BOSTON PUBLIC LIBRARY 3 9999 06317 776 8 7/ LETHAL DIETARY TOXICITIES OF ENVIRONMENTAL POLLUTANTS TO BIRDS iTORY UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE Special Scientific Report— Wildlife No. 191 Library of Congress Cataloging in Publication Data Main entry under title: Lethal dietary toxicities of environmental pollutants to birds. (Special scientific report-wildlife; no. 191) Includes bibliographical references. Supt. of Docs, no.: I 49.15/3:191 1. Pesticides and wildlife. 2. Pesticides— Toxicology. 3. Pollution- Toxicology. 4. Game and game-birds— Diseases. I. Hill, Elwood F. II. Series: United States. Fish and Wildlife Service. Special scientific report-wildlife; no. 191. SK361.A256 no. 191 [QH545.P4] 639'.97'90973s [639'.97'82] 75-619346 UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE LETHAL DIETARY TOXICITIES OF ENVIRONMENTAL POLLUTANTS TO BIRDS By Elwood F. Hill, Robert G. Heath James W. Spann, and Joseph D. Williams Patuxent Wildlife Research Center Laurel, Maryland 20811 ^8 mst^ U.S. Fish and Wildlife Service Special Scientific Report — Wildlife No. Washington, D.C. 1975 191 CONTENTS ABSTRACT iv INTRODUCTION 1 PROCEDURES 1 RESULTS AND DISCUSSION 3 Comparative Toxicity in Relation to Chemical Class 3 Comparative Toxicity in Relation to Species 4 CONCLUSIONS 5 ACKNOWLEDGEMENTS 6 REFERENCES 7 Table 1 8 Table 2 37 Table 3 38 Table 4 40 Table 5 42 APPENDIX 43 in ABSTRACT This report is a compilation and analysis of the results of nearly 10 years of testing the lethal dietary toxicities of pesticidal and industrial chemicals to young bobwhites (Colinus virginianus) , Japanese quail (Coturnix c^ japonica) , ring-necked pheasants (Phasianus colchicus) , and mallards (Anas platyrhynchos) . A total of 131 compounds were tested. Toxicities are expressed as median lethal dietary concentrations (LC50) and are based on 5 days of dietary exposure to the test compound followed by 3 days of untreated feed. From these data statistical comparisons between toxicities are possible for a given species. Certain classes of pesticides — organochlorine compounds, organo- phosphates and organometallic compounds — contained most of the compounds judged "highly toxic". The most frequent order of species response was bobwhite > Japanese quail > ring-necked pheasant > mallard. This order correlates with their body sizes at the ages tested. IV INTRODUCTION This report is a compilation of the results of nearly 10 years of testing the subacute toxicities of pesticides and industrial chemicals to young bobwhites (Colinus virginianus) , Japanese quail (Coturnix c_. japonica) , ring-necked pheasants (Phasianus colchicus) and mallards (Anas platyrhynchos) . It supersedes our earlier publication (Heath et al. 1972). A total of 131 compounds were tested, including 30 organochlorine compounds, 39 organophosphates, 17 carboxylates, 15 carbamates, 12 metallic compounds, 5 organonitrogen compounds, 4 organosulfates, 4 ureas, 3 ketones, and 2 nitrophenols . Our objectives were two-fold: to provide a readily referable source of subacute toxicity data for the species and chemicals we tested, and to compare the responses of different species to different classes of chemicals. A detailed exposition of mathematical procedures and a list of chemical and common names (with often-used synonyms) are included in appendices. PROCEDURES Subacute toxicity tests were designed to measure a median lethal dietary concentration (LC50) of chemical to young birds during an 8-day test, including 5 days of treated diet followed by 3 days of untreated diet. Five or six geometrically arranged concentrations of toxicant were used per test at levels expected to kill from 10 to 90% of the test population. An equal number of positive and negative controls accompanied each test. Using shared controls, several compounds could be tested simultaneously. A completely randomized experimental design was used. Feed treated with dieldrin dissolved in corn oil served as the positive control (standard) and feed treated with corn oil, the diluent used for most compounds tested, was the negative control. Each test group (one test group per toxic concentration) consisted of 10 birds. Ages of test birds were 14 