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Some effects of partial substitutions of lithium for environmental sodium on Limnoria species

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Some effects of partial substitutions of lithium for environmental sodium on <italic>Limnoria</italic> species

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Content SOME EFFECTS OF PARTIAL SUBSTITCTIONS OF LITHIUM FOR ENVIROmENTAL SODIUM ON LIMNORIA SPECIES A Thesis Presented to the Faculty of the Zoology Department the University of Southern California In Partial Fulfillment of the Requirements for the Degree Master of Arts in Zoology by Lerner B* Hinshaw January 1953 UMI Number: EP67209 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI EP67209 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 This thesis, written by Lerner—R*-- - Hinshaw................. under the guidance of h±^...Faculty Committee, and approved by all its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fullfill- ment of the requirements for the degree of -Master—of—Ar-ts-in..Soolo.gy. ........ Date 19 .53 ..... Faculty Committee Chairman ...... ...... PREFACE Special indebtedness is expressed to the following persons for assistance and guidance: The late Dr. Frederick P. Shelden for the inception of the study and his inspiration. Dr. Walter S. Martin, head of the Zoology department, for departmental advisement. Dr. John L. Webb for valuable technical guidance. Mr. Duncan Thompson for direction. Drs. Robert J. Menzies, John L. Mohr and Laurens Barnard of this department and of the Southern California Marine Borer Council for assistance early in the study. Drs. Norris W.Rakestraw, Clifford V. Harding, and Robert M. Chew !for helpful advice and evaluation. TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION, ............................... 1 A general discussion of the organism .... 1 The purpose of the study.................. 3 The function of sodium chloride............ 5 Lithium and the alkali metal group......... 6 The interaction of cations........... • • 8 The physiology of the heart................ 11 Experimental work involving the use of lithium............................... II. MATERIALS AND METHODS....................... 16 III. OBSERVATIONS................................. 22 IV. DISCUSSION........... 50 V. SUMMARY ............................. 54 LITERATURE CITED................................... 56 LIST OP TABLES TABLE page I# Effect of Altered Concentrations of sodium Chloride in Artificial Sea Water.............23 II. Effect of Lithium as a Substitute for, or in Combination with. Sodium ........ 25 III. Effect of Lithium as Compared with the Effect of Low Sodium Concentration ................27 IV. Effects of Electrolytes and Non-Electrolytes in Low Sodium Concentrations..................29 V. Effect of Magnesium on Lithium Toxicity. .... 31 VI. Effect of Calcium on Lithium Toxicity..........* 33 VII. Effect of Potassium on Sodium and Lithium. ... 35 VIII. (kjmparison of Heart-beat Rates in Various Solutions . . . . . . . . . . . . . . 37 IX. Heartbeat Rates of Single Isolated Animals in Varying Conditions........................40 X. Heartbeat Rates of Single Isolated Animals in Various Solutions. ....... ......... 42 XI. Illustration of Prolonged Effects of Lithium . . 43 XII. Effect of Temperature on Toxic Concentration of Lithium................................... 44 XIII. Prolonged Effect of Toxic Concentration of Lithium..................................... 46 TABLE PAGE XIV. Effect of Conditioning by Toxic Exposure to Lithium................................... 47 XV. Effect of Conditioning by Sub-toxic Exposure to Lithium..................... 48 XVI. PH Changes in Various Solutions of Artificial Sea Water................................. 49 CHAPTER I INTRODUCTION In the light of recent work with living organisms con cerning the effects of the lithium ion as a substitute for the sodium ion, the present study was begun in 1951 using the marine isopod Limnoria. A general discussion of the organism. Limnoria, a crustacean (order Plabellifera), is commonly known as the gribble. It is represented locally by the species Limnoria quadripunctata Hoithins and Limnoria tripunctata Menzies. Menzies (1951a,b) gives a clear description of Limnoria tripunctata. Limnoria appears from field data and from'limited ex perimental work (Menzies 1951a) to be mesohaline, that is, moderately tolerant to changes in salinity, particularly for short periods, and more to abnormally high than to un usually low salinities. Limnoria is small, being from 2.5 to 5.0 millimeters in length and from 0.4 to 1.7 millimeters in width (Menzies 1951a). The body of the animal is char acterized by three principle regions; the head (cephalon), the thorax (peraeon) and the abdomen (pleon). These re gions are further divided into smaller areas called seg ments or somites, each possessing a pair of jointed appen dages and paired ganglia. The tough shell or exoskeleton 2 enclosing the body is partly chitinous and contains a form