined by subtraction.

Reference 5. Standard Methods for the Examination of Water and Wastewater, 13th Edition, American Public Health Association, Washington, D. C., 1973. in 90 percent acetone with 1   cm light path 4. DETERMINATION OF DRY WEIGHT 47 SECTION III EXPERIMENTAL RESULTS AND ANALYSES 1. ALGAE TP. EAIED WITH SODIUM AZIDE A pure culture of the unicellular green algae, Chiorella vulgaris, was treated with 0.0511 sodium azide. This compound is a potent inhibitor of oxida   tive phosphorylation and is thus poisonous to respiring cells. As Figure A 3 shows, the amount of ATP in the ~ ulture declined by 94 percent over a 48 hour period; the number of intact cells declined by only 24 percent. The experiment clearly shows that the effect of a poison on an algal culturecan be sensitively measured by the assay of AlP content.. These data also suggest that more than simple cell death was measured. Although viability of the algal cells after azide treatment was not directly assayed, unicellular organisms generally undergo rapid cell ° lysis after death. It is probable that the cells counted after treatment were not dead. If this assumption is true, the measurements show a decrease in the amount of AlP per cell after the poison treatment. It would be useful to be able to ~ eterminethe health and vitality of a periphyton community or a single larger animal simply by measuring its ATP content.

2. DAPHNIA TREATED WITH FC   206 The possibility that ATP content might change in a sublethal fashion as a result of treatment with an aquatic pollutant was tested. Adult Daphnia were treated with various concentrations of FC 206 for 24 hours. The amount of ATP in a sample of the organisms, along with the dry weight of organisms in that same sample, was subsequently determined. A number representing the pg ATP/ mg dry weight was calculated for each sample. As Table A 2 shows, the amount of ATP was decreased by approximately 50 percent in the samples treated for 24 hours with 30, 120, 600, and 12,000 mg/ C FC 206. Ninety six hour static tests with FC 206 demonstrated an LC   5O for adult Daphnia of about 4,000 mg/. C. The AlP data indicate that a toxicant concentration 100 fold less than the LC   50 caused a 24 hour effect equal to that of the LC 50. It is tempting to conclude that this very sensitive method has detected a subtle disruption of the 48 0.1~~ 24 48~ Time in NaN , hr  3 Note: Sodium azide ( 0.0511) was added to a culture of Chiorella. Aliquots of the culture were pulled at 3, 24, and 48 hours for determination of the number of cells and AlP content.

Figure A. 3. Azide Poisoning ` of Chloreila vulgaris Table A 2. TWENTY FOUR   HOUR EFFECT OF FC   206 ON ATP CONTENT OF DAPHiVI. 4 MAGNA Concentration of FC 206, mg/. C , ATP pg/ mg Cont dry ent, weight  0.86 0.75 30 0.38 0.42 120 0.74 0.50 600 0.41 0.51 12,000 0.38 0.24 Note: There were no deaths during the course of this experiment Daphnia were removed from the FC 206, divided into two roughly equal groups ( about 100 organisms in each), and immersed in boiling 0.0211 Tris buffer for 5 minutes. The dead organisms were dried for measurement of dry weight while the AlP content of the buffer was determined. O. 3~     S.

L)

L4~ 0 c'J C, 0.2  1.6 1 .4 1.2   ~ __

0.8 0. 1   to x E ( I,

Ia L)

ft ~

  u. u. 0 5 ~ ~` ci) o.' t ~

E ATP Content     Growth 0 12 0.2 36 49 organism's metabolism. Until a great deal of additional work has been done on the range of control variability and the effects of environmental changes not due to the toxicant, however, such a conclusion must be considered premature

3. FATHEAD MINNOWS TREATED WITH FC 206 A similar experiment was performed with whole fathead minnows. Young fish were kept for 72 hours in 10 gal tanks containing FC 206 in concentrations of 1000 and 1500 mg/ C ( 96 hr LC   50 = 2500 mg/ C); a control tank was also maintained.

