'v‘l‘ ? a" ,.'"‘ '6 swsrd- "“ ’ ... ,.,.3:»{-?’¢x+. . . 0 - I ‘- uni-'Ii'eifiiiét'31~.~tz-2-;z'eir:.»-az,3~:-3; :- . ' Niki)”; ‘9} "u" - I. w. .W—‘f"_~q" CHLORINE TOXICITY AND ITS EFFECT ON GILL TISSUE RESPIRATION OF THE WHITE SUCKER Catostomus commersoni (Lacepede) Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY RDNALD L. FOBES 1 97 1 ABSTRACT CHLORINE TOXICITY AND ITS EFFECT ON GILL TISSUE RESPIRATION OF THE WHITE SUCKER ‘Catostomus commersoni (Lacepede) BY Ronald L. Fobes The purpose of this investigation was to help determine the mechanism of chlorine toxicity to freshwater teleosts. White suckers of a relatively large size range were exposed to a lethal concentration of chlorine (one ppm total residual chlorine) for 30 and 60-minute periods. Following the assump- tion that normal filamental and lamellar gill tissues active- ly use oxygen while metabolizing, it was hypothesized that any damage to such tissue would alter its respiration rate. Subsequent to chlorine exposure, complete gills (arch and filaments) were excised from the fish and their respiration rate (002) determined with a Gilson differential respirometer. An estimate of "normal" 002 for white sucker gill tissue ranged from 1.5 to 1.7 p1 Oz/mg dry gill weight/hr. Statis- tical analysis indicated no significant difference between QOZ means of the control gills and those exposed to chlorine. It was concluded that death resulting from relatively short exposures to lethal chlorine concentrations was not caused by gill damage and that gills were not the primary site of chlorine toxicity. CHLORINE TOXICITY AND ITS EFFECT ON GILL TISSUE RESPIRATION OF THE WHITE SUCKER Catostomus commersoni (Lacepede) B r \ '1‘ Ronald LSLFobes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1971 ACKNOWLEDGMENTS I wish to thank fellow graduates Dave Rosenberger for use of his toxicant dilution system and Dean Eyman for his manuscript criticisms and helpful comments. My sincerest appreciation is extended to Dr. E. W. Roelofs, Dr. P. O. Fromm and Dr. N. R. Kevern, the members of my graduate committee, for their academic, moral and financial support. My heartiest thanks to Dr. W. H. Conley and Dr. J. H. Stapelton for their guidance in statistical analysis. Sincere thanks are extended to my wife, Karen, for her tolerance, understanding and continuing moral support. This research was financed by the Federal Water Quality Administration training grant STl-WP-109 and the Environ- mental Protection Agency Office of Water Pollution grant 5P3-WP-264. Use of the Michigan State University computing facilities was made possible through support, in part, from the National Science Foundation. ii TABLE OF LIST OF TABLES. . . . . . . LIST OF FIGURES . . . . . . CONTENTS INTRODUCTION. . . . . . . . Need for Study . . . . Purpose and Scope of Study METHODS . . . . . . . . Fish Holding and Feeding . Toxicant Dilution System . Dissection Procedures. Gill Tissue Respiration Measurements Data Collection. . . . Water Chemistry . Chlorine Determination. FiSh. O O O O O O Respiration Rate. Statistical Analysis . RESULTS 0 O O O O O O O 0 Water Chemistry . . . . Chlorine Determination. FiSh. O O C O O O Respiration Rate. Statistical Analysis . DISCUSSION AND CONCLUSION . LITERATURE CITED. . . . . . APPENDIX. . . . . . . . . . iii Page iv viii LQFJH LIST OF TABLES Table Page 1- Range of pH, means and standard errors for determinations of temperature, dissolved oxygen, alkalinity and hardness in holding (H), acclimation (A), control (C) and test (T) tanks . . . . . . . . . . . . . 15 2. Means and standard errors (S.E.) for the different chlorine residuals during the 30 and 60-minute exposures . . . . . l7 3. Means and standard errors (S.E.) of total length, total weight and dry gill weight for test (T) and control (C) fish exposed for 30 and 60-minutes. . . . . . 20 4. Log 0 transformations of 002 means and fisE weights (g) for two test (T) and two control (C) fish at each 30-minute exposure . . . . . . . . . . . . . . . . 22 5. Log 0 transformations of Q02 means and fish weights (g) for two test (T) and two control (C) fish at each 60-minute exposure . . . . . . . . . . . . . . . . 