of calcium carbonate. In order for the animal to grow or increase in size, the exoskeleton must be sloughed off or molted periodically. The digestive tract is complete, con sisting of a fore-, raid-, and hind- gut. The coelom is re duced and the body spaces are occupied by a fluid contain ing blood cells. The heart is dorsal and pleonal. Res piration is effected by the pleopods or swiramerets which unlike those of related peracaridans are flattened to thin lamellae. The brain, a grouping of anterior dorsal gan glia, is joined by clrcumesophageal connectives to the ven tral chord of paired segmental ganglia. The cephalon con sists of five fused somites. These have two pairs of an tennae, the first and the second, a pair of jaws called mandibles, and two pairs of foliaceous or phyllopodial ap pendages, the first and second maxillae. The body of the crustacean terminates with the tel son which bears the anal opening. The body is divided into nineteen somites, the cephalon has five, the peraeon eight, and the pleon six. Including the telson. Each somite of the peraeon of the isopod bears a pair of legs or peraeopods. The somites of the pleon each have a pair of appendages called pleopods. The terminal appendages of the pleon are called uropods. 3 The food of Limnoria consists mostly of plant material. The animals ingest wood or algal stems and derive nourish ment from them. Specimens kept from wood die in thirty to fifty days.(Menzies 1951a). Limnoria live in burrows in the wood (except the algal dwelling species) and these are formed entirely as the re sult of gnawing and ingesting the wood into which they burrow. The wood is probably torn into minute chunks by the action of the mandibles, and is then ingested passing through the digestive tract and out the anus. For cen turies Limnoria has attacked wooden structures. To date no entirely effective commercially feasible means of protection, against them has been found. (Menzies 1951a). Limnoria swim by a rapid beating of the pleopods which also serve as gills. Swimming varies considerably from short rapid spurts to more or less prolonged periods of swimming at a uniform rate. They frequently swim on the back with the ventral surface directed upwards. Except for the fairly rapid movement of swimming, Limnoria move from one place to another by a relatively slow crawl. The purpose of the study: This study has several functions: (1) It is desirable to better understand the ionic mechanisms of the animal; for such understanding this work lays a foundation. Work which will tell us more 4 about the physiology of the animal may be of value in curb ing the tremendous damage done by Limnoria# Menzies (1951a) states that in the United States alone it is estimated that the animal annually destroys #1 ,0 0 0 , 0 0 0 of marine piling. (2) The substitution of lithium for sodium has Interesting implications in nerve-muscle physiology. This study lays a foundation for further work on isolated nerve and muscle tissues, especially the heart of Limnoria. More explicitly the study has the following purposes; (1) To obtain an understanding of some effects of altered concentrations of sodium chloride on Limnoria; (2) to sub stitute lithium for sodium in an isosraotic medium to deter mine a minimum toxicity level; (3) to determine some effects of lithium on Limnoria when used as a substitute for, or in combination with, sodium, using artificial sea water solu tions which are isosmotic, hypotonic and hypertonic with respect to natural sea water; (4) by the use of the non electrolytes, sucrose and sorbitol, in substitution for sodium chloride in low sodium chloride concentrations to determine whether the deaths of Limnoria, upon the addition of toxic concentrations of lithium, are due to low sodium concentrations, hypotonicity, or lithium toxicity; and (5) to more clearly define the lithium effect by an experimen tal study of the additive or antagonizing effects of sodium, 5 potassium, magnesium and calcium upon lithium. The main criteria of substitution effects were: (1) mortality rate and (2) rate of heart beat. Other aspects studied were the effect of temperature on lithium toxicity, the prolonged effect of toxic concentrations of lithium af ter the organisms were returned to normal solutions, the ef fects of conditioning to lithium in sub-toxic and toxic con centrations; and the changing behavior of the pH in various solutions of artificial sea water. The function of sodium chloride. In the vertebrate animal sodium, as sodium chloride, contributes toward the acid-base balance of the body and is responsible in large measure for the total osmotic pressure of the extracellular fluids (Harrow, 1950). In the vertebrate animal about 90 per cent of the total base in blood serum is due to sodium . The extent of the excretion of sodium is dependent upon the amount of intake, and although an animal can maintain it self on a very small quantity, there results a loss of ap petite, retarded growth, disturbance of the reproductive function, and ultimate death. The removal of the adrenal