THE CALCULATION OF ACUTE TOXICITY OF FREE CHLORINE AND C'HLORAMINES T0 COHO SALMON BY MULTIPLE REGRESSION ANALYSIS Thesis for the Degree of M ., S. MICHIGAN STATE UNIVERSITY DAVID R. ROSENBERGER 1971 LIBRARY University IIIIIIIIIIZIIIILIIIILIIIIIIIIIIIIIZIIIIIIIII ~ I, .- . Michigan ‘7 swims av T? . HUM; & SUNS'E" i 30% ”marl“. t'rfi.‘ .17 BINDERS ABSTRACT THE CALCULATION OF ACUTE TOXICITY OF FREE CHLORINE AND CHLORAMINES T0 COHO SALMON BY MULTIPLE REGRESSION ANALYSIS By David R. Rosenberger Coho salmon, Oncorhynchus kisutch (Walbaum), were exposed to con- centrations of free chlorine, monochloramine, and dichloramine in hard water (total alkalinity of 321 mg/L as CaC03) and at a temperature of 16 C. The median response-time (LTSO) was determined by probit analysis and an equation to predict the LT value was then calculated by multiple 50 regression analysis. The equation formed was f - 2.19123 - 49.46504F - 5.61668M - 16.90604D + 115.80039FM + 715.71820FD + 55.3237MD - 1514.80481FMD - 1.68220(1og10)w, where T - loglo median time of death (hrs + 1); F - free chlorine; M - monochloramine; D = dichloramine; and W - fish weight. The equation formed was 95.98% confident of predicting the true LT50 for the samples studied. The equation indicates that free chlorine is the most toxic form of chlorine studied followed by dichloramine and monochloramine in respective order. Graphical representation of the degree of toxicity attributable to each form of chlorine suggests that monochloramine has a different mode of action from that of either free chlorine or dichloramine. David R. Rosenberger Three dimensional graphs are drawn to show all combinations of free chlorine, monochloramine, and dichloramine necessary to kill 50% of a sample of fish for both 24 and 96 hr. 'The graphs indicate that low con- centrations of chlorine (0.20 ppm total chlorine) may be fatal to fish. However, fish are able to withstand high concentrations (1.00 ppm total chlorine) when the chlorine to ammonia ratio is approximately 2:1 (break- point chlorination). Chlorine dioxide, trichloramine, nitrous oxide and organic chloramines may be present at break-point. One or more of these chemicals may act as an inhibitor of chlorine to enable a fish to survive the high concentrations. Small fish (0.5 gm) survive longer than larger fish (1.0 gm) at any given concentration of chlorine. This is thought to be a result of the gill surface area-body weight ratio. Small fish have a proportionately greater gill surface area for their size than a large fish. THE CALCULATION OF ACUTE TOXICITY OF FREE CHLORINE AND CHLORAMINES TO COHO SALMON BY MULTIPLE REGRESSION ANALYSIS By David RIWRosenberger 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 would like to express my thanks to all the people that contributed to my research through their help and comments. I give.special thanks to Dr. E. Roelofs, Dr. H. Johnson, and Dr. W. Porter, the members of my graduate committee, for their continued assistance. Lastly, I thank FWQA Training Grant 5Tl-WP-109 and IT3-WP-264 for their support in this research. ii INTRODUCTION . . . . . . . Water Analysis. . . Dosing Apparatus. . Test Fish . . . . RESULTS. . . . . . . . . Fish Mortality. . . Multiple Regression DISCUSSION AND CONCLUSION. LITERATURE CITED . . . . APPENDIX 0 O O O O O O O 0 TABLE OF CONTENTS Analysis. . . . . . iii Page 10 10 ll 18 32 34 Table LIST OF TABLES Chemical characteristics of experimental water. . . . . . . Experimental concentrations of chlorine, median response- time (LT50), and fish weight employed in the calculation of the regression equation. . . . . . . . . . . . . . . . . Analysis of variance and correlation coefficients for the overall regression. . . . . . . . . . . . . . . . . . . . . Regression coefficients and statistical analysis of bi' . . Observed median lethal time (LTSO), estimated median lethal time (LTSO), and error of estimation. . . . . . . . . . . . iv Page 12 15 16 17 Figure LIST OF FIGURES Amperometric titrator used in the analysis of chlorine. Diagram of dosing apparatus . . . . . . . . . . . . . . . . Estimated median lethal time (LTSO) of l-gm coho salmon exposed to concentrations of free chlorine, monochloramine, and dichloramine. . . . . . . . . . . . . . . . . Contour map of the amount of free chlorine necessary to produce a 24-hr TLSO to l-gm coho in concentrations of monochloramine and dichloramine . . . . . . . . . . . Contour map of the amount of free chlorine necessary to produce a 96-hr TL50 to l—gm coho in concentrations of monochloramine and dichloramine . . . . . . . . . . . . . . Three dimensional response surface of the amount of free chlorine necessary to produce a 24-hr TL50 to l-gm coho in concentrations of monochloramine and dichloramine. Three dimensional response surface of the amount of free chlorine necessary to produce a 96~hr TLSO to l-gm coho in concentrations of monochloramine and dichloramine. Median lethal time (TLSO) to a l-gm coho salmon for combina- tions of monochloramine and dichloramine (—-- addition, -——- TLSO) O O O O O O O O O O O O O O O O I O O O I 0 Calculation of the median lethal time (LT50) of coho salmon exposed to concentrations of free chlorine, mono— chloramine, and dichloramine. Resistance time is plotted against the