23 6. Two-way analysis of variance testing the effects of chlorine exposure (30 and 60 minutes) and fish type (test and control) upon gill tissue 902 without regard for fish weight . . . . . . . . . 24 7. Analysis of covariance for data in Tables 4, S. . . . . . . . . . . . . . . 25 iv LIST OF TABLES - Continued TABLES F-tests for difference between two regression coefficients; test fish = (T), control fish = (C), exposure time = 30 or 60 minutes. . . . . . . . . Water chemistry data: pH, temperature, dissolved oxygen, alkalinity and hard- ness readings for holding (H), acclima- tion (A), control (C) and test (T) tanks. . . . . . . . . . . . . . . . . . Chlorine and chloramine concentrations (in ppm) measured midway through (A) and immediately after (B) 30-minute exposures Chlorine and chloramine concentrations (in ppm) measured midway through (A) and immediately after (B) 60-minute exposures Total length, weight, gill wet weight and gill dry weight for suckers used during the 30-minute exposures to 1 ppm total residual chlorine. . . . . . . . . Total length, weight, gill wet weight and gill dry weight for suckers used during the 60-minute exposures to 1 ppm total residual chlorine. . . . . . . . . Correction factors (CF), fish weights (9), oxygen uptakes and 002 rates of white sucker gill tissue following a 30-minute exposure to 1 ppm total residual chlorine, August 2, 1971 . . . . . . . . . . . . . Correction factors (CF), fish weights (9), oxygen uptakes and Q02 rates of white sucker gill tissue following a 30-minute exposure to 1 ppm total residual chlorine, August 27, 1971. . . . . . . . . . . . . Page 27 44 46 47 48 50 53 LIST OF TABLES - Continued Page TABLES B-3. Correction factors (CF), fish weights (9), oxygen uptakes and Q 2 rates of white sucker gill tissue follow1ng a 30-minute exposure to 1 ppm total residual chlorine, August 29, 1971. . . . . . . . . . . . . 54 B-4. Correction factors (CF), fish weights (9), oxygen uptakes and Q 2 rates of white sucker gill tissue follow1ng a 30-minute exposure to 1 ppm total residual chlorine, August 30, 1971. . . . . . . . . . . . . 55 B-5. Correction factors (CF), fish weights (9), oxygen uptakes and Q 2 rates of white sucker gill tissue follow1ng a 30-minute exposure to 1 ppm total residual chlorine, September 1, 1971. . . . . . . . . . . . 56 B-6. Correction factors (CF), fish weights (9), oxygen uptakes and Q 2 rates of white sucker gill tissue follow1ng a 30-minute exposure to 1 ppm total residual chlorine, September 4, 1971. . . . . . . . . . . . 57 8-7. Correction factors (CF), fish weights (9), oxygen uptakes and Q 2 rates of white sucker gill tissue follow1ng a 60-minute exposure to 1 ppm total residual chlorine, July 26, 1971. . . . . . . . . . . . . . 58 B-8. Correction factors (CF), fish weights (g), oxygen uptakes and Q rates of white sucker gill tissue follow1ng a 60-minute exposure to 1 ppm total residual chlorine, August 10, 1971. . . . . . . . . . . . . 59 8-9. Correction factors (CF), fish weights (g), oxygen uptakes and Q rates of white sucker gill tissue follow1ng a 60-minute exposure to 1 ppm total residual chlorine, August 21, 1971. . . . . . . . . . . . . 60 vi LIST OF TABLES B-lO. TABLES - Continued Correction factors (CF), fish weights (g), oxygen uptakes and Q02 rates of white sucker gill tissue following a 60-minute exposure to 1 ppm total residual chlorine, August 22, 1971. . . . . . . . . . . . . Correction factors (CF), fish weights (g), oxygen uptakes and Q 2 rates of white sucker gill tissue follow1ng a 60-minute exposure to 1 ppm total residual chlorine, August 23, 1971. . . . . . . . . . . . . Correction factors (CF), fish weights (g), oxygen Uptakes and Q02 rates of white sucker gill tissue following a 60-minute exposure to 1 ppm total residual chlorine, August 24, 1971. . . . . . . . . . . . . vii Page 61 62 63 FIGURE 1. LIST OF FIGURES Page Regression coefficients for test (t) and control (c) fish during the 30- minute exposure (solid lines). Dashed line represents estimated slope for all treatments given: all regression coefficients equal . . . . . . . . . . . 