cortical hormone is fol lowed by a considerable loss of sodium from the body. Loss of sodium chloride from the blood as produced ly extreme sweating due to high temperature or much exertion 6 may result in the development of leg and abdominal cramps. Sodium intake may relieve prolonged vomiting (chloride loss) diarrhea (sodium loss), adrenal cortex insufficiency (disturbance of salt metabolism), and shock due to loss of blood volume. Lithium and the alkali metal group. Element Symbol Atomic Number Atomic Weight Li thium Li 3 6.940 Sodium Na 11 22.997 Potassium K 19 39.096 Rubidium Rb 37 85.48 Cesium Cs 55 132.91 Francium Fr 87 223 Lithium, discovered in 1817, is the lightest and most reactive metal known. It has physical and chemical proper ties which are similar to those of sodium and potassium, but the lithium atom differs from the atoms of other alkali metals in weight and size. The name is derived from the Greek word for stone, and lithium was so called because it was considered never to be present in plant and animal matter. However, it does occur in both in minute amounts. The salt, lithium chloride, has a molecular weight somewhat lower than that of sodium chloride. It occurs as white, deliquescent crystals. 7 As a substitute for sodium chloride in humans lithium was shown to be toxic by Stern (1950), Hanlon et al. (1949), Corcoran, Taylor and Page (1949), Talbott (1950), and Masson (1949) wrote about its toxicity in rats. Sollraann (1948) states that the medicinal uses of lithium in the human have no rational foundation. Concer ning some general effects of lithium ingestion, in many animals lithium appears to have a depressant action on the motor nerves and weakens muscular contraction. This effect is probably due to the lithium ion itself. Lithium acts much less powerfully on thq mammalian heart than potassium but has some effect in weakening it. Cushny (1940) stated that its chief effects are exercised in the alimentary tract, since gastroenteritis and extravasations of blood j into the stomach and bowel are induced by its subcutaneous I or intravenous injection. These effects were also empha sized by Sollmann (1948). Potassium is found largely in body cells, whereas sod ium is distributed in body fluids. Potassium penetrates rapidly into most of the tissues of the body and only a small quantity is found in the plasma. The growth of rats is retarded when the diet contains less than 15 milligrams of potassium daily, and the animals develop lesions of the heart and renal hypertrophy (Harrow, 1950). Thus far the 8 requirement of body potassium is not known* Rubidium appears to act on the frog’s heart and on muscle cells in much the same manner as potassium* It is slowly excreted by the kidneys, traces are found in the feces, especially in diarrhea* Cesium resembles lithium in causing inflammatory reactions in the alimentary tract, leading to vomiting and diarrhea either when injected hypodermically or given orally. It is partly excreted by the alimentary tract in mammals. In the frog it induces weakness of the muscles and paralysis. Cesium salts have an action on the excit ability of the frog’s heart similar to that of calcium but on its contractility an action similar to that of potassium. The interaction of cations. In this study the concen trations of sodium, potassium, lithium, magnesium and calcium were all shifted with respect to one another. Cer tain general relationships appear to exist. One cation may be antagonistic to another in that it tends to counteract the effect of the other, or it may be additive to, or act synergistically with, another cation, in that it appears to add to or increase the effects of the former. The mechan isms behind these actions are not well understood. Heilbrunn (1943) shows the following relationships: (a) One type of cation antagonism depends on the 9 valences. In sea urchin eggs, magnesium and calcium tend to prevent the toxic effects of sodium and potassium. (b) Antagonism does not always depend on valence* The anesthetic effect of magnesium is antagonized best by calcium. (c) Monovalent cations may be antagonistic. Potassium and sodium show this relationship. (d) Each cation may act antagonistically or syner- gistically with any other cation. Potassium and sodium are antagonistic, calcium and magnesium antagonize sodium but together are synergistic. (e) Ions which are antagonistic to each other, in relation to a given biological process, may act in the same way in relation to another process. Magnesium and calcium, and potassium and calcium illustrate this. (f) An ion which is antagonistic to a second ion at one concentration may act with it at another concentration. Magnesium and calcium act this way. In the nervous system it is believed that excitability is conditioned by the ion equilibrium. It appears that monovalent and bivalent cations are antagonistic to each other. Variations in the ratio K plus Na/Ca plus Mg may cause a change in excitability. A relative or an absolute increase in monovalent cations lowers the threshold and 10 decreases