days for quail and 10 days for pheasants and mallards. In the 1973 tests, mallards were 5 days old. Testing commenced at midday. Mortality and feed consumption were recorded at 24-hour intervals thereafter. Fresh feed was added to all pens daily. After the 5th day, all feed, including that of controls, was replaced by untreated feed. Occasional deviations from the basic procedure were necessary because of shortages of facilities or birds. As few as five birds per test group and four toxic concentrations per test were sometimes used. Before standardization in 1970, age of birds sometimes varied as much as 1 week between tests. All test birds were incubator-hatched progeny of randomly outbred Patuxent colonies. Bobwhite and pheasant colonies originated from the Pennsylvania Game Commission's game farm stock; Japanese quail from Auburn University; and mallards from wild stock. Parent colonies were outbred to maximize individual variation and to more closely approximate characteristics of wild populations. Gallinaceous birds were tested in six-tiered brood units with tiers divided into four pens measuring 35 x 100 x 24 cm. Floors and external walls were of wire mesh; ceilings and common walls were galvanized sheet- ing. Tiers were equipped with thermostatically controlled heat and fluorescent lighting. Mallards were tested in wooden, walk- in pens on concrete slabs. The pens measured 1.5 x 3.0 x 2.1 m. The upper half of the lee wall of the pen was screened. Heat (infra-red), straw litter, and running water were provided. Test diets were prepared by blending a toxicant-carrier solution into commercial starter mash in the ratio of 2:98, by weight. Corn oil was the usual carrier, although propylene glycol was substituted when compounds were insoluble in oil. Chemicals were dissolved in the carrier, over heat when necessary. Some chemicals were first dissolved in minimal quantities of acetone. If extremely large quantities of a compound were required, or if the compound had a talc base, it was mixed directly into the feed and appropriate amounts of the carrier were added to the mixture as a supplement. Unstable compounds were mixed immediately before the test. Control diets contained corn oil in the ratio of 2:98, by weight. The LC50's and associated statistics were derived by methods of probit analysis described by Finney (1952) and were programmed for com- puter by the system of Daum and Killcreas (1966). The 50% response level was chosen because it can be estimated more precisely than extreme percentage levels (Finney 1952; see Appendix 1 for statistical details). Positive and negative control data were included in the probit analysis with every set of compounds tested simultaneously. Compounds with LC50's exceeding 5000 ppm in preliminary range finding experiments were not tested further. Estimates for preliminary data were made graphically by the method of Litchfield and Wilcoxon (1949) and are presented as approxi- mate values that are considered to be provisional. In Table 1, the toxicity of each compound to each species is compared ,with the toxicity of the dieldrin standard to the same species as deter- mined by concurrent tests. The resultant value (relative toxicity of dieldrin or RTD) is a direct ratio of the LC50's of the two compounds and represents the difference in toxicity between those two compounds under a single condition. Therefore, toxicities of different chemicals in relation to each other can be estimated statistically by using the RTD's as described in Appendix 2. Lethal concentrations in addition to the LC50's can also be estimated from data in Table 1 as described in Appendix 2. RESULTS AND DISCUSSION Five-day subacute dietary toxicities of 131 compounds were deter- mined for young bobwhites, Japanese quail, ring-necked pheasants and mallards. In Table 1, results are arranged alphabetically by common name of the test compound. Chemical names, purity, chemical classes, and principal uses are shown in Appendix 3. Comparative Toxicity in Relation to Chemical Class Although interest in toxicity data tends to center on specific comparisons between chemicals, broader generalization is also useful in understanding the toxicity process, especially