29 Regression coefficients for test (t) and control (c) fish during the 60- minute exposure (solid lines). Dashed line represents estimated slope for all treatments given: all regression coefficients equal . . . . . . . . . . . 31 Regression coefficient for combined 30 and 60-minute control (c) fish, solid line. Dashed line represents estimated slope for all treatments, given: all regression coefficients equal. . . . . . 33 viii INTRODUCTION Need for Study Beneficial facets of chlorination have been explored and expounded in previous studies. Chlorination has helped control or eliminate odors and noxious tastes, improved Operation of sedimentation tanks, abolished psychoda flies, decreased pooling on trickling filters, reduced BOD and killed harmful bacteria (Scott and Van Kleeck, 1934). Chlorine also destroys or modifies decomposable organic wastes and reduces chemical oxygen demand (COD) (Moore, 1951). BOD reductions of 62%, bactericidal efficiences from 90-95%, and dissolved oxygen (DO) increases of 147% have been reported for sewage chlorinated to an average residual of 2.0 ppm (Baity 2: 31., 1933; Eddy, 1934; Faber, 1944). Nearly every major industry, domestic waste treatment facility and water treatment plant throughout the United States incorporates some aspect of chlorination in their processes. The extent and scope of research concerned with possible deleterious effects of chlorination is in no way proportionate to the quantity of work expended on its beneficial oxidative and bacteriocidal properties. Reports on the toxicity of chlorine and its derivatives to stream biota include those by Enslow, 1932; Doudoroff and Katz, 1950; Merkens, 1958. Enslow discovered that chlorinated organic waste products were not as assimilable to stream biota as the original material. In fact, at times the chlorinated products were toxic, even when highly diluted. Representative early toxicity work was reported by Allen gt al_(1946, 1948). They determined that sewage plant effluents chlorinated with quantities much smaller than those required to give residual chlorine detectable by the ortho-tolidine test were highly toxic to stream fish. It was later discovered that the toxicity was caused by form- ation of cyanogen chloride from the reaction between chlorine and cyanates in the effluent. More recently, chlorine concentrations within permis- sable limits for municipal water systems were found to be toxic to fingerling brook trout and fingerling smallmouth bass (Pyle, 1960). Merkens (1958) investigated toxicity of chlorine and chloramines to rainbow trout and could only theorize that a safe concentration might be very low -- less than 0.08 ppm. Tsai (1968) and Hynes (1960) agreed that chlorinated sewage acts toxically on aquatic organisms. Tsai found chloramines to be more toxic to fish and they retained their toxicity longer than the free chlorine fraction of residual chlorine. He also theorized that DO and pH values, which are employed as primary water quality parameters for stream pollution assessment, actually are not decisive factors for fish mortality in areas immediately below chlorinated sewage outfalls. Although chlorine toxicity studies on stream biota have increased, very few deal with the relative toxicity of chlorine and chloramines. In addition, there is a real lack of quantitative and qualitative measurements of chlorine and chloramine concentrations used in experiments. Lastly, and most importantly, there has been no investigation into physiological mechanisms of chlorine toxicity to freshwater teleosts. Purpose and Sc0pe of Study The purpose of this investigation was to develop on a macrosc0pic level some understanding of the mechanism of chlorine toxicity to freshwater teleosts. Gill tissue was chosen for this study because of the sensitivity of this tissue to toxicants and its close proximity to water born pollutants. Also, even though toxicants may effect a fish through gut or skin, it is more probable that they act on or through the gill and, finally, the physiological aspects of gill tissue are well documented. Five major objectives comprise the basis of this re- search: 1. Establish an estimate of the "normal" tissue respiration rate for a complete gill. 2. Determine effects of a lethal concentration of residual chlorine on the respiration rate of a complete gill. 3. Help reveal whether death by chlorine toxicity is attributable to gill failure. 