the chronaxie; the predominance of bivalent cat ions has the opposite effect; removal of the parathyroid glands diminishes the blood Ca-ion and therefore, the threshold of excitability is lowered and tetany occurs. The integrity of the nerve is dependent on little-known metabolic processes. Respiratory exchanges are necessary to maintain the physico-chemical structure of the axon membrane. The stability of this membrane is conditioned by the ionic equilibrium. Any change that causes an increase in K-ion or a shift of the acid-base equilibrium toward the alkaline side, diminishes stability. An increase in Ca-ion or a shift toward the acid has a stabilizing effect. A considerable change in either direction can suppress con ductivity and block the nerve. Hober (1945) described the antagonistic action of pairs of cations. An especially strong support of the colloid theory of physiological ion activity comes from the study of pairs of salts. The antagonism existing here was be lieved to be due to the rule that the flocculating power of an electrolyte depends upon the valence of the ion whose charge is opposite to that of the colloidal particles, and that the influence of valence rises nearly in a geometrical progression* In electrical excitability extent the following 11 relationships exist: Na > Li > Cs > HH4 > K. Another difficulty in attempting to interpret the effects of some cations is that in different conditions they act in different ways. Sodium or potassium can either cause a muscle to contract or prevent it from contracting. Potassium can increase the excitability of a nerve, or can prevent the passage of a nerve impulse. Heilbrunn provides an explanation: If one assumes that when sodium or potas sium replaces the calcium of the cell cortex, it liberates calcium which may pass from the cortex into the cell inter ior, one has an explanation of why sodium or potassium on short exposure acts like calcium. On longer exposure to sodium or potassium, there is an antagonism to calcium, and the exact mechanism, although not understood, is described by Heilbrunn as typical. The problem of cation interaction which includes anta gonistic or additive characteristics, is a very difficult one, and the writer will only point out some of his obser vations of their relationships, not the mechanisms behind their activities. The latter is beyond the scope of this study. This work lays a foundation for a further one in which the mechanisms of inter-cationic activities will be disclosed. The physiology of the heart. In a discussion on the 12 circulation of body fluids, Prosser et al. (1950) states that the hearts of most crustaceans have neurons on the dor sal surface which initiate the excitation wave for the heart beat. These are described as neurogenic hearts as contrast ed to myogenic hearts, in which the heart beat originates in muscular tissue. Isopods have been mentioned as having a ganglionic pacemaker and it is believed that Limnoria has a neurogenic heart. The heart of Limnoria appears as an enlarged tube near the dorsal surface of the pleon between, and dorsal to, the (digestive pouches. Its rate, rhythm and amplitude of beats are altered by varied cation concentrations. In general, speaking of the animal kingdom, the effects of a cation on the heart are multiple, depending on the condition and previous history of the heart. The effects of an ion also vary with the concentrations of other ions, e.g., calcium antagonizes some effects of sodium, and potassium antago nizes some sodium action. Ionic ratios such as Na plus K/ Ca may be more significant than the absolute concentrations of each ion. In general, sodium has a stimulating action on heart pacemakers and favors contraction of heart muscle. Sodium initiates a fast but an irregular rhythm, and when sugar is substituted for sodium the rate of heart beat in most hearts declines. Prosser (1950) states that potassium 13 probably acts differently on invertebrate hearts. It may act predominantly on pacemaker, conducting, or contracting mechanisms. It appears that calcium antagonizes the effect of potassium and sodium on the pacemaker, and slows the heart beat rate. In the arthropods the action of calcium on the pacemaker is predominant, and high calcium slows the heart and stops it in diastole, whereas low calcium acceler ates the heart, and the lack of calcium stops it in systole. Magnesium seems essential for many marine invertebrate hearts, but it. lacks the muscle-contracting effect of calcium. Like calcium in excess it inhibits the pacemaker bringing about diastolic arrest. Many effects on hearts are due to ionic ratios not to single ions. I In Limnoria, some definite relationships appear to exist and these will be discussed later. Experimental work involving the use of lithium. A number of investigators have studied the effects of lithium on biological phenomena. It would seem helpful to briefly summarize these studies even though they may bear uncertain relationship to the present work. In 1902 Overton showed that the effect of lithium ions upon the