percentage mortality (probit scale) Page 19 22 23 25 26 28 34 INTRODUCTION Chlorine has gained widespread usage in many areas in which a strong oxidizing agent or disinfectant is essential in a manufacturing process. Industries which employ chlorine as an oxidizing agent are tanneries, textile mills, pulp mills and paper factories. Sewage treatment plants use chlorine as a disinfectant in their final effluent to prevent the spread of water-borne diseases. Electric power plants utilize chlorine as a scrubbing agent to discourage the formation of slime growth in the cooling columns. Sawyer and McCarty (1967) present an excellent description of the chemistry of water chlorination. Chlorine combines with water to form hypochlorous and hydrochloric acids: Cl2 + H20 2 HOCl + HCl (1) The hypochlorous acid can dissociate as a function of pH and, to a lesser extent, of temperature: HOCl z H+ + 001‘ (2) Chlorine, hypochlorous acid, and hypochlorite ion are collectively referred to as "free chlorine residuals". Chlorine is also added to water as either the sodium or calcium salt of hypochlorous acid whereby it dissociates to form the hypochlorite ion: NaOCl + Na+ + 001- (3) Ca(oc1)2 + Ca-H' + mm" (4) This ion then establishes equilibrium with a hydrogen ion as shown in equation (2). 2 Chlorine and hypochlorous acid react readily with a wide variety of impurities in water. They combine with reducing agents and organic matter which decreases the amount of free chlorine in the system. The amount of this reduction is described as a ”chlorine demand". Ammonia, another chemical commonly found in natural bodies of water, reacts with free chlorine to produce monochloramine, dichloramines, and some tri- chloramines. These are collectively referred to as ”combined residual chlorine": NH3 + HOCl + NH2C1 + H20 monochloramine (5) NHZCl + HOCl + NHCl2 + H20 dichloramine (6) NHCl2 + HOCl + NCl3 + H20 trichloramine (7) The relative amounts of monochloramine and dichloramine produced are determined by the ratio of chlorine to ammonia in the system. At a 1:1 ratio, monochloramine will be the predominant chloramine; as the chlorine to ammonia ratio approaches 2:1, dichloramine becomes the dominant form. When the chlorine:ammonia ratio reaches 2:1, trichloramine is formed and part of the ammonia is oxidized to free nitrogen and nitrous oxide (N20). The formation of the accessory reactions is referred to as "break—point chlorination". The early words on the effect of free and combined chlorine on aquatic life have been summarized by Doudoroff and Katz (1950) and McKee and Wolfe (1963). These authors indicate a wide discrepancy in reports of the degree of toxicity of these compounds. The values range from a concentration of 5.0 ppm cited as being nonfatal to goldfish to a concen- tration of 0.05 ppm claimed to kill trout in 48 hr. Much of the work was conducted under static conditions and little distinction was made in the difference in toxicity of free residual chlorine and combined residual chlorine. 3 Merkens (1958), working with rainbow trout (SaZmO gairdherii Richardson), attempted to differentiate the chlorine forms and assess their respective toxicities. He concluded that the toxicities of free and combined chlorines were all of the same order--free chlorine was the most toxic, followed by dichloramine and monochloramine respectively. Merkens speculated from his studies that chlorine in a concentration of 4 ug/L would in one year's time be fatal to 50% of a sample of test fish. Holland at al. (1960) reasoned that the degree of toxicity with respect to time, placed dichloramine first, chlorine second, and mono— chloramine last. He also expressed the Opinion that dichloramine and monochloramine "must be considered as different causative agents... since a smaller amount of dichloramine killed more rapidly than a larger amount of monochloramine". The objective of the present study is to derive a predictive equa- tion that will estimate the median survival time of a group of fish to any combination of free chlorine, monochloramine, and dichloramine; and to determine the degree of toxicity attributable to each form of chlorine. The test fish employed was the coho salmon, Oncorhynchus kisutch (Walbaum), exposed continuously to varying concentrations of free chlorine, mono- chloramines, and dichloramine. MATERIALS AND METHODS Water Analysis The source of water used in this experiment was obtained from the East Lansing, Michigan, potable water supply, filtered through a 50-gal water filter containing activated charcoal and gravel. During the course of the experiment the filter was backflushed at least once a week until all visual traces of filtrate had been cleared. Routine water-chemistry analysis was conducted at least once for each test concentration studied (Table l). Alkalinity, total hardness, and dissolved oxygen were measured using standard methods of the American Public Health Association (1965). Water temperature was measured to the nearest tenth (0.1) of a degree Centigrade with a hand thermometer and the pH determined to the nearest tenth (0.1) of a pH unit on a Leads- Northrup pH meter. Ammonia determinations were conducted by direct nesslerization (APHA, 1965). Water samples were collected prior to the introduction of chlorine to guard against interference from organic chloramines. This procedure was checked periodically by the distillation method to insure accuracy. Analysis for heavy metals was conducted at the end of the experiment on a Jerrell Ash model 82-800 atomic absorption instrument. The recorded values were as follows: element Fe in Cu Cr Pb mg/L _o> Eocflom N 50.5 coo.