4. Assist in resolving the location of the primary site of chlorine toxicity. 5. Observe behavioral and physical changes in the test animal. It is aspired that correlation of the five preceding objectives and their results will establish a base from which more in-depth studies into the exact mechanism of chlorine toxicity may be carried out. METHODS Fish Holding and Feeding Advantages in choosing the white sucker Catostomus commersoni (Lacepede) follow: 1. Available from local private ponds. 2. Easily maintained under laboratory conditions. 3. A good test fish: not as sensitive as trout or salmon and not as resistant as carp or catfish. 4. Easy to work with: little fish smell, no spines or pointed fins, lack of teeth, and not excessively slimy. Capture was effected by both glass and wire minnow traps from January to June, 1971. A total of 134 fish were collected and held in a 190-gallon metal tank interiorly coated with a non-toxic grey, epoxy paint. One-third of the tank was covered to afford a place of fish concealment. A single standpipe and one siphon hose provided drainage. Flow rate was about 2 gal per min of filtered water. East Lansing municipal water was passed through a 50-ga1 charcoal and gravel filter and then through a one-gal Nalgene container packed with polyethelene filter floss. The latter became necessary because forceful back flushing of the 50-gal filter tended to disintegrate the charcoal. Two air pumps oxygenated the water through one 11-inch air stone and seven smaller 1-inch stones. PhotOperiod was not a factor because lighting was continually on. The fish received daily feedings of salmon starter food produced by Aktiebolaget Ewos Co. of Sodertaljie, Sweden. The preceding diet was occasionally augmented by shredded frozen horse heart. Toxicant Dilution System The dosing apparatus employed during this study was developed for earlier studies at Michigan State University (Rosenberger, 1971). Rosenberger modified the basic design of Alabaster and Abram (1965) by incorporating a three- way electrical timer, solenoid valves, and various other building materials such as plastics, vinyls, and glass. Filtered tap water piped into an elevated head tank was gravity fed to the constant head vessels. Chronologically, the first valve would open and allow the filtered water to fill the l-liter mixing flask to a level even with the constant head standpipe. Valve two released the toxicant, which finished filling the flask up to l-liter as determined by the height of the toxicant filled Marriotte bottle. Valve three then permitted the 1-1iter of diluted toxicant to flow into the 5-gal test aquarium. The previously described system recycled every six minutes giving a fill time of 2 hr. and a 90% replacement time of 4.5 hr. The latter was more rapid than Sprague's (1969) suggested replacement time of 8-12 hr. The afore- mentioned fill time was well below APHA's (1971) recommended time of 6.5 hr. Three aquaria were utilized in this study. The first aquaria served as an acclimation chamber for the four test fish of any given run. Test fish were acclimated overnight. The following day two fish were placed in the control tank and two into the toxicant tank. Duration of exposure to approximately 1 ppm total residual chlorine was 30 or 60 minutes. Toxicant was made from approximately 10 g of technical grade calcium hypochlorite (Ca(OCl)2) dissolved in 20 liters of deionized, distilled water, which gave a concentration of about 200 ppm. Sulfuric acid helped bring the Ca(OCl)2 into solution. The final solution had a pH of about 7.0, was filtered and placed in a 20-liter Marriotte bottle which, along with the toxicant reservoir, was covered with black plastic to help prevent chlorine breakdown due to light ex- posure. Dissection Procedures Following exposure to chlorine, each fish was pithed through the brain and anterior portion of the spinal column. Both gill membranes were severed anteriorly to a point just forward of the isthmus, which