excitability of sodium free nerve develops in two phases: during the first phase lithium ions restore to the nerve fibers the ability to conduct impulses; during the 14 second phase lithium ions again render the nerve fibers inexcitable* ‘ Runstrom (1935) analyzed the effect of lithium on sea- urchin development. He showed that when lithium is added to natural sea water the cells of the larvae are deformed. Pyocyanine antagonizes this effect. It is believed that lithium effects the structure of protoplasm and respiration, The alteration of development caused by lithium was also shown by Lindahl (1942) in echinoderms. Hall (1942) has demonstrated the action of lithium salts in amphibian dev elopment. He showed that microcephalic defects were pro duced by subjecting the eggs of Rana piplens and other amphibians to lithium salt. This action was intensified by increased time, temperature and lithium concentration and was antagonized by calcium and potassium ions. The defor mation of development of the sand dollar by exposure to lithium chloride before and after fertilization was shown by Rulon (1946). Rulon (1948) wrote on the modification of reconstitutional development in planarians by lithium chloride. Lithium chloride was used before and after section of the planarians and reconstitution was modified in that there was an increased head frequency and increased scale of organization. MacLeod, et al. (1949) demonstrated that li.thium 15 chloride in low concentration inhibited the aerobic and an aerobic glycolysis .of human spermatozoa and destroyed their motility. He furthur showed that the toxic effects of lithium induced in rats were similar to those found in humans. Masson (1949) and Radomski (1950) determined the toxic level of lithium in rats and dogs. Radomski (op. cit.) described the excretion and distribution of lithium chloride as did Talso and Ciarke (1951)>in the dog* Gallego and Lorente de No have published two papers (1947, 1951) on the effect of lithium upon frog nerve with and without sodium. Lithium ions when present in the ex ternal medium of the nerve fibers of a frog at a high con centration were shown to cause a depolarization of the jnerve fibers. In the latter paper they indicated how lithium can partially substitute for sodium. Poulks (1952) demonstrated the excretion of other cations influenced by the presence of lithium. It has therefore been shown that lithium (1) cannot be satisfactorily substituted for sodium, (2 ) interacts with other cations and influences their distribution, (3) alters the development of certain larval forms, (4) may alter normal respiration and glycolysisrahd (5) is toxic in varying concentrations. chapter II MATERIALS AND METHODS A number of artificial sea water solutions were pre pared and tested. It is impossible to prepare solutions that exactly duplicate the properties of sea water. The salts in which the elements occur in sea water are not al ways known, and elements that occur in sea water In small amounts are present as contaminants in other compounds in quantities which may far exceed those that should be added. Many of the salts which must be added in fairly large amounts are hygroscopic or contain water of crystallization and are difficult to weigh accurately. After experimenting with solutions which contained no natural sea water, and after consulting with Prof. Norris W. Rakestraw, physical chemist at the Scripps Institution of Oceanography, it was decided to follow suggestions by Lyman and Fleming (1940) and Rogers (1938). Substances were added which existed in smaller amounts in natural sea water and there was included a one per cent total volume of natural sea water to the artificial sea water solutions in order to assure micro-nutrient requirements. The percentage of the concentrations of many impuri ties which existed in salts labeled "CP" were ascertained and found to be negligible as far as they contributed to 17 sources of error for this study. After running a series of experiments using six dif ferent solutions, the following modification was adopted; Compound Molecular Weight Moles/Liter Millimoles Grams NaCl 58.454 0.447 447 26.129 MgClg^GHgO 203.234 0.025 25 5.081 MgS0 4 120.38 0.025 25 3.010 CaClgiSHgO 146.994 0 . 0 1 0 10 1,470 KGl 74.553 0 . 0 1 0 10 0.746 NaHCOs 84.015 0 . 0 0 2 2 0.168 NaBr 102.913 0 . 0 0 1 1 0.103 E3BO3 59.844 0 . 0 0 1 1 0.060 SrClg 158.544 0 . 0 0 0 1 .1 0.016 NaP 41.997 0 . 0 0 0 1 .1 0.004 NaCl, MgClg.ôHgO, CaClg.SHgO, and LiCl were kept in a desiôcatôrï'. All substances were purchased in sealed con tainers from the chemistry laboratory supply department at the University of Southern California, and all were as pure as could be obtained. At first, the magnesium and calcium chlorides were not weighed directly, but were put in solu tions and the normalities were determined by the Mohr method (titration with AgNO^ with KgCrO^ as an internal indicator). However this was found not to be necessary if the compounds were desiccated._____________________________ 18 An analytical balance was used for the weighings whose accuracy was compared with a Braun chainomatic (with mag netic damper) reading to 0.0001 grams. The analytical bal ance was found to be in error by 0 . 