“— o» I N JL 3 N 4. Ln = _ comEopa 9.32 . .2 4 a 2:32.05 .5 o H £2.33 m o u mtofiofi sotmmmm u ........... II F 3 om /. JL L H _0wmo> I I I. IIIIIIIIIIIII I Om a _E 0: $6“ch 30 c J L . 52—h?” 8:25 8 polyethylene plastic, tygon tubing, stainless steel solenoid valves and a three-way electrical timer were used in the construction. Filtered tap water was piped into the head tank and then gravity fed to the constant head vessel. The solenoid valves opened and closed as indi- cated in the numerical sequence. Valve I allowed the filtered tap water to flow into the mixing chamber up to the same height as the constant head standpipe. Valve II permitted the toxicant to complete the filling of the mixing chamber to a volume of l L as determined by the height of the Mariotte bottle. Valve 111 then delivered 500 ml of the toxic solu— tion to each of the duplicate 5-gal test tanks. The electric timer was set to recycle the system every 6 min giving a fill time of 4 hr and a 90% replacement time of 9 hr. This was within the 8- to 12-hr range suggested by Sprague (1969) and the 4-hr fill time was below the 6 hr recommended by APHA (1971). The source of chlorine used in the experiment was a 5% aqueous solu- tion of reagent grade sodium hypochlorite. This solution was diluted with double distilled water to represent 100 ppm and placed in the Mariotte bottle. Both the Mariotte bottle and reservoir were covered with a black plastic sheet to protect the chlorine from light. The experiment was divided into two parts, the first with only chloramines present, and the second with mixtures of free chlorine and chloramines. To produce only chloramines, reagent grade ammonium chloride was added to the dilution water at a rate sufficient to give 1 ppm ammonia as N. Varying the concentration of chlorine formed dif— ferent monochloramine:dichloramine ratios. The second portion of the experiment was conducted using only the ammonia normally found in the tap water (0.15-0.20 ppm as N). With these low ammonia:chlorine ratios, free chlorine was formed along with the chloramines. Test Fish Lake Michigan coho salmon eggs were collected from the Platte River Hatchery and brought to the Michigan State University campus where they were hatched. When the swim-up stage was reached, the fish were fed salmon starter food produced by the Aktiebolaget Ewos Compant of deertaljie, Sweden. The fry were transferred to the experimental holding tank and at this time measured approximately 4 cm total length and had a wet weight of 0.42 gm. Water in the holding tank was obtained from the same source as that in the rearing tank. The experiment was begun one week after the transfer. Concentrations of toxicants were run in duplicate. Each concentra- tion was randomly assigned one of the six dosing units to minimize the possibility of position effect. Ten fish were used per test concentra— tion with half placed in each of the duplicate test tanks. Numbered slips of paper drawn at the same time a fish was removed from the hold- ing tank determined its destination. As each fish expired, the time of death was recorded and the dead fish immediately removed to a formalin solution. Death was defined as the point at which mild stimulation would not elicit a response. The total wet weight of all fish was then taken and the mean fish weight determined. Time and percentage effect were plotted on logarithmic-probability paper and a straight line fitted by eye. Estimates of the median response- time (LTSO) and its 95% confidence limits were calculated from a nomo- graph according to Litchfield (1949). A predictive equation of the median survival time for combinations of free chloramine, monochloramine, and dichloramine was then derived by multiple regression analysis. RESULTS Fish Mbrtality When the fish were placed in the toxic solutions, an immediate jaw-snapping response was observed. This response appeared to be an effort by the fish to rid the gills of the irritant. Fish in high con- centrations of total chlorine had a greater rate of jaw-snapping than did fish in low concentrations. The fish were also excitable and showed a high degree of random movement. Fish in the control tanks did not demonstrate either of these characteristics and their behavior patterns were not noticeably different from those of fish maintained in the holding tank. The fish showed signs of listlessness under continued exposure and had difficulty maintaining their position in the water. Without ceaseless swimming they would tend to sink to the bottom; and they had to swim upwards continually to maintain a position in the tank. At the onset of this condition, the fish were near the bottom of the tank; but as the symptoms intensified, they moved toward the surface. Simul— taneously, a slight production of mucus was seen forming on the ventral pairs of fins and, to a lesser degree, over the entire body surface. Abdominal contractions were