was transversely cut. The isthmus was separated from the underlying gills and pulled posteriorly. Each opercle and cheek was torn and pulled anteriorly and the gills, now exposed laterally and ventrally, were deftly excised taking care not to injure individual filaments. Es0phageal tissue attached to the excised gill was carefully removed. The isolated gills (arch and filaments) were rinsed with distilled water and placed in the respirometer reaction flasks. Gill Tissue Respiration Measurements A Gilson Differential Respirometer employing the constant pressure method of measurement was used to monitor oxygen consumption of gill tissue. Each of the 14 reaction flasks had a capacity of approximately 16-m1. The reference flask, or thermobarometer, was 235 ml. All flasks were cleaned by a modification of the nitric acid method described by Umbreit 35 31 (1964) as follows: l. Soak flasks in gasoline. Remove remaining grease with gasoline on a cotton swab. 2. Wash in a mild Alconox detergent solution; about one tablespoon Alconox per 2 gal water. 3. Rinse well with tap water. 4. Soak in a solution of equal parts H2804 and HNO3 for at least 30 min. 5. Wash several times with tap water. Rinse twice using distilled water. All fittings were then sealed with a high vacuum grease. After randomly choosing four flasks for the test tissue, the remaining 10 flasks and reference flask were prepared. Four ml of distilled water and 6N NaOH-saturated filter paper (displacing 0.5 ml) were placed in each of the remaining 10 flasks. By adding distilled water, the reference flask gas volume was adjusted to approximate the cumulative gas volume of the reaction flasks. All 10 flasks and the reference vessel were then connected to the respirometer. Readying the respirometer consisted of activating the stirring motor, shaking motor and setting the water bath at 23 C. Temperature equilibration was achieved while the test fish were exposed to the toxicant and dissected. Immediately prior to dissection, 4 m1 of Ringer solution (Stokes and Fromm, 1964) was added to each randomly chosen flask. After dissection, prepared gills of each fish were 10 placed in one of the four test flasks. Next, NaOH-soaked filter paper was lodged inside the inner well of each flask. The four vessels were connected to the respirometer. While the entire system equalized for 15 min. prior to the re- cording of oxygen consumption, manometer index lines were aligned and initial micrometer readings set at convenient, uniform values. Data Collection Water Chemistry Approximately once a week pH, temperature, DO, alkalinity, and hardness were quantified for holding, acclimation, control and test tanks. The pH was measured to the nearest 0.1 and temperature recorded to the nearest 0.5 C. Alkalinity, DO and hardness were all measured in accordance with APHA (1965) standards. Chlorine Determination The APHA (1965) method for differentiation of mono- chloramine and dichloramine by amperometric titration was employed for all chlorine determinations. Free chlorine, monochloramine and dichloramine were determined twice for each run, midway through and immediately after exposure. 11 Concentrations were recorded to nearest 0.01 ppm. The amperometric titration apparatus consisted of the following parts. The silver-silver chloride billet type reference electrode was immersed in a saturated NaCl solu- tion, which was attached to the sample cell by a 10% NaCl agar bridge. A readily polarizable platinum electrode was spun in the sample cell. The electrodes were connected to a recorder sensitive to 0.01 milliamps. Fish Data on length and weight were collected subsequent to dissection. Total length was determined to the nearest millimeter. Fish wet weight without gills was measured on a t0p loading balance sensitive to 0.01 9. After moni- toring tissue respiration, wet gill weight was determined to the nearest 0.0001 g on an analytical balance. Total fish wet weight was calculated by adding gill wet weight to wet weight of fish without gills. After drying for 48 hr at 100 C, dry gill weight was determined in the same manner as wet weight. 