At the end of the 3 day period, it was clear that the fish kept at concentrations of 1000 mg/' C had been affected by the toxicant; several in this group had died, and others were gasping and showing unusual swimming behavior.

Individual living fish from each group were immersed in 10 mt of boiling buffer to extract ATP and then weighed. The pg AlP/ mg dry weight for fish in each group was calculated. The average value of this ratio for fish in both treatment groups was 0.54 ( Table A 3), 32 percent less than the average control value of 0.80. Again, it is tempting to speculate that the AlP methodology had detected a subtle effect. However, the very slight decrease measured and the many overlapping individual values in each group indicate that the measured change is not significant.

Initially it was hoped that measurements of ATP content would be useful in assessing the health of an organism being affected by a toxicant. The work has not verified this hope. In fact, ATP is not stored by animal tissues. In a healthy cell, the amount of AlP produced is determined by the amount of adeosine diphosphate ( ADP) generated. When more ADP is present, more of it is rephosphorylated to restore the ATP used. This very fine regulation occurs in seconds in the mitochondria; thus, it is not surprising that the overall ATP content is so constant. Severe perturbations from normal patterns of energy use need not necessarily be reflected in a detectable change in ATP content.

4. POLLUTION MONITORING METHODOLOGY The main goal in the development of this technique was that of defining a 50  Table A   3. SEVENTY   Tt! O~ OUREFFECT OF FC   206 ON ATP CONTENT OF WHOLE FATHEAD MINNOWS Concentration of FC 206, mg/ C ATP Content, pg/ mg dry weight Mean ± S. D.

    1,000 1,500 0.59, 1.35, 1.16, 0.68 0.50, 0.74, 0.64, 0.69 0.54, 0.59, 0.59, 0.51, 0.46 0.55, 0.67, 0.46, 0.46 0.55, 0.63, 0.60, 0.51 0.41, 0.65 0.79 ± 0.30 0.54 ± 0.07 0.56 ± 0.09 Note: At 1,500 mg/ C fish were obviously affected and some died. Only living fish were used for the AT? assay. ATP was extracted by immersing each live fish in boiuinn 0.0211 Iris ( pH 7.8) for 10 minutes. The fish were then removed from the buffer, dried at 70 ° Cfor 6 days and weighed. ATP content of the buffer was assayed. The approximate dry weight of the fish was 100 mg.

methodology for monitoring the effects of toxicants on a natural fresh water community. It was hoped that a capability could be developed for assessing damage to the populations in an artificial stream stressed with toxicants.

This same methodology might be useful in the systematic monitoring of natural waterways.

A surface periphyton community was chosen to test this methodology.

Specifically that means those protozoans, algae, and small invertebrates which attach themselves to, or are closely associated with, growth on a glass or plexiglass slide suspended just below the surface of the body of water being tested.

Samplers used consisted of a plastic support that holds eight standard microscope slides approximately 1 inch apart. This unit was attached to a wire frame having two foam floatation pieces on a stabilizing loop; this allowed the slides to remain suspended just below the surface of the water. The whole unit can move around with currents, however, so the orientation to sunlight is variable on a day   to day basis. The slides were harvested at specific time intervals or when the amount of growth on each slide seemed sufficient for an assay.

Slides were removed from the sample holders outdoors and dropped into individual petri dishes containing enough water to cover them. They were then brought into the laboratory, where the growth on both sides was immediately scraped off 51 by hand. The edge of a clean slide was used as the scraping instrument. It was assumed that these samples were equivalent unless visual inspection clearly showed this to be untrue ( e. g., a large snail egg case on one sample).