also noted on the stressed fish. This first contraction presented a constriction of the entire abdominal cavity. Later, as the fish began moving towards the surface, there became visible a peristaltic contraction which originated at the posterior portion of the abdomen and progressed slowly forward. The 10 11 speed and magnitude of these contractions increased and culminated in violent regurgitation and head shaking. Subsequently, the fish lost all control of their equilibrium, alternating between erratic swimming and quiescence. This was followed by a complete cessation of movement and, finally, death. The LT50 values and their 95% confidence limits are presented in Table 2. Corresponding to each LT 0 is the concentration of free chlorine, 5 chloramines, and total chlorine used in the trials. The ranges in con- centration of each chlorine fraction in this study are: free chlorine - 0.000 to 0.280 ppm monochloramine — 0.053 to 1.280 ppm dichloramine - 0.004 to 0.093 ppm Multiple Regression Analysis At the onset of this experiment it was not known whether the various forms of chlorine had interactions which might affect their combined toxicity. The relationship of chlorine to the time of death for various sizes of fish was also an unknown factor. These unknown factors made advisable the inclusion of as many variables as possible to analyze their contribution to the predictive model. The independent variables considered for regression analysis were: Xl= F (free chlorine) X7 = F-M-D X2a M (monochloramine) X8 = F2 X3= D (dichloramine) X9 = M2 X = FoM X = D2 4 10 X5: F-D x11: W (fish weight) X = M-D X = W2 6 12 12 00.0 00.0 000.0 000.0 000.0 000.0 0.0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0.0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0.0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.000 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0.0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0.0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 00.0 00.00 000.0 000.0 000.0 000.0 0.0 00.0 00.00 000.0 000.0 000.0 000.0 0.0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.0 000.0 000.0 000.0 000.0 0-0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 00.0 00.00 000.0 000.0 000.0 000.0 0-0 000 0.000 00\0sv A0\0a0 A0\0a0 A0\0av 00000 uanmB swam omHA mGHEmuoasoHQ mafiamuoanuoaoz mawuoazo mwum oawuoaso HmuOH aofiumnvm cowmmmuwmu ecu mo GOHumH300mo mnu s0 pmzoaaEm unw0o3 nmflw mam .AomHAV mefiulmmcommmu defipoa .ocfiuoHno wo mc00umuucoocoo amusmafluomxm .N nanny 13 The overall regression equation that predicts the time of death to a papulation of coho salmon exposed to varying concentrations of free chlorine and chloramines is expressed as: Y = BOXO + lel + 82X2 + ... + 812x12 The dependent variable (Y) is the time of death in hours for 50% of the population. Free chlorine, chloramines, and weight of the fish are the independent variables (X's). For a population sample the equation is: Y 8 b0 + lel + b2X2 + ... + blZXIZ The estimate of time of death for the sample is Y. If a variable did not contribute significantly (0 = .1) to the regression equation, it was deleted and the equation recalculated. The data were analyzed in four stages, employing as the linear model: Y vs x's (1) and as log transformed models: log (Y + 1) vs X's (2) Y vs log (X + l)'s (3) log (Y + 1) vs log (X + 1)'s (4) The final equation was chosen for its ability to predict Y with the smallest error of estimation. Transformation (2) best met this criterion. The final equation is given as: Y = 2.19123 - 49.46504F - 5.61668M — 16.90604D + 115.80039FM + 715.71820FD + 55.33237MD - 1514.80481FMD - 1.68220 (loglO)W* *Fish weight appears in the equation as a normalizing factor to account for the variation of the LT50 resulting from a change in fish size. Log weight is used in the final equation instead of arithmetic weight. Experimental trials using 3.0 gm fish indicated the time to death of fish was related to fish weight by a log function. The results from 14 The analysis of variance for the overall regression is given in Table 3. From Table 2, 28 observations were used to form the predictive equation. Three observations could not be used for analysis since no death was recorded, which made an estimate of the LT impossible. F 50 is an F-statistic for testing the null hypothesis that the entire group R of independent variables (X‘s) does not account for any of the variation in the dependent variable. The FR—statistic in this experiment was 56.7290 which had an approximate significance probability for FR of <0.0005. Table 3 also lists the correlation coefficients of the regression equation. R2, expressed as a percentage, reflects the amount of the variance of Y accounted for by the variance of the X's. Thus, in the present equation, R2(100) = 95.98%, which represents the percent reduc- tion in the sum of squares of Y attributable to the combined effect of the X's (Steele and Torrie, 1960). The multiple correlation coefficient (R) measures the combined effect of the independent variables (X's) on the dependent variable (Y) (Steele and Torrie, 1960) and is essentially the corre— lation of all the independent variables with the dependent variable. The regression coefficients are given in Table 4 together with their