12 Respiration Rate The Q02 rate is expressed as pl of 02 uptake per mg of dry gill tissue per hour. For six hours, each half-hour cumulative and incremental amount of 02 consumed was re- corded to the nearest 0.1 p1. A correction factor (CF) was applied to each half-hour increment of 02 consumption. This factor was obtained by averaging the fluctuations in the 10 "normal" reaction flasks for each half hour. For example, if average fluctuations of the 10 flasks over a 30-min span was +1.5 pl, this indicated outside factors were increasing all 14 readings to that degree. Thus, 1.5 pl was subtracted from each of the four half-hour tissue readings. If CF were negative, it was added to the 30-min tissue readings. Each corrected half-hour tissue 02 up- take reading was divided by its corresponding gill tissue dry weight and doubled to give the final 002 hourly rate. Statistical Analysis Basic statistics such as means, standard deviation and standard error are presented with the corresponding data in the Appendix Tables. A model I, or fixed effects model, randomized complete- block design with 12 observations per experimental unit was used in this investigation. Covariance analysis was chosen for interpretation of results primarily because the independent variable (total fish weight) fluctuated widely and influenced the dependent variable (002). This analysis was also chosen because it combines the concepts of analysis of variance and regression to furnish a more discriminating analysis than that afforded by either componet (Ostle, 1954). Ostle (1954) and Steel and Torrie (1960) discuss in detail the assumptions, models and mathematical procedures used in covariance analysis. 13 RESULTS Water Chemistry Data and statistical description concerning the five water parameters monitored are presegned in Appendix Table A—1. A summary of means and standard errors for determin— ations of pH, temperature, dissolved oxygen, alkalinity and hardness in holding (H), acclimation (A), control (C) and test (T) tanks is found in Table 1. There were no differences between control and test tanks in parameters quantified (T=0.064, P>;9). Therefore, it was assumed that water quality was constant and not an error factor in the experiment. Chlorine Determination Chlorine and chloramine determinations along with their complete statistical description are in Appendix Tables A—2, A-3. Free chlorine residual usually includes free chlorine, hypochlorous acid and hypochlorite ion whereas combined chlorine residual refers to chloramines (Moore, 1951; Saw- yer and McCarty, 1967). In the present study total residual 14 15 TABLE 1. Range of pH and means and standard errors for determinations of temperature, dissolved oxygen, alkalinity, and hardness in holding (H), acclimation (A), control (C), and test (T) tanks. Tank Temperature D.O. Alkalinity Hardness TYPe PH (C°) (ppm) (ppm CaC03) (ppm CaC03) H 7.5-7.6 l3.40:0.10 6.78:0.36 306.3:5.5 318.7:1.0 A 7.5-7.8 15.20:0.12 7.67:0.14 306.7:3.5 322.3:0.8 C 7.8 17.25i0.14 8.03:0.03 312.0:2.9 321.5i1.0 T 7.7-7.8 17.2010.12 8.15:0.03 319.0:1.3 323.0:1.3 16 chlorine is the sum of free chlorine and combined chlorine residuals. Mean total residual chlorine (ppm) during the 30 and 60 min exposures were respectively 0.970 1 0.024 (S.E.) and 1.008 1 0.033 (S.E.). The two means did not signifi- cantly differ from 1.000 ppm (T=0.094, P>;9). Following pilot studies to determine a toxicant level lethal within one to two-hour exposure, the 1 ppm concentration was chosen. Total residual chlorine was chosen as the measure of toxicant because its concentration could be controlled. Combined chlorine and free chlorine residuals were in a constant state of flux as fish-excreted ammonia united with chlorine to form monochloramine and dichloramine. Full in-depth discussions of chlorine and its chemistry are pre- sented by Moore (1951) and Sawyer and McCarty (1967). By comparing means in Table 2 certain trends may be distinguished concerning the changing proportions of combined and free chlorine residuals with time. The following trends could not be proven significant and thus do lie in the realm of chance. During both exposure periods mean total residual chlorine decreased over time. This suggests a slight overall loss of chlorine, possibly due to an initial chlorine demand of the fish, loss to the atmosphere, or formation of trichlor- amine which could not be quantified. 