0 0 1 grams. The solutions were prepared in one liter amounts and were stored in glass stoppered one-liter bottles. The effect of the sodium in the glass which would diffuse into the solutions was considered negligible. All glassware was washed in a special laboratory detergent soap, washed and rinsed four times in tap water, four times in distilled water and air dried. The distilled water was purchased by the Zoology Department and any existing contaminating substances were believed to be in very small concentrations which would not be detrimental to the study. However it was reported by a i department worker that a drop of silver nitrate would bring down a precipitate from this water. The equipment needed Included finger bowls, a binoculai^ dissecting microscope, a large aquarium, an aerating unit, bottles of one liter capacity, an analytical balance, a Beckman pH meter, a centigrade thermometer, a large desic cator, measuring cylinders, 1 0 0 and 1 0 0 0 milliliters, a 1000 milliliter beaker, pipettes, 10 and 50 milliliters, a wash bottle, glass stirring rods, small pipettes, rubber 19 bulbed, a concave culture slide, and a stop watch* Limnoria specimens were obtained from the 28th Ave* pier in San Pedro, California* At this collecting point the animals are Limnoria quadripunctata* At the beginning of the study in 1951 a discussion was undertaken with Dr* Shelden (see Preface) concerning species identification* Since thousands of animals were used, and since according to Mr* Laurens Barnard species identification would be burden some, Dr* Shelden believed the study might be completed using the two species Limnoria tripunctata and Limnoria quadrIpunctata* However this was not necessary since the animals were collected at a location almost specific for Limnoria quadripunc tata * The animals were maintained in a large aquarium with an aerator pump* The water level was kept at two inches in depth, natural sea water obtained at the 28th Ave* pier being used* Specimens were allowed to remain in their wood burrows in the tank, and the temperature was not regulated* For each test run in the experiments described in Table I through Table VII, 25 animals were carefully placed in each of four finger bowls after having been rinsed by large volumes of the test solution twice previously, and 50 milliliters of the test solution was added* It was be lieved that most of the contaminating substances were 20 removed from the animals by this rinsing procedure* Small rubber-ended pipettes were used in transferring the speci mens so as not to injure them* Average sized specimens found swimming in the first bowl of natural sea water were selected to avoid using injured animals * A segregation by sex was not made* Each test run included one hundred ani mals with the exception of the runs shown on Tables VIII through XVI, where the numbers of animals used are indicated on the tables* Temperature and pH determinations were made regularly, and observations of the animals were made at 24 hour intervals* Each experiment was considered concluded at the end of a 14 day period although some were carried on for another week or ten days. The few specimens lost by crawling up the sides of finger bowls were not counted as dead from the solution's effect. The range of numbers of animals lost in this manner was from none to four per test solution, which was considered a negligible amount* Of the four per cent total lost this way a certain percentage might have remained alive had they stayed in the solution, so the total error might actually run less than four per cent* Solutions were changed each week because of debris (moultings and feces) which had accumulated* Animals were not given any wood on which to live during the tests, but 21 this was not believed to be a significent factor since the control animals appeared to be quite normal after 21 days in natural sea water. It was not known how much nourishment if any, was provided by the one per cent natural sea water added to each test solution. Animals were counted as dead upon assuming an opaque appearance or on showing an absence of heart beat. CHAPTER III OBSERVATIONS The range of the studies to be emphasized in this work were previously presented in the introduction. After a suitable artificial sea water was adopted, the first experiment undertaken was to determine the effect of altered concentrations of sodium chloride in artificial sea water. This was done so as to understand the animal’s re actions to hypertonic, isosmotic and hypotonic solutions of artificial sea water, in relation to sodium levels. If the organism had shown itself to be exceptionally sensitive to changes in the concentration of the sodium ion, this would be considered in the interpretation of the lithium effect. In all experiments conducted, the normal concentration of NaCl was considered as 450 millimoles. This concen- ; tration was slightly