accompanying statistics. The standard error of the coefficients is calculated as one standard deviation. The beta weights are the con— tributions attributable to each of the independent variables in explain- ing the variance of the dependent variable, while holding the remaining Xi's constant. An Fb -statistic for each of the betas is given together i the 3.0 gm fish weight trials were not incorporated in the equation, however, since the chlorine concentrations in the test tanks could not be held constant using fish of this size. 15 Table 3. Analysis of variance and correlation coefficients for the overall regression Analysis of Variance Sum of Degrees of Source Squares Freedom Mean Square FR Sig. of FR Regression (about mean) 6.17614488 8 0.77201811 56.7290 <0.0005 Error 0.25856893 19 0.01360889 Total (about mean) 6.43471381 27 Multiple Correlation Coefficients R2 R 0.9598 0.9797 2 with accompanying significance probability of F R delete is that b . portion of the sum of the squared deviations forithe mean of the depend- ent variable which can be accounted for by all the independent variables (including b0) except variable Xi (MSU Agriculture Experimental Station, description No. 7. LS; unpublished data). The experimentally observed LT50 values are listed in Table 5 with their transformed log (Y + 1) values. The estimated median time of death, as predicted from the regression equation, is also given with its error of estimation [10810(Y + 1) - est. loglO(Y + 1)]. l6 .uz Aoawoav qqaom.o mooo.0v mmam.oq macm0.o mooqm.o ommmo.HI omqmm.o 000.0 moqm.oa mumma.oal mNHMH.mom quow.qamal szxm omomw.o mooo.ov mmqm.am ommmo.~ m0000.0 NMNmm.mm Q02 mmmmm.o moo.o mwmo.m omqoo.m mawoo.000 omwam.mam me mmHHm.o mooo.0v omno.~m ommoo.oa mmomm.0m mmoow.maa zxm Hmwmm.o mooo.ov m0mo.mw wamm.on 000mm.0 qooom.oal m wwomm.o mooo.0v 0000.00 qmcmm.m1 Hammo.o wooao.ml z mmamm.o moo.o a0mm.m mammq.m| m0mm~.m~ qomoe.mql m II mooo.0v enmq.mom In wmmm0.o MNHmH.N ucmumcou mmumaoa m cm mo .m0m om muzwfimz wumm mquHUHMMmou mo muso000mwooo moanm00m> N uouum mumwsmum scammmummm 0a mo mammamam Hmu0um0umum can quM0oflwmmoo COHmmmuwmm .q mHDmH 17 Table 5. Observed median lethal time (LTSO), estimated median lethal time (LT50), and error of estimation Estimated LT50 Estimated LTSO (hr) (hr) Error of Log Trial y (log y + 1) y (log y + 1) Estimation 1-2 22.00 1.36173 17.02 1.25658 0.10515 1—3 57.50 1.76716 89.96 1.95886 -0.19l70 1-4 7.20 0.91381 10.18 1.04854 -0.l3473 1-5 9.35 1.01494 11.61 1.10611 -0.09117 2-1 50.00 1.70757 57.38 1.76626 —0.05869 2-2 65.50 1.82282 55.49 1.75201 0.07081 2-3 50.75 1.71391 53.28 1.73466 -0.02075 2—4 21.75 1.35698 11.20 1.08645 0.27053 2-6 31.50 1.51188 22.94 1.37910 0.13272 3—1 11.25 1.08814 12.28 1.12335 -0.03521 3-2 4.40 0.73239 5.39 0.80571 -0.07332 3—3 41.00 1.62325 37.55 1.58604 0.03721 3-4 5.10 0.78533 5.71 0.82671 -0.04138 3-5 112.50 2.05499 81.51 1.91650 0.13849 3—6 6.70 0.88649 7.58 0.93351 -0.04702 4-1 8.33 0.96988 11.76 1.10636 -0.13648 4-2 1.05 0.31175 1.05 0.31258 -0.00083 4-4 2.50 0.54407 2.10 0.49176 0.05231 4-6 5.60 0.81954 5.25 0.79579 0.02375 5—1 7.62 0.93551 6.72 0.88745 0.04806 5—2 1.22 0.34635 1.22 0.34576 0.00059 5-4 2.38 0.52892 1.74 0.43739 0.09153 5-6 3.63 0.66558 3.58 0.66048 0.00510 6-2 3.57 0.65992 4.99 0.77792 -0.11801 6—3 10.90 1.07555 9.04 1.01640 0.05915 6-4 4.69 0.75511 5.25 0.79609 -0.04098 6-5 6.25 0.86034 6.98 0.90196 -0.04162 6-6 1.50 0.39794 1.52 0.40152 -0.00358 DISCUSSION AND CONCLUSION The equation formulated is 95.89% confident of predicting the LT50 for the coho salmon used in this study. The equation also enables one to make inferences of the degree of toxicity and the interactions between the various forms of chlorine comprising the toxicity. It is recognized that extrapolation of the data beyond the ranges of concentrations of chlorine and fish weight used in the experiment may not be statistically sound. The regression equation was formulated to fit data within a specific range and combination of free chlorine, monochloramine, dichlor- amine, and fish weight. Deletion of one or more X variable results in a marked increase in the error of estimation as was shown by the R2 deletes of Table 4. Recognizing these limitations, such extrapolations will be presented as hypotheses along with the following discussion. By equating two of the forms of chlorine tested to zero, and plot— ting the remaining chlorine concentration vs time to fish death, the toxicity of the remaining chlorine can be calculated (Figure 3). Such an analysis suggests that free chlorine is the most toxic form of chlorine. Dichloramine appears to be slightly less toxic than free chlorine and has a similar slope. This agrees with that preposed by Merkens (1959) in his study of the toxicity of chlorine and chloramines to rainbow trout. The amount of free chlorine and dichloramine required to kill the fish studied by Merkens is not comparable to that used in the present study. The two studies were conducted at a different pH and, as Merkens showed, pH affects the degree of toxicity of the chlorines. 18 l9 .mswamuoanofiv mam .mswamuoanoosoa .oaauoano moum mo macaumuuamusoo ou wmmomxo 0050mm onoo amid mo Aomagv oEHu Hmfiuoa smfiwoa voumafiumm .m muawfim AEQQV cozotcoucou 02.0 m N 0.0 09.0 mm 0.0 omod m «0.0 0 .2 1 _ T 0.. . u. . 