if judgments of relative toxicity are desired. Several rating systems have been developed for this purpose (Hodge and Sterner 1949, Radeleff 1964, and Melnikov 1971). These systems classify LD50's (median lethal dose) into categories of relative toxicity from "highly toxic" (<50 mg/kg) to "practically nontoxic" (>5000 mg/kg) with class divisions arranged geometrically. We developed a similar system for rating subacute data in which five toxicity classes were recognized. The classes are: I, <41 ppm; II, 41-200 ppm; III, 201-1000 ppm; IV, 1001-5000 ppm; and V, >5000 ppm. Table 2 illustrates differences in general order of toxicity among chemical classes for Japanese quail, mallards, and rats. The rat data were derived from previously published acute toxicity tests (Gaines 1960, 1969; Melnikov 1971). Japanese quail and mallards showed similar responses, except that mallard results fell slightly more often into the least toxic class, V. Both species responded similarly to organochlorine and organophosphorus compounds. These chemical classes tended to be most toxic to birds, as they contained the greatest proportion of compounds rated class I and II. All carboxylates and most "miscellaneous" compounds fell in the least toxic class. The toxicity ratings for rats were different because a much higher percentage of compounds were judged to be highly toxic (classes I and II) . With the possible exception of carboxylates, it is clear that lethal hazard cannot be predicted solely on the basis of chemical class. Nor can it be assumed that relative hazard of chemicals based on acute study with rats will follow the same order when tested subacutely on birds. In Table 3 relative toxicities of different classes of chemicals to birds are given in more detail by subdividing the major classes according to their structural properties. Here, toxicities of similar compounds follow more definable patterns. Organochlorines tested are halogen derivatives of either alicyclic or aromatic hydrocarbons. Nearly all compounds in class I and II are alicyclic. In contrast, most aromatic hydrocarbons are in class IV or V. There are exceptions, however. For example, Starlicide, an aromatic hydrocarbon bird control agent, is surpassed in subacute toxicity to Japanese quail only by endrin, an alicyclic hydrocarbon, and azodrin, an organophosphorus compound (Table 1) . Tests with rats followed a similar toxicity pattern (Gaines 1960, 1969). Alicyclic hydrocarbons generally produced LD50's of less than 100 mg/kg (class I and II) whereas aromatic hydrocarbons were above 800 mg/kg (class III-V) . Starlicide was generally in line with other aromatics in rat tests as the reported LD50 is 1170 mg/kg (Christensen 1973) . Organophosphorus compounds are derivatives of four phosphorus acids: phosphoric, thiophosphoric, dithiophosphoric, and phosphonic. All organo- phosphates that fell into class I were derivatives of either phosphoric or thiophosphoric acids, except Mocap (bobwhite LC50, 33 ppm) , a dithio- phosphoric acid. Phosphoric and thiophosphoric acids produced few LC50's above 1000 ppm (class IV and V), although nearly all LC50's of dithio- phosphoric and phosphonic acids were over 1000 ppm. Among the phosphoric acids, azodrin (the most toxic compound tested), its close relative Bidrin, and phosphamidon were consistently among the most toxic of all compounds tested. Several thiophosphoric acids — Dasanit , famphur, fenthion, methyl parathion and thionazin — also were highly toxic. In general, the order of toxicity of these phosphorus acids to birds was: phosphoric _> thiophosphoric > dithiophosphoric _> phosphonic. Phosphoric and thiophosphoric acids also were highly toxic to rats (Gaines 1960, 1969). Abate, a thiophosphoric acid, provides an interesting contrast because it was quite toxic to birds (Table 1 and Hill 1971), but not to rats (Gaines 1969) . The metallic compounds tested varied widely in chemical composition and permitted only general comparisons. Organic forms tended to be more toxic than inorganic forms. The organomercurials Ceresan M and Morsodren consistently gave LC50's less than 100 ppm (class II). LC50's for other metallics usually exceeded 1000 ppm (class IV and V) . Comparative