higher than that recently reported by Menzies {1951a) in Los Angeles Harbor in which the sal inity was listed at about 55.8 ,o/oo. The salinity of the normal artificial sea water solutions used in these experi ments was about 36.5 o/oo (Sverdrup et al., 1942). Table I indicated that (1) 36 per cent of the animals died in the 14 day period in a solution in which 300 milli moles of sodium were substituted for the normal 450, but that only 12 per cent died in the same period when 300 m w s fOA/ rm(iJsLZ3 T ' / r s # 0 0 24 millimoles of sodium was added to the normal 450. (2) 100 per cent of the animals died in one day in a solution lack ing 400 millimoles of sodium, but the death rate over a 14 day period was only 28 per cent in a solution containing an excess of 400 millimoles of sodium. (3) The animals could not very well cope with an additional 500 millimoles of sodium. The total effect appeared to be that the or ganism was much more able to accommodate to a hypertonic concentration of sodium, but at a high level of concen tration the death rate increased markedly. Heilbrunn(ppiC105-107, 1943)uses the term "0" value. "O" values are numerical equivalents of osmotic strengths of various salts. Since lithium and sodium (chlorides) have very similar ”G-” values, substitutions were made on a mole for mole basis. The substitutions were made in order to ascertain the effect of lithium as a substitute for, or in combination with, sodium. Sixteen different solutions were prepared and 1 0 0 animals were run in each concentra tion. The solutions ranged from isosmotic to hypertonic a and hypotonic. I s ' ^ / h e n 50 millimoles of lithium were sub stituted for 50 millimoles of sodium in an artificial sea water solution, 88 per cent of the animals died in 14 days. The arbitrarily taken toxic concentration of lithium was chosen as this 50 millimolar level of lithium. (Table II) ~r t / ( ^ / k ' / y ^ À \?h^T*ÿ^ f ^ y t i c TG BZE3W gcr4— * > ' » ’ g f / : ^ 0 / j ^ t ~ 4 " 4 ' ^ ' ^ ' SyA _ / / w/k»L_ , # zp_Æ^(L_: - , S a f 7 J - - r J v > r f e ' € - Y^yo //g^ry 26 From this experiment several facts appear to stand out; (1) A concentration of 75 millimoles of lithium was extremely toxic, whereas the animal could fairly well tol erate 25 millimoles. Thus the toxic concentration of lithium was bound by narrow limits. (2) There appeared to be antagonism of sodium for lithium in that the animal could survive the toxic 50 millimolar lithium level much better if an excess of sodium was present. The more sodium the better able the animal was to withstand the effects of lithium up to a level* when 300 millimoles of sodium were added and beyond this level sodium had no antagonizing ability. Beyond this level the deleterious effects of sodium as such or as an osmotic force outweighed any ad vantage. Table III summarizes some of the results of the pre vious tables and is intended to demonstrate the effect of lithium as compared with the effect of a low sodium con centration. It can be seen that at any level of sodium concentration the result of adding the same toxic concen tration of lithium was more detrimental than when the sodium levels were present without the lithium. Even though sodium antagonized lithium in hypertonic solutions the antagonism was not so complete as to result in a com plete nullification of the effects of lithium. fpP'i [ Ü . m j T o M . i X - . ^ « p S L _ WWH^/r/W/f 3 pQ y v / i , f T z '^<U. _ T f : _ ^ < ) _ _ ^ Y _ C X - — Î O L l^yV//^yUS_Æ "éizdstiz: /7C3 28 To more clearly demonstrate whether the deaths were due to the low sodium concentration, hypotonicity, or lithium toxicity, an experiment on the effects of electro lytes and non-electrolyte8 in low sodium concentrations was completed. Table IV shows that in the same hypotonic concentra tions of sodium more animals died from the effects of 50 I millimoles of lithium added than when lithium was not added and that the isosmotic concentration resulting from the adding of sucrose and sorbitol did not lower the death rate in the hypertonic solutions of sodium in the absence of lithium. It appeared that deaths resulted mainly from the toxic concentration of lithium, but that the number of deaths also increased in lowered concentrations of sodium. Moderate hypotonicity seemed to be a less important factor than the loss of sodium and the presence of lithium. In lowered sodium concentration it might be indicated that sodium was not present in sufficient concentration as to antagonize the lithium. After the effect of lithium on sodium had been ascer tained, experiments were run to determine the action of other cations on the toxic action of lithium. It was be lieved that magnesium might be antagonistic to lithium in that it is antagonistic to sodium. ■60 ' * = ^ / i y ~ /ypWA^ / f / ( ' 4 - z7 ( M ëM - ï f c V i L 7 r - ^ ÆÆÇ L - 4 ^ ' d-Cid^ oa k&( r / j 3w& : - Up. >. A f/icx.. 3 Z zJdS 30 Table V illustrates the effect which magnesium(as its chloride) had on toxic concentrations of lithium. The main control in this experiment was the solution