0 1.. 0 $00 5 .-._ L30 9 ) u: Ian ..J loo ( 20 Monochloramine is the least toxic of the three forms of chlorine studied. The slope of the toxicity curve for mhnochloramine differs greatly from those of free chlorine and dichloramine. This difference suggests that the mode of action of monochloramine differs from that of free chlorine and dichloramine. It may be postulated that monochloramine acts as a receptor* site in the organism which is not the same as that for free chlorine and dichloramine. On the basis of this hypothesis, it can then be concluded that free chlorine and dichloramine, with similar slopes, Operate on the same or homologous receptors. Merkins (1969) postulated that monochloramine was the least toxic form of chlorine, but calculated that its slope was the same as that for the other forms. Holland et a2. (1960) considered monochloramine and dichloramine to be "different causative agents...since a smaller amount of dichloramine killed more rapidly than a larger amount of monochloramine". It is difficult to determine from Holland's data the meaning of "causative agent", since no definition is included in the text. A "causative agent" suggests that the monochloramine and dichlor— amine have a different mode of action, but the same hypothesis could result if the two substances had a different degree of toxicity. The oxidative and reactive properties of chlorine to ammonia may partially explain the difference in the mode of action of monochloramine. The oxidative and reactive properties of the chlorines could result in the mass destruction of cells in gill tissue and fins, as reported by Mann (1950). Chlorine (free chlorine, monochloramine, or dichloramine) that did not become bound to the cell membranes would pass to the * A receptor is defined in this text as being the element (an ion, molecule or tissue) in the living organism with which the chemical combines. (See also - Goldstein at al., 1969; or Lewis, 1970). 21 interior of the cell. Here ammonia produced by the fish would combine with the free chlorine and dichloramine and convert them to monochlor— amine. This would mean that monochloramine would be the form of chlorine found inside the fish. Green and Stumpf (1946) postulated that the receptor site of chlorine in bacterial cells is an enzyme involved in the oxidation of glucose. Knox et a1. (1948) later confirmed that the effect of chlorine is the result of its oxidative action of the sulfhydryl groups of enzymes. Monochloramine, as the only form of chlorine found inside the fish, would be the form involved in enzyme action. The equation predicts that there are two-way and three-way inter- actions among the three forms studied. To analyze the significance of these interactions, the equation was solved for combinations of free chlorine, monochloramine, and dichloramine necessary to kill 50% of a test sample of l-gm fish in 24 and 96 hr. [Solving the equation for a specific time is equivalent to the tolerance limits of 50% survival (TLSO) described by APHA (1971).] Time of death and fish weight were held constant; monochloramine and dichloramine were incremented by units of 0.050 ppm and 0.005 ppm respectively; the equation was solved for values of free chlorine. Calculations were made with monochlor- amine ranging from 0.00 ppm to 0.65 ppm and dichloramine from 0.00 ppm to 0.10 ppm. This resulted in a grid pattern of 294 values of free chlorine for both 24 hr and 96 hr. The grids were then contoured for values of free chlorine between 0.00 ppm and 0.30 ppm (Figures 4 and 5). The equation predicted that concentrations of free chlorine were possible above and below the values contoured. These values are not included in the figures, since negative concentrations are not possible and concen— trations above 0.30 ppm were not used in the experiment (i.e., they are outside the bounds of prediction). 22 .osaamuoaso0v use mafiamuoanoosoa mo msoaumuusoodoo :0 once awla ou oqu uzlqm n 0090000 0» 00mmmooms msauoano 0000 no assoam onu no gas usousou .0 ouswfim 00.0 p p . p,pIp-. . p p . 0-0 0 00.0 p F. 0. r «Eng 035000200932 3... . 8.0 8.0 36 8. P—p-ppnppPp—pprb-pn-.—-P-rpr-p—P-bP-Frp— oo~o° 10‘0 90‘0 OD‘O CO‘O Dichloramine (ppm) L0°O 00‘0 00‘0 .oafiamuoanowv 0cm os0amuoanoosoa mo macaumuusoo Idea :0 onoo BMIH ou omqa uslom m 0050000 on mummmoom: onwuoaso 0000 Mo unsoam onu mo mma “sousoo .m ouswwm A803 oEEocoIoocoi 8.0 8.0 2.0 on... 3.0 2.0 8.? LLb-bb-k-PbbbL—pr—pp FI——b.~L—-——up.ppm-pn—LL-b-nth-knphp_nlp_on :0 1.. 