Material from one slide was used for determination of chlorophyll a, dry weight,

or ATP. The periphyton material was concentrated by a short spin in a table   top centrifuge. The supernatant was discarded, and the loose pellet could then be dropped into 5 nit of 90 percent acetone for chlorophyll extracting, 5 rn.~. of boiling 0.0211 Tris for ATP extraction, or a tared crucible for dry weight determination A small aliquot of the material was also examined microscopically to obtain a qualitative picture of the population being measured.

Measurements of this kind were performed in a largc outdoor tank over a period of several months. All of the data collected on this tank is shown in Table A 4. The extremely preliminary nature of this data must be stressed, for it was gathered while the methodology was being developed. The purpose was to determine whether° this method could be used to docurnentthe presence, health,

and composition of a periphyton community. Several things were learned.

The first series of measurements was performed on slides harvested from the tank on successive days. The data show that the amount of material, ATP,

and chlorophyll a accumulated on the slides had probably reached its maximum before day seven and did not change substantially during the next seven days.

In addition, the measurements have a respectable degree of reproducibility ( pg ATP/ mg dry weight was between 0.1 and 0.3).

A number was developed that is the ratio of pg ATP/ mg chlorophyll a measured per square meter of slide surface harvested. This ratio is similar to, but represents a major improvement upon, the Autotrophic Index suggested by Weber ( Reference 3). The Autotrophic Index is the ratio of dry weight of organic matter to chlorophyll a. Because it is a measure of the amount of material that contains chlorophyll, it can assess only the proportion of the sample that is autotrophic. The number we have developed uses ATP as the numerator, thus avoiding the problems inherent in dry weight measurements. By comparing ATP content to chlorophyll a content, we are comparing two parameters which are present only in living material; accumulation of dead organisms and inorganic material will not bias the result. Since AT? is contained by autotrophic and heterotrophic 52 Table A 4. ASSAY OF PERIPHYTON COMMUNITY FROM ONE OUTDOOR TANK Harvest Growth, Date days Dry weight,' mg AT?, pg ATP/ rng, pg ATP/ rn2, pg Chlorophyll/ rn2, mg ATP/ rng chlorop pg of

hyll,

8/ 18 7 18.8 2.00 0.110 533 7.30 73 8/ 19 8 16.2 4.10 0.253 1100 8.50 130 8/ 20 9 15.5 3.90 0.250 1033 6.50 159 8/ 21 10 19.1 4.80 0.251 1280 12.50 102 8/ 22 11 11.7 2.60 0.222 702 7.70 91 8/ 25 14 20.2 3.40 0.170 907 6.40 142 10/ 8 7 12.6 5.20 0.410 1400 10.50 133 10/ 8 30 5.5 8.80 0.160 2400 19.40 124 10/ 14 7 2.5 7.90 0.320 2121 14.20 149 10/ 14 36 76.0 5.10 0.067 1300 11.70 111 10/ 31 17 34.0 2.00 0.059 535 15.50 34 11/ 7 24 50.0 4.40 0.088 1197 24.00 50 11/ 14 14 22.0 3.40 0.160 913 18.00 51 11/ 18 11 14.2 2.00 0.143 548 14.00 39 11/ 25 7 3.0 1.60 0.530 214 5.10 ` 42 1/ 13 36 7.6 0.28 0.037 37 0.89 42 1/ 14 34 1.8 0.22 0.120 30 0.29 100 1/ 21 57 3.1 0.28 0.090 38 1.26 30 2/ 15 34 2.7 0.15 0.056 20 0.38 50 4/ 23 50 38.0 3.60 0.095 973 24.00 40 4/ 30 27 47.0 2.30 0.049 624 9.30 67 53 organisms in approximately similar amounts per unit of dry weight, while chlorophyll a is found only in autotrophic organisms, the ratio will be an expression of the proportion of autotrophic living material in the sample. This ratio has been tested with data from different periphyton communities and appears to be potentially useful as a predictor of the composition of the community For example, measurements presented in Table A 4 were from a tank that contained fish that were being fed daily. The high nutrient condition of the water provided food for a variety of heterotropic organisms, which were obvious whenever samples were examined microscopically. The brown, scurnmy growth appearing on the slides consisted primarily of diatoms; however, associated with them were numerous amoebae, ciliates, nematodes, and rotifers. The ATP/ chlorophyll ratio for these communities ranged from 100 to 150. In November, when the cold weather became severe, this ratio dropped to about 50. Observation of the periphyton during this period showed that along with the very much reduced rate of algal accumulation, a change in the composition of the population had also occurred. The small invertebrates were no longer present; the protozoan popu   la. tion was greatly reduced and was now composed primarily of flagellates. The lowered ATP/ chlorophyll ratio indicated that an increased percent of the popu   lation was now autotrophic. In midwinter, the amount of growth, as measured by accumulation of mass and AlP on the slides, was extremely small; but the population that did accumulate had an ATP/ chlorophyll ratio and an observed composition similar to those of the late fall. In April, the return of warm weather brought a bloom of algal growth, which was measured as an increase in periphyton ATP and, particularly, chlorophyll. Other tanks which were not receiving fish food had highly autotrophic populations. The AlP/ chlorophyll ratio of samples from these tanks ranged from 25 to 50, even during the summer months.