Toxicity in Relation to Species Comparisons of the susceptibility of Japanese quail, ring-necked pheasants and mallards to different chemicals are shown in Table 4. Quail appeared to be most sensitive to the comparatively toxic alicyclic hydro- carbons and mallards the least sensitive. Pheasants were most sensitive to aromatic hydrocarbons and mallards appeared least sensitive (based on response rating and median LC50) . Species sensitivity to organophosphorus compounds followed the order: quail > pheasant > mallard. For carbamates, the order was quail > mallard > pheasant. This indicates a change in the pheasant-mallard relationship, even though organophosphates and carbamates both are chlorinesterase inhibitors. Meaningful comparisons were not possible for other classes of chemicals, but some observations are pertinent. Only pheasants produced LC50's less than 5000 ppm for carboxylates. Pheasants appeared most sensitive and mallards least sensitive to inorganic metallics, but the reverse was true for organic metallics. Overall comparisons showed the probable order of sensitivity to be Japanese quail > ring-necked pheasant > mallard; this relationship occurred 31°' of the time. The opposite order occurred least frequently (6%). The second most frequent order of sensitivity (27%) was pheasant > quail > mallard. Because all possible variations in order of species sensitivity occurred in all chemical classes, it is clear that an LC50 for any of these species probably would not permit prediction of that chemical's toxicity to either untested species. A similar conclusion was reached by Tucker and Haegele (1971) from the results of tests of acute pesticidal toxicity to six species of birds. Although accurate prediction of the sensitivity of one species to a given compound from data for a different species appears unlikely, there are positive correlations between LC50's for different species within a given chemical class. Table 5 shows correlation coefficients for paired LC50's for the species we tested within the major chemical classes. All correlations of LC50's for Japanese quail and mallards were statistically significant (P<0.05 or P<0.01). Correlations between LC50's for ring- necked pheasants and either mallards or Japanese quail were significant in three of four comparisons with each species. Only three of eight comparisons between bobwhite and other species proved significant. Because all correlations are positive, 17 of 20 are either significant or very nearly significant at P=0.05, and one-half are highly significant (P<0.01), it is clear that the test species responded similarly, in a relative sense. CONCLUSIONS We have measured the dietary susceptibility of two to four species of birds to 131 toxic compounds. From the data provided in Table 1 the toxicities of different compounds to the same species can be tested statistically; thus, toxicity rankings are possible. Most of the more toxic compounds were halogen derivatives of alicyclic hydrocarbons, derivatives of phosphoric and thiophosphoric acids, and organomercurials. Carbamates, often of extreme acute toxicity to rats, were only moderately toxic when fed to birds. Most carboxylates, ketones, organonitrogen compounds, organosulfates and ureas were of a relatively low order of toxicity. Interspecies comparisons showed the overall order of susceptibility to be quail > pheasant > mallard which is size related. All combinations of species order occurred. Although the order of susceptibility of species varied, a characteristic order usually prevailed within a given class of chemicals and the LC50's for any two test species were strongly correlated. This suggests that, regardless of test species, the relative toxicities of different chemicals in the same class would be similar if test conditions were constant. However, unpredictable differences in species' suscepti- bility occur often enough that tests of at least two species are desirable. ACKNOWLEDGMENTS We are grateful to the many persons who contributed to this study. Special recognition is extended to the following: William H. Stickel was an invaluable source of information relating to our selection of test compounds. Clyde Vance supervised aviculture