3 38‘0 ED‘O we Dichloramine (ppm) (3‘0 - .IO‘I‘II-IIE-I If 24 To aid in the interpretation of Figures 4 and 5, three-dimensional graphs drawn with the aid of a computer are included. Figure 6 corresponds to Figure 4 and Figure 7 to Figure 5. The plateaus visible in Figures 6 and 7 at 0.30 ppm free chlorine are false plateaus drawn per instruc- tions to the computer to equate all values above 0.30 ppm equal to 0.30 ppm (e.g., the equation predicts that at concentrations of monochloramine equal to 0.350 ppm and dichloramine of 0.055 ppm, the free chlorine necessary to kill a l-gm fish in 24 hr is 0.820 ppm). The contour maps (Figures 4 and 5) indicate that at low concentra— tions of monochloramine and dichloramine, free chlorine is also necessary to obtain a fish kill in the respective time indicated. This is not evident in the three-dimensional graphs (Figures 6 and 7). The resolu- tion of the plotting scale and the inclination of the graphs mask the visibility of the chlorine concentration. For chloramine concentrations of 0.00 ppm, the amount of free chlorine necessary to cause fish mortality in 24 and 96 hr should be considered to be present in the figures at concentrations of 0.016 ppm and 0.004 ppm respectively. As viewed from the graphs (Figures 4 through 7), the equation predicts that free chlorine is more toxic to the coho at low concentrations than at high concentrations and that free chlorine is actually necessary for fish survival in high concentrations of monochloramine and dichloramine. For example, the 24 hr TL (Figure 6) shows that a monochloramine con- 50 centration of 0.05 ppm and a dichloramine concentration of 0.01 ppm will not prove fatal to the fish unless free chlorine is also present in excess of 0.01 ppm. At concentrations of monochloramine equal to 0.20 ppm and dichloramine of 0.05 ppm, the fish cannot survive even in the absence of free chlorine. However, the fish can survive in higher concentrations of monochloramine and dichloramine as long as chlorine is added which does not exceed that shown by the plane in Figure 6. 26 (unde-I 001'0='U! 91/8) Figure 7. Three dimensional response surface monochloramine and dichloramine. a 96-hr TLSO t0 1 27 The reason for this apparent disparity may be explained by the phenomenon of break-point chlorination. The inflection point on the graphs at which a rapid increase in the amount of free chlorine occurs at a point where the chlorine:ammonia ratio is 2:l--the ratio necessary for break—point (e.g., dichloramine = 0.00 ppm, monochloramine = 0.43 ppm, and free chlorine = 0.30 ppm produces a chlorine:ammonia ratio approximately 2:1). Trichloramine, nitrous oxide, chlorine dioxide and organic chloramines may be present in the water at break-point. APHA (1971) reports that of these, trichloramines, chlorine dioxide, and organic chloramines may also titrate partly as free chlorine. If one or more of these chemicals are either non—toxic or less toxic than the chlorine studied and able to combine with the same receptor sites as free chlorine, monochloramine and dichloramine, the result would be that the fish would be able to survive in higher concentrations of the toxicant due to its inhibition of the receptor sites. The effect of this inhibi- tion on the time of death is seen by comparing the amount of chlorine necessary to cause death at 24 hr and 96 hr with monochloramine of 0.60 ppm and dichloramine of 0.00 ppm (front surface of Figures 6 and 7). More chlorine (inhibitors?) is necessary to keep the fish alive for 96 hr than for 24 hr. The inflection point of free chlorine concentration occurs at the same point for the 24—hr and 96-hr curves. The similarity in the shape of the two curves results from the non-predictability of the equation in determining the time of death at break-point chlorination. There is also interaction between monochloramine and dichloramine in systems in which free chlorine is absent. Figure 8 represents all combinations of the two amines that kill 50% of fish weighing 1 gm in the times Specified graphically. If there were no interactions between 28 .AomAH I .aowuawwm IIIV 00008000000300 0am 90080000050258 00 2000900000000 How 6080mm. onoo awn." m 3 3de mafia H.053” 08.2002 .0 0.000me 5 0. Dichloramine (ppm) _ L _ _ 9 8 7 6 o o 0 0 10- AEQQV EECEZuOcOE 29 the amines, a straight line would result (Gaddum, 1948). The curvature of the line indicates that more of the amine combination is required to produce death than if their toxic effect were additive. As predicted from the equation, fish weight also has an effect on the time of death. The negative b coefficient indicates that as the 8 log weight of the fish increases, the time to death decreases. This means that larger fish will die faster than small fish. Arthur and Eaton (1971), studying the effects of chloramines to fathead minnows, Pimephales promelas Rafinesque, stated that the 96-hr TL50 value for adults was between 85 and 154 ug/L total chloramines, while 38% survival of larvae (0.014 gm) occurred in 108 ug/L for a 30-day study, which shows that small fish can survive longer in concentrations of chlorine than can large fish. The fact that large fish are more susceptible to chlorine than are small fish is explained by the ratio of gill surface area to body weight. Muir and Hughes (1960) reported the general equation for this relation— ship as: or log A = log a + b log W where A = total secondary lamellar area in mmz, a = total secondary lamellar area for a l-gm fish in mmz, W - weight of fish in gm, b = regression coefficient (slope). They added that for skipjack (Katsuwonus pelamis), yellow fish (Thunnus albacares) and blue fin (T. thynnus) the equation was: log Y = log 0.00883 + 0.5164 log W Prince (1931) states that in smallmouth bass (Micropterus dblomieui), the total area was related to body weight by a power of 0.78. As cited 30 by Hughes (1970), Gray (1954) made comparative studies on tuna, Cbryphaena, Microplerus, toadfish and the roach, and showed that the average slope was about 0.8. Thus, a large fish has less gill