17 TABLE 2. Means and standard errors(S.E.) for the different chlorine residuals during the 30 and 60-minute ex— posures. Chlorine 30 60 Form Mean and S.E. Mean and S.E. (ppm) (ppm) Total Residual ’ A1 0.995 + 0.036 1.052 + 0.036 32 0.945 I 0.030 0.965 T 0.054 c3 0.970 f 0.024 1.008 f 0.033 Free Chlorine ' A 0.737 + 0.082 0.640 + 0.064 B 0.638 1 0.062 0.582 $'0.075 C 0.688 E 0.051 0.612 5 0.048 Mono- Chloramine A 0.148 + 0.051 0.273 + 0.090 B 0.182 1 0.033 0.228 $ 0.044 C 0.165 f 0.029 0.251 E 0.048 Di- Chloramine A 0.110 + 0.007 0.135 + 0.020 B 0.125 I 0.008 0.155 I 0.012 C 0.118 E 0.006 0.145 5 0.011 éDeterminations midway through exposure. 3Combined determinations. Determinations immediately after exposure. 18 Free chlorine also decreased over time during both exposures. This was expected as free chlorine would react with the excreted ammonia. Dichloramine increased during both exposures probably due to the continuing reaction between monochloramine and hypochlorous acid. The fluctuations of combined chlorine residuals may be due to inability to coordinate chlorine determinations with the 6-min recycling of the toxicant diluter system. When a water sample was being taken for chlorine determin— ation, the dosing apparatus may have been adding fresh toxicant, just finished or just ready to add, etc. Fish Data on total length, weight, wet gill weight and dry gill weight along with pertinent statistics are recorded in Appendix Tables A-4, A-5. Total length for all test fish ranged from 80 to 185 mm. Total weight ranged from 3.25 to 52.21 g. The large variation in size of experimental fish was unavoidable due to the collecting method. The wire minnow traps selected against only very large and very small fish. Since fish size is closely related to metabolic rate (Fry, 1957; Muir and Hughes, 1969; Prosser e£_al, 1952; Winberg, 1960), the wide range in size dictated the choice of an 19 appropriate statistical analysis. Inspection of Table 3 shows no differences (T=0.08, P>.9) between means for total length, weight and gill dry weight in the 30-min group and those in the 60-min ex- posure. In addition, both groups showed no differences (T=0.03, P>.9) between the means of test and control fish measurements. Respiration Rate Appendix Tables B (1-12) present data on correction factors (CF), fish weights, half-hour oxygen uptake readings and the corresponding calculated 002's. Inspection of these data reveals two main relationships. First, the total volume of oxygen consumed by gills varies directly with total fish weight. Secondly, there is an inverse relation- ship of Q02 to fish weight. These two observations agree ‘ with those of Winberg (1960). In addition, it is also generally apparent that all gills tested remained viable over the six hours and that Q02 and oxygen uptake were fairly constant, decreasing less than 10 percent over time. 20 TABLE 3. Means and standard errors (S.E.) of total length, total weight, and dry gill weight for test(T) and control (C) fish exposed for 30 and 60 minutes. 30 60 Measurement Mean and S.ET' Mean and S.E. Total length (mm) T 119.6 + 5.0 115.2 + 8.0 C 116.5 E 3.8 114.7 E_7.2 Total weight (g) T 13.097+ 1.806 13.469+ 4.053 c 12.1293 1.124 11.7135 2.818 Gill dry weight (mg) T 33.17 i 4.98 32.917: 5.397 C 33.08 i 3.49 29.917: 3.736 Statistical Analysis Individual fish 002 means, total weights and corre- sponding logslo appear along with pertinent statistics in Tables 4 , 5 . A preliminary two-way analysis of variance ignoring fish weight differences was performed to test the hypothesis that all four treatment means were equal; H0:Y1=Y2=Y3=Y4 (Table (1). The null hypothesis was ac- cepted, inferring that all treatments were from the same population. After initial examintion of scatter diagrams plotting 002 against fish weights and Q02 against time, it was hypo- thesized that logarithmic transformation of Q02 and weight would provide a better fit. With the transformation to loglo (Tables 4 , 5 ) , the correlation coefficient (R) was increased from .60 to .71 and the coefficient of determina- tion (R2) increased from .36 to .51. The mathematical covariance model employed was: Yijk=u+ti+sj+(ts)ij+BXijk+eijk where Yijk=logloof mean 002 reading for fish k, for fish type i (test or control) and strength j 21 22 mnsa»o nonuno ~mom.a wao.o H¢~H.H oamo.o hmmo.o mhvm.o thKmv .m.m memo.o mmma.ma mamo.o HHhm.o mmmo.o mmma.mm mmvo.o mmmn.o A mv .Q.m Homo.H HmNH.