and consistently provided high quality test birds on schedule. Marshall Hynson, David Jaquith, Norman Kruhm, Claude Mills (deceased), Frank Polak, David Prevar, Richard Rowlett, and Perry Waters serviced test facilities and assisted with data gathering. William L. Reichel, T. Earl Kaiser and Thair G. Lamont advised on various chemical considerations. Helen L. Young conducted the computer analyses. Lucille F. Stickel and J. Larry Ludke critically reviewed the manuscript. Special thanks go to the various chemical companies who provided the compounds for testing. REFERENCES Bliss, C. I. 1952. The statistics of bioassay. Pages 445-628 in Vitamin methods, Vol. II. Academic Press, New York. Casewell, R. L. , D. E. Johnson, and C. Fleck. 1972. Acceptable common names and chemical names for the ingredient statement on pesticide labels, 2d ed. U. S. Environ. Prot. Agency, Washington, D.C. 243 pp. Christensen, H. E. , editor. 1973. The toxic substance list. U. S. Dep. Health, Educ, Welfare, Rockville, Md. 1001 pp. Daum, R. J., and W. Killcreas. 1966. Two computer programs for probit analysis. Bull. Entomol. Soc . Am. 12 (4) :365-369. Finney, D. J. 1952. Probit analysis, 2d ed. Cambridge Univ. Press. 318 pp. Finney, D. J. 1964. Statistical method in biological assay, 2d ed. Hafner Publ. Co., New York. 688 pp. Gaines, T. B. 1960. The acute toxicity of pesticides to rats. Toxicol. Appl. Pharmacol. 2(l):88-89. Gaines, T. B. 1969. Acute toxicity of pesticides. Toxicol. Appl. Pharmacol. 14 (3) :515-534. Heath, R. G., J. W. Spann, E. F. Hill, and J. F. Kreitzer. 1972. Compara- tive dietary toxicities to birds. U. S. Fish Wildl. Serv. , Spec. Sci. Rep. Wildl. 152. 57 pp. Hill, E. F. 1971. Toxicity of selected mosquito larvicides to some common avian species. J. Wildl. Manage. 35 (4) : 757-762. Hodge, H. C, and J. H. Sterner. 1949. Tabulation of toxicity classes. Am. Ind. Hyg. Assoc. Quart. 10(4):93-96. Litchfield, J. T. , Jr., and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96(2): 99-113. Melnikov, N. N. 1971. Chemistry of pesticides. Residue Rev. 36:1-480. Radeleff, R. D. 1964. Veterinary toxicology. Lea and Febiger, Philadelphia. 314 pp. Snedecor, G. W. , and W. G. Cochran. 1967. Statistical methods, 6th ed. Iowa State Univ. Press, Ames. 593 pp. Tucker, R. K. , and M. A. Haegele. 1971. Comparative acute oral toxicity of pesticides to six species of birds. Toxicol. Appl. Pharmacol. 20 (1): 57-65. 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XI 14-1 vo o O CO w &- cn xi XI C3> CU X) CU C X) CU 3 4-1 rH X O CU c •H CO cfl 2 C cfl O CO •H CU 4J 1-1 Cfl CU > O l-i CU 1-4 CO o 43 C4-I o CO 4-) - c o h3 cfl cu i-i +J cfl o •U iH 4-1 43° CO o o 1-1 (3, 36 Table 2. Percentage frequency distribution by toxicity class for pesticidal compounds tested subacutely against Japanese quail and mallards as compared to acute rat toxicities . Chemical class Species Toxicity class II III IV V Organochlorine Quail (n = 18) Mallard Rat Organophosphorus Quail (n = 35) Mallard Rat Carbamate Quail (n = 12) Mallard Rat Carboxylate Quail (n = 7) Mallard Rat Metallic Quail (n = 9) Mallard Rat Miscellaneous Quail (n = 14) Mallard Rat 11 11 6 11 11 33 9 26 6 6 43 31 8 33 25 14 22 22 22 56 21 33 33 11 28 28 28 33 6 17 31 23 11 40 26 23 17 6 3 25 33 42 25 33 33 42 100 100 43 29 14 11 56 11 11 11 56 22 14 7 79 7 21 71 50 29 Only compounds providing complete data for each species are included. Avian data are from Table 1; rat data are from Gaines (1960, 1969) or Melnikov (1971). ^Bounds of toxicity ratings: I = <41, II = 41-200, III = 201-1000, IV = 1001- 5000, V = >5000. Toxicologic parameters are LC50's as ppm compound in diet of birds and LD50's as mg compound per kg body weight for rats. "Miscellaneous includes nitrophenol, ketone, organonitrogen, organosulfer and urea compounds. 