surface area per body weight than a small fish. Since chlorine attacks the gill tissue (Mann, 1950), small fish apparently have a pr0portionately greater ability to withstand the chlorine. The predictive equation suggests that the gill surface area to body weight relationship is in the order of 1.68220 which is much higher than the values cited in the literature. This results in the equation becoming non-predictive for fish weights that are much above that used in the experiment. A truer value might have been obtainable if a wider range of fish weights could have been used in the study. This was not possible with the experimental design employed. Larger fish (3.0 gm) produced excessive amounts of ammonia, which resulted in wide fluctuations of the titratable forms of chlorines. Direct application of the predictive equation to other systems and other test fish cannot be determined at this time. Further research is necessary to determine a more accurate value of 88 for the coho pOpula— tion. Similarly, it is not possible to predict the effect different test fish would have on this factor. Other systems with different chemical water parameters, such as pH or ammonia, might also be suspected as being able to alter the predictive equation. Merkens (1969) showed that by changing the pH of his test water, a different time to death was seen. Low level changes in the ammonia concentration, which would still enable break-point chlorination to occur, might cause a shift in the inflection point that allows fish to withstand higher concentrations of the chlorines than at low concentrations. 31 The chlorine complexes formed during water chlorination should be evaluated individually rather than employing the former methods of check- ing only the free and combined chlorine. The structural differences of each of the molecules suggest that each chlorine should be regarded as possessing potential biological differences. LI TERATURE CI TED LITERATURE CITED Alabaster, J. S., and F. S. H. Abram. 1965. Development and use of a direct method of evaluating toxicity to fish. In: Jaag, 0., ed. Advances in Water Pollution Research. 1:41-57. Proceedings of the Second International Conference, Tokyo. Aug. 1965. APHA et al. 1965. Standard methods for the examination of water and waste water including bottom sediments and sludges. Am. Pub. Health Assoc., New York. 12th ed. 769 pp. . 1971. Standard methods for the examination of water and waste water. Am. Pub. Health Assoc., New York. 13th ed. 974 pp. Arthus, J. W., and J. G. Eaton. 1971. Chloramine toxicity to the amphipod Gammarus pseudblimnaeus Bousfield, and the fathead minnow, Pimephales promelas Rafinesque. J. Fish. Res. Bd. Canada. (in press). ' Doudoroff, P., and M. Katz. 1950. Critical review of literature on the toxicity of industrial wastes and their components to fish. I. Alkalies, acids, and inorganic gasses. Sewage Ind. Wastes. 22:1432-1458. Gaddum, J. H. 1948. Pharmacology. 3rd ed. 0. U. P., London. Goldstein, A., L. Aronow, and S. M. Kalman. 1969. Principles of Drug Action The Basis of Pharmacology. Harper & Row, New York. 884 pp, Gray, 1. E. 1954. Biol. Bull. mar. biol. Lab. Woods Hole. 107:219. From: Hughes, G. M. 1970. A comparative approach to fish respiration. Experientia. 26:113-121. Green, D. E., and P. K. Stumpf. 1946. The mode of action of chlorine. Jouro A. W. W. A. 38:1301-13050 Holland, G. A., J. E. Lasater, E. D. Neumann, and W. E. Eldrige. 1960. Toxic effects of organic and inorganic pollutants on young salmon and trout. State of Washington, Dep. Fish. Res. Bull. 5:203-215. Hughes, 0. M. 1970. A comparative approach to fish respiration. Experientia. 26:113-121. Knox, W. E., P. K. Stumpf, D. E. Green, and V. H. Auerbach. 1948. The inhibition of sulfhydryl enzymes as the basis of bacteri- cidal action of chlorine. J. Bacteriol. 55:451-458. 32 33 Lewis, J. J. 1970. Lewis's Pharmacology, 4th ed. Rev. by J. Crossland. E. & S. Livingstone, Edinburgh. 1359 pp. ‘ Litchfield, J. T. 1949. A method for rapid graphic solution of time— per cent effect curves. J. Pharmac. exp. Ther. 97:399-408. Mann, H. 1950. Die einwerkung von chlor auf fische und fischnahrtiere. Dtsch. Aquarien-u. Terrarien—Ztschr. 3:119-120. McKee, J. E., and H. W. Wolfe. 1963. Water quality criteria. Calif. State Water Quality Control Bd., Sacramento Pub. No. 3A. 548 pp. Merkens, J. C. 1958. Studies on the toxicity of chlorine and chlor- amines to the rainbow trout. J. Water Wast. Treat. 7:150-151. Muir, B. S., and(;.bi. Hughes. 1969. (lill dimension for three species of tunny. J. Exp. Biol. 51:271-285. Prence, J. W. 1931. Growth and gill development in the small—mouthed black bass, Micropterus dblomieu Lacapede. Studies, Ohio State lhiersity. 4:46 p. Sawyer, C. L., and P. L. McCarty. 1967. Chemistry for Sanitary Engineers, 2nd ed. McGraw-Hill, New York. 518 pp. Sprague, J. B. 1969. Iieasurement of pollutant toxicity to fish. I. Bioassay methods for acute toxicity. Water Res. 3:793-821. Steel, R.(}. D., and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill, New York. 481 pp. APPENDIX APPENDIX Figure 1. Calculation of the median lethal time (LT ) of coho salmon exposed to concentrations of free chlorine, monochlor— amine, and dichloramine. 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