substitu ting 50 millimoles of lithium for 50 millimoles of sodium. In this case, 8 8 per cent of the animals died in 14 days. When 50 millimoles of magnesium was added to this solution, the death rate in 14 days decreased to 40 per cent, so the mortality rate was cut at least to half the original amount. The resulting hypertonic level of the second solu tion was not deemed significantly important in contributing to the decreased death rate. ^Vhen 50 millimoles of lithium was substituted for 100 millimoles of sodium the death rate in 14 days was again 8 8 per cent. However when 50 millimoles of magnesium was j added to this solution, the death rate in 14 days dropped ' to 20 per cent. When only 5 millimoles of magnesium was added to this solution the mortality decreased to 80 per cent. In this experiment the greatest antagonism resulted when a large concentration of magnesium was added making the solution nearly isosmotic. As Table VI indicates, considerable time was under taken to determine the effect if any, of calcium on lithium toxicity. As in the previous experiment, it was O M . I r /»OÀ<èSj jx £ O/0!jfc«^L Æ'ëzf pA^Â<î,c.^ ^h-ûJd 70 52 believed that calcium might prove antagonistic to lithium. The striking results seen with magnesium were not obtained. The two main controls in this experiment were the solutions in which (a) 50 millimoles of lithium were sub stituted for 50 millimoles of sodium and (b) 100 millimoles of lithium were substituted for 1 0 0 millimoles of sodium. An artificial sea water and a natural sea water control were also run. An antagonistic action was apparent when 35 millimoles of calcium were added to a solution containing 50 millimoles of lithium as a substitute for 1 0 0 millimoles of sodium. This solution was nearly isosmotic and the death rate in 14 days was decreased from 88 to 76 per cent. It was also noted that magnesium tended to cause a delay in death rate at the onset of the experiment, a delay of one or two days before any animals died, as com pared with the 50 millimolar lithium control solution. A definite antagonism existed in the solutions con taining 1 0 0 millimoles of lithium as a substitute for sodium. This was a very toxic concentration of lithium and all the animals died within 24 hours. Yet when 100 millimoles of calcium was added only 56 per cent were dead by the end of 6 days, and it took 1 2 days to kill all of the animals. A àoépx^- 04- cbe -. is j0 :rm ’ . ' ' M t/: 4 L A . _ S 3 < S > * __: é yomféfX^Æ L < 3 5 gfc t ^ r 7 s i □ r ï: eu 0 < 7 / O J 2 i / î 4 4 ^ - ^ . . i 5 t 34 Prom an analysis of the remainder of the curves it is clear that calcium did not have as strong an antagonistic action as magnesium. After studying the action of calcium and magnesium on lithium, the concentration of potassium was shifted in a few solutions. Results were not conclusive, although the animal appeared quite sensitive to any significant changes of potassium concentration. In artificial sea water, the total concentration of potassium was 10 millimoles. If this quantity was omitted from a solution containing 50 millimoles of lithium, all of the animals died within 24 hours, but if only half of this quantity was omitted 80 per cent of the animals died within 14 days. If 50 milli moles of potassium was added to solutions containing or not containing lithium, all animals died within 24 hours. Studies were made on the effect of various solutions on the heart rates of the animals. Hypertonic, isosmotic, and hypotonic solutions were used. The heart beat rates of 25 animals were counted in each solution. The average sized animal used.was about 2.5 millimeters in length. The rate of heart beat was counted by trapping the animal dorsal side up in a small piece of toweling paper and covering the body with the solution to be used. A concave slide was used for this purpose. The animals had been x p . . i - - . 3 i a / ? - . u S - 4 , « y _ . o oa re _ SiÆ ÿ K — /Wî^ZT ~~ - 7 Z5^ l _ ^£Z < , _ ---5 W 36 placed in the solution to be studied for one day or more. A dissecting microscope was used at 40 power with a lamp directed at the animal’s body. The light was shifted until the heart could be seen to beat and a pencil and stop watch were used to tap out the beats for periods of time up to one minute. After counting the number of dots made on the paper an average number of beats for each animal in the solution was determined. In hypertonic solutions it was difficult to keep the animals quiet since they were ex tremely active and counting was difficult. Sometimes debris covered the body making it difficult to see the heart. The heart appeared as an enlarged tube, pinkish in color and lying between, and dorsal to, the digestive pouches. Looking down, the lateral edges of the heart seemed to overlap the median edges of the digestive sacs, especially when the heart was at its largest size, (diastole), Table VIII indicates that the heart beat most rapidly in hypertonic sodium solutions in the absence of lithium, up to an additional amount of 400 millimoles of sodium, making a