37 Table 3. Frequency distribution by toxicity class for organochlorine, organophosphorus , carbamate and metallic compounds tested subacutely against birds . Species Toxicity i c class Chemical class I II III IV V ORGANOCHLORINE COMPOUNDS Derivatives of alicyclic hydrocarbons BW 3 1 5 1 JQ 2 2 5 1 1 PH 1 2 5 3 ML 1 2 4 2 2 Derivatives of aromatic hydrocarbons BW 5 6 3 IQ 1 1 9 6 PH 4 9 3 ML 10 7 ORGANOPHOSPHORUS COMPOUNDS Derivatives of phosphoric acid BW 1 1 JQ 2 2 2 1 1 PH 1 2 2 1 1 ML 1 1 1 3 1 Derivatives of thiophosphoric acid BW 2 6 3 JQ 7 5 1 1 PH 5 7 2 ML 1 2 8 1 2 Derivatives of dithiophosphoric acid BW 1 4 1 JQ 1 4 5 3 PH 1 4 5 3 ML 3 5 4 Derivatives of phosphonic acid BW JQ PH 1 2 2 1 2 3 ML 1 2 1 38 Table 3. Frequency distribution by toxicity class for organochlorine, organophosphorus , carbamate and metallic compounds tested subacutely against birds — continued Chemical class Species Toxicity class II III IV CARBAMATE COMPOUNDS Derivatives of carbamic acid BW JQ PH ML 1 3 3 3 2 5 6 6 1 3 3 2 Derivatives of dithiocarbamic acid BW JQ PH ML 1 1 2 3 3 3 METALLIC COMPOUNDS Inorganic JQ PH ML 3 2 1 1 4 Organic BW JQ PH ML 1 2 2 2 1 4 4 1 1 3 Basis for frequency distribution is Table 1. Species: BW, bobwhite; JQ, Japanese quail; PH, ring- necked pheasant; and, ML, mallard. "Bounds of toxicity ratings, I = <41 ppm, II = 41-200 ppm, III = 201-1000 ppm, IV = 1001-5000 ppm, and V = >5000 ppm. 39 Table 4. Comparative responsiveness among young Japanese quail, ring- necked pheasants and mallards to pesticidal compounds when tested subacutelya. Chemical class Species Response rating" Median Most Least LC5Q (Extremes) ORGANOCHLORINE Derivatives of alicyclic hydrocarbons (10)d Derivatives of aromatic hydrocarbons (15/12) Quail 50% 0 388 ( 18->5000) Pheasant 30% 20% 495 ( 14- 1540) Mallard 20% 80% 669 ( 22->5000) Quail 17% 50% 3165 ( 568->5000) Pheasant 84% 8% 2078 ( 311->5000) Mallard 0 42% 3572 (1651->5000) Quail 67% 0 286 ( 2->5000) Pheasant 33% 0 246 ( 3->5000) Mallard 0 100% 1991 ( 32->5000) ORGANOPHOSPHOROUS Derivatives of phosphoric acid (7/6) Derivatives of thiophosphoric acid (13) Derivatives of dithiophosphoric acid (12/10) Derivatives of phos phonic acid Quail 69% 8% 211 ( 47->5000) Pheasant 15% 23% 240 ( 49->5000) Mallard 15% 69% 640 ( 43->5000) Quail 50% 15% 2067 ( 100->5000) Pheasant 40% 20% 2230 ( 118->5000) Mallard 10% 65% 3300 ( 248->5000) (4) Quail 25% 0 972 ( 295- 1901) Pheasant 50% 25% 708 ( 270- 3401) Mallard 25% 75% 1430 (~330->5000) 40 Table 4. Comparative responsiveness among young Japanese quail, ring- necked pheasants and mallards to pesticidal compounds when tested subacutely3 — continued Chemical class Species Response rating" Median Most Least LC-rt (Extremes) CARBAMATE Derivatives of carbamic acid (11/9) Quail Pheasant Mallard 56% 22% 1427 ( 381->5000) 11% 67% 2874 ( 573->5000) 33% 11% 2300 ( 190->5000) Comparisons are restricted to compounds providing comparable data among the species as shown in Table 1. Mallard values used are mainly for 10-day-old birds. Percentage of times each species produced the lowest (most responsive) or highest (least responsive) LC50 for compounds within each chemical class. c Derivation of median toxicities were restricted to LC50's of compounds used in construction of this table. Where two numbers are shown, the first represents total compounds used for determination of median toxicities and the second is the total compounds upon which response rating percentages are based. 41 Table 5. Relation between four avian species in subacute responsiveness to pesticidal compounds3. Chemical class Species compared Statistic OC OP CB IMfOM Bobwhite - Japanese quail n r Bobwhite - ring-necked pheasant n r Bobwhite - mallard n Japanese quail - ring-necked n pheasant r Japanese quail - mallard n Ring-necked pheasant - mallard n 5 13 6 0.704 0.900** 0.491 I.D. 11 0.561 6 13 0.759 0.543 12 30 I.D. 6 I.D. 0.675** 6 I.D. 0.958** 11 7 0.679* 0.941** 0.486 0.836* 14 28 11 11 0.918** 0.853** 0.633* 0.774** 12 28 13 0.897** 0.902** 0.902** 0.622 Correlation coefficients are for paired LC50's from standardized data in Table 1. Standardized data includes only results for tests of 12-18-day-old quail and 8-12- day-old pheasants and mallards. n, number of paired LC50's; r, correlation coefficient; I.D., insufficient data. C0C, organochlorine; OP, organophosphorus ; CB, carbamate; IM, inorganic metallic; OM, organometallic. *Correlation coefficient statistically significant (P<0.05). **Correlation coefficient highly significant (P<0.01). 