~H vmva.o voom.H ommo.a wmmo.ma meH.o ommm.a «mv cmmz vmhm.o m¢.m memm.o oHHH.~ Hwom.o >~.m mvm~.o movm.a mmHo.H. mv.oa Noom.o hmmm.H HHQH.H vm.ma mHNH.o ommm.a quEmummm v HwNN.H mm.ma nmmo.o mm-.H meom.a ma.o~ memo.o hmHN.H mm-.H mw.wH omma.o N~m¢.H mmHu.H om.m~ mhma.o omvm.H umnfimummm H Hmnn.o mm.m omav.o HNmm.~ mmmh.o on.m vomm.o moov.m ommh.o Ha.m mmmm.o mmmv.~ mHHm.o mN.m osmm.o mmmm.m umesd om mmNH.H mm.mH mmmo.o omm~.H Nmmm.o No.m ommN.o mmhm.a memo.a Hw.HH mmoa.o mom~.H mmmo.a NN.HH mmNH.o mmwm.a umsms< mm maqo.a Ho.HH mma~.o mamm.a vaH.H m~.va mmmo.o omea.a mth.H Hh.va mvno.on umwm.o ~0oa.a hh.NH v~m~.o mnon.a umsmsd 5N Heno.a mm.HH mmHH.o1 mmwb.o mmmo.a mH.~H heed.01 Nth.o mmvm.a Nm.hH mamo.o| vmmm.o mmmm.a mH.mH mmma.on anon.o umsms¢ N Ahma unwflmz unmam3 cmmz cam: unmwmx unmwmz com: com: mumn swam mos swam moo mos moo swam mos swam «00 mos moo omIU omue .mHSmomxm mussHEIom some um swam AUV Houusoo 03» can ARV ummu o3u new Amy musmflmz nmau can magma No0 mo mcowumEH0mmcmuu camoq .v mange 23 .GOwuuom comm co momE mGOHDMCwEHmumc moo .03» 2H uso madam “momma oou nmflme tho.o vmam.~ mamo.o moHH.o. oooa.o mmmo.e mneo.o mmva.o om»\mv.m.m mamo.o vamm.hm mmao.o ommH.o Homa.o ~mmo.oma mnmo.o mmmm.o A my .Q.m ommo.a hNHh.HH moma.o mmwm.a anho.a omw¢.ma woom.o ~mo>.a Amy cmmz onwm.a mvoo.o1 nmmm.o th~.H mmmfl.on nomw.o osmm.a «om.mm hHHH.o mmm~.H whah.a «H~.~m homo.ou mamm.o umsmsd em ommm.o oo.m mmmm.o mNNH.N vmnm.o mh.¢ vomm.o memm.~ vmmm.o m~.v hmmm.o HHhH.N monm.o mo.v Hmmm.o o¢HH.~ umsmsm mm vaeo.a oo.HH omma.o mmvm.a ommo.H mm.~a mmmm.o mmam.a ohmo.a mm.OH oNHH.o Nvm~.H v~¢H.H mm.m~ mma~.o Hmww.a umsmsm mm ehmm.o o¢.m «mma.o mmhm.a -oo.a mo.oa mmma.o omhm.a mmom.o wo.m vmha.o mHHm.H mmmm.o mo.m onma.o mmmv.a umsmsg Hm mmom.o om.m vmom.o mvom.H omam.o m~.m avom.o mvao.m wmom.o mH.m NamH.o Hamm.H Hmom.o vm.w maam.o nuvo.~ umsms< oH mmmm.o mh.w maem.o Hmma.m mamm.o vm.m voem.o omma.~ Nmom.H oa.o~ mmeo.o HmHH.H mom~.H ~m.na omnm.o ohmm.a hasn mu Huma unmwmz unmflmz cam: com: unmwmz unmamz com: com: mama gnaw moq mumm, woo mom moo smmm moq nmmm moo moq moo omuu owns .mnsmomxm muscwfilom some um swam ADV Houucoo 03» can ABC ummu ozu mom Amv mungwm3 :mflm can magma moo mo msoflumEH0mmcmuu camon .m mamma 24 TABLE 6. Two-way analysis of variance testing the effects of chlorine exposure (30 and 60 minutes) and fish type (test and control) upon gill tissue 002 without regard for fish weight. Degrees of Sum of Mean sum Source freedom squares of squares F P1 Exposure 1 0.0530 0.0530 0.138 .50‘rupas pesto: a sore mmPQEam Lao» mcwzmcu an mmzpm> -l tuneup mcwc_mpao we mmwu_p_amaoca_ mo.¢ u mme.PHmo a Ef A. voma N_.o oae_oo.o oaepoo.o _ =o_suaems=a mxp umumzncm mcwummu LP... mucmgmmazo somamm.o as oammm~._ mmmamm._- mmmam~.m me m+mxc &om.v a meQ _N.P meaepo.o amaeso.o F memos gumcmgum umpmsnem mcwummp Lo... mucmgmwh—E momoem.o as ommFNN.F oemmew..- aoemm~.m me u + m any 2 v9.3 mF.F mmeepo.o mm¢¢_o.o P memes “swagger“ umumanue m:_ummp so» mucmgmwms ~aeoem.o as oomoe~.F mem_mm._- maawm~.m me u + c mmmwpo.o mmoomm.o me emmmm~._ emmmmm._- anammm.m as «my aorta Neoooo.o ameooo.o semeoo.o F xmxpv .Lap=H ammmpo.o _eoeoo.o- mmmooo.o _ Amv gsmcmtpm mooaoo.o mmeooo.o mammoo.o F lav mash call mammkm._ Neemmm.1- somee~.m as Peach 1 m: mm .Hm IN» xx Nx .Hm mascara) x Low umumzwum > muuzueq $0 Ezm Lb mugaom .m .e mmpnm» cw aunt com mucmwrm>oo we mwmxpoc< .u m4m.75. This infers that acceptance of H0:BZ=B4=B5 is reasonable. Under the assumption that the three regression 32 TABLE 8. F-tests for difference between two regression coefficients; test fish = (1), control fish = (C), exposure time = 30 or 60 minutes. Treatment comparison Calculated F-value P1 C-30 VS. T-30 0.59 .25