Samples of pure green algae ( both mats of filarnentous Iilothrix and the unicel   lular ChZ. orella) had a ratio of 12  to 20.

5. SIDESTREAM POLLUTION The AT? analysis technique was developed so that a methodology for assessing the toxicity of certain compounds on an aquatic ecosystem could be obtained.

The feasibility of the methodology was given a preliminary trial during the pollution of one sidestream system i~ an artificial stream. One arm of a 54 sidestream was polluted with FC 206. Four days prior to the initial addition of FC 2O6, the entire contents of an 8 inch cross section of the sidestream at approximately station 3 ( see Figure 3 in body of report) was siphoned into a bucket. The sides and rocks were rubbed free of attached material.

Approximately 8 £ . of material was then poured through two layers of nylon screening to separate coarse from fine material. Dry weight, chlorophyll a, and AT? content of the coarse material were analyzed. The AT? content of the fine material was also measured. These data ( top line of Table A 5) show that twice as much AlP ~° ias present in the coarse material as in the fine; that the pg AlP/ mg dry weight was in the anticipated range; and that the Alp/ chlorophyll ratio was very high, indicating the presence of many nonphotosynthetic organisms. After FC 206 had been administered for 8 days, similar transects were siphoned off from the middle of the polluted and the unpolluted arms of the sidestream.

Similar analyses of these samples showed that the mass of material on the polluted side had not been decreased significantly, but that the AlP content had been reduced by 87 percent, indicating that most of the previously living material had been killed. The chlorophyll content also dropped 76 percent. The Table A 5. ASSAY OF SIDESTREAM TRANSECTS BEFORE AND AFTER POLLUTION WITH FC   206 Dry Chioro h 11 ATP, pg ATP/ mg of ATP/ mg of weight, ~ ` dry weight, chlorophyll,

______________ g pg Coarse Filtrated pg pg Before addi 1.410 667 78.0 47 0.090 188 tion of FC   206 Polluted arm 1.388 160 2.5 14 0.011 106 after addition of FC 206 for 8 days Unpolluted arm 2.860 2346 7.0 22 0.012 12 after addition of FC 2O6 to opposite side for 8 days  _____________ ______ __________ ____________ _____________

Note: The entire contents of the sidestream for an approximate length of 8 inches were siphoned into a bucket and filtered through a nylon screen; aliquots of the coarse material were assayed for dry weight, chlorophyll a, and AT? content; aliquots of the filtrate were also assayed for AT? content.

55 ratio of pg ATP/ mg dry weight was low, probably because of the dead material now represented in the denominator. The ratio of ATP/ chlorophyll was over 100,

indicating the presence of many nonautotrophic organisms. Observation of this sidestream indicated that virtually all the normal stream organisms had been killed or had migrated to a less polluted location in the system. The general appearance was that of greyish   brown scum devoid of vertebrate or invertebrate animals and containing very little floating algal material.