42 APPENDIX 43 Appendix 1 Toxicity Statistics The principal statistical reference point is the LC50, as determined by computerized probit analysis. The LC50, as used under our procedure, is ppm toxicant (based on active ingredient) in an ad libitum diet produc- ing 50% mortality in 8 days (5 days of toxic diet followed by 3 days of untreated diet). The probit analysis program calculates the following maximum likeli- hood statistics: LC50 and its 95% confidence limits; slope of the weighted linear regression of probits on log-concentration and its standard deviation; and relative toxicity, with 95% confidence limits, of any two compounds after testing regression lines for parallelism and heterogeneity. The program permits simultaneous analysis of all compounds tested in any single experiment. Comparison of toxicities between compounds is by determination of their relative toxicity or "toxicity ratio." The toxicity ratio may be expressed unconditionally as the ratio between LC50's of two compounds provided the level of tolerance of test populations is the same and probit regression lines are parallel. The level of tolerance can be assumed comparable only if both test populations are drawn from the same population and are tested concurrently in a completely randomized exper- iment. Because this condition is obviously restrictive, adjustment for tolerance differences between experiments is possible with the positive control, according to the procedure presented in Appendix 2. Parallelism is assumed if slopes of regression lines are not shown to be different at a specified level of significance. Lethal concentrations other than the LC50 may be useful. These values can be estimated from data in Table 1 by the procedure described in Appendix 2. Estimates of this type should be derived from especially designed experiments, however, because extrapolation from a standard probit regression line can be misleading if the true regression equation has some curvature (Finney 1952) . 44 Appendix 2 Calculation of Some Significant Toxicity Values Toxicity ratios: The RTD values listed in Table 2 are used to calculate the toxicity ratio of two compounds for a particular species as follows: 1. Compute the toxicity ratio of "Compound 1" to "Compound 2" by dividing the RTD of Compound 2 by the RTD of Compound 1. For example, if dieldrin is 4 times as toxic as Compound 1 (RTDi = 4) and 6 times as toxic as Compound 2 (RTD2 = 6) , then Compound 1 is 1.5 times as toxic as Compound 2 (i.e., RTD2/ RTD j = 6/4 =1.5). An algebraic argument for the procedure was previously presented (Heath et al. 1972). The calculation of confidence limits for potency ratios require more data than could reasonably be included in this paper. 2. Test the slopes of the probit regression lines of the two chemicals for parallelism using a 2-tailed t-test. Let b and b2 be the estimated slopes and Sj and s2 their standard devia- tions. (The s values are actually in standard error form.) Also let n1 and n2 equal the number of concentrations used in the respective determinations. Then t = (bi - b2) / J71 2 + s22 Since s 2 and s 2 have n1 - 2 and n - 2 degrees of freedom, t is given n-. + n2 - 4 degrees of freedom, provided si2 and 9 9 s„ can be considered estimates of a common a . We expect homogeniety of variances in most instances; however, procedures for testing the equality of two variances and the significance of the difference of two means (i.e., b^ and b2) when variances are unequal are presented in Snedecor and Cochran (1967) . LC's for the percentage of response: Lethal concentrations for percentages of mortality other than the median can be estimated from the data in Table 2 as follows: 1. Transform the LC50 to its common logarithm and the desired percentage of mortality to its probit, the probit of 50% being 5. If we let k equal the new percentage of mortality for which we wish to estimate the lethal dietary concentration (i.e., the LC ), and b equal the particular slope value from Table 2, then k log LC = log LC50 + (probit k -5) /b . K. 45 The antilog of LCk is the desired estimate. Tables for transforming percentages to probits can be found in various statistical texts, including Finney (1952, 1964). 46 to •H co cj CU C co •H 3 U B2 < •H 6*« 3 P-. 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