The unpolluted arm had not been assayed prior to pollution; thus, a strict before and after comparison was not possible. Our visual impression was that the two arms of the sidestream had similar amounts and types of growth before pollution. After FC 206 was administered, the mass of material in the unpolluted arm was increased, and the chlorophyll content was greatly increased ( nearly 4 times). The amount of AlP in the unpolluted side was low, as was the ratio of AlP/ chlorophyll. This low ratio is typical for cultures of pure algae. The high chlorophyll and low AlP/ chlorophyll ratio confirm the visual observation that a massive bloom of algae had occurred in that part of the sidestream receiving very low levels of the toxicant. At this point, it is not possible to state that the low toxicant level was a cause of the bloom,

but it would be a feature to watch carefully in subsequent testing. FC   206 was added to laboratory cultures of Chiorella vulgar is in concentrations ranging from 200 to 10,000 mg/ L. It did not kill this algal species, even at the highest concentration. At the lower concentrations, culture growth and morphology were indistinguishable from those of the controls. At concentrations above 1500 mg/ Z the cultures soon became quite cloudy and the growth rate was reduced. Closer observation revealed a heavy growth of rodshaped motile bacteria The pH of the medium dropped to 5.2. This pH change alone could have been responsible for the reduced growth rate.

Four days before the FC 2O6 was administered, plexiglass slides ( total surface = 125 cm2) were suspended in the sidestream at stations 1, 3, 5, 10,

12, and 14 ( see Figure 3 in body of report). They were removed after 8 days of exposure to the pollutant. The periphyton attached to the slides was scraped off and analyzed for AlP and dry weight. These data are presented in the top half of Table A   6. Since this was the first time that these slides and this technique had been used, it can be concluded only that the amount of AT? at stations 56  Table 1 6.

01 ASSAY OF PERIPHYTON COMMUNITY ATTACHED TO PLEXIGLASS SLIDES IN SIDESTREAMS DURING ADMINISTRATION OF AND RECOVERY FROM POLLUTION WITH FC 206 Stationa ATP/ slide, Dry weight/ slide,

~ ".~`~" ATP/ mg,

pg Chlorophyll a/ r~ 2,

pg ATP/ rng of chlorophyll a,

pg Four Days Prior Plus Eight Days During Addition of FC 206 ( 10/ 24  11/ 5)

Polluted 1 35 Unpolluted 10 12 14 0.075 0.150 0.200 0.200 1.130 17.0 13.0 5.4 `

` 9.1 5.0 45.0 5.3 0.0043 0.0120 0.3000 0.0200 0.1100  1.8 Fifteen Day Recovery ( 11/ 5  11/ 20)

Polluted 1 35 Unpolluted 10 12 14 3.800 2.500 4.000 1.000 1.400 2.600 31.0 17.0 27.0 14.0 60.0 `

29.0 0.1200 0.1500 0.1500 0.0700 0.0240 0.0890 ` 4.0 1.8 6.5 , 4.4 12.8 10.6 76 112 49 18 9 20 aSee Figure 3 in body of report for location of stations. 1 through 10 was low and dry weights were extremely variable. Inconsistencies in growth on different slides suspended at the same station were visual ly obvious therefore, the variations measured are not surprising. That so very little AT? was measured in the 17 mg of material taken from station 1 indicates that most of the killed organisms remained attached, at least at this point.

New slides were positioned at each station after additions of FC   205 had been halted and the sidestream had been flushed with water from the main stream.

The slides were left in place for 15 days and then harvested ° for analysis of AlP, mass, and chlorophyll content. These data show that the amount of AT?

in the polluted arm was substantial     greater, in fact, than that in the unpolluted arm. The dry weights, though variable, were all in the same range. The greatest differences. occurred in measurements of chlorophyll a where it was clear that stations 12 and 14 had a very high chlorophyll content. The ATP/

chlorophyll ratio indicated that stations 1 through 5 had supported the attachment of a heterotrophic community; stations 10 through 14 had supported virtually pure algal communities.

These data were at first very puzzling. Visual inspection suggested that everything in the polluted arm was dead; while at the same time the unpolluted arm contained an unchanged or perhaps enhanced periphyton community. The measurements however, suggested that the polluted arm was supporting a nearly normal mixed periphyton community. The numbers alone would lead one to conclude that FC 206 had had no detrimental effect on that ecosystem. Microscopic examination of material from both arms solved the puzzle. The scumniy, brown material in the polluted arm proved to be covered with a grey layer of bacteria These bacteria were attached to everything and were multiplying rapidly In this scum of bacteria and dead algal material, there were few living algae and even fewer living protozoans. Invertebrates were absent. Thus,

the high AlP content and high ATP/ chlorophyll ratio had resulted from the rapid growth of bacteria. These bacteria are probably the same as those found in the laboratory tests of Chiorella ( species identification was not done) and had probably been introduced into the sidestream with the FC 206.

During the initial recovery period, the abundant nutrient available from the organisms killed by the FC   206 created conditions favoring growth of the bacteria It is unfortunate that it was not possible to further analyze this 58 sidearm to document the time and progression of the organisms responsibie for the eventual recovery from the FC 2O6 pollutant.

Although these results are preliminary, measurement of AT?, dry weight,

and chlorophyll a clearly offers an attractive tool for assessing the effects of a toxicant on an aquatic ecosystem. It should be stressed again, however,

that such measurements must be closely correlated with the intelligent observation of the population being measured. The numbers alone would indicate that FC 206 had caused only temporary damage to the periphyton community.

In fact, a kill followed by a bacterial takeover occurred, and complete recovery probably took months.

59 SECTION IV CONCLUS IONS There exists a sensitive, reliable method of quantitating AT?. This compound is present only in living organisms and can be used to assess the living biomass of a particular sample. The method is potentially valuable in the field, where the biggest problems will be reproducible sampling and transportatIon of samples to the laboratory without loss of AT?.

The ratio pg ATP/ mg dry weight seems to decrease in organisms subjected to FC   206 in sublethal concentrations. A great deal more work on the range of variability in control situations must be done before this method will be useful in assessing the health of treated organisms.

The ratio pgATP/ mg chlorophyll a can provide information about the composition of the population assayed. It is superior to the Autotrophic Index because only living material is included in the numerator. The ratio will be 100 to 150 for mixed periphyton communities and about 20 for populations of pure algae. This number, taken alone, is not meaningful as a measure of pollution.

Measurement of AT? content, chlorophyll a, and dry weight of periphyton samples, when combined with intelligent observation, can give useful quantitative information when the effects of a pollutant on an aquatic ecosystem are to be assessed.

60 REF ER EflC E S 1. Brezonik, P. L., Bowne, F. X., and Fox, J. L., " Application of AT? to Plankton Biomass and Bioassay Studies," Water Research, 9, 1975, pp. 115   162.

2. Holm   Hansen, 0., " AT? Levels in Algal Cells as Influenced by Environmental Conditions," Plant Cell Physiol. , 11, 1970, pp. 689 700.

3. Weber, Cornelius I., " Recent Developments in the Measurement of the Response of Plankton and Periphyton to Changes in Their Environment," Bioassay Techniques and Env iron'nental Chemzstry, Ann Arbor Science Publishers 1973, pp. 119 138.

4. JRB ATP Photorneter Instrumentation Manual, JRB Associates, Inc., La Jolla, California, August 1972.

5. Standard Methods for the Examination of Water and Wastewater, 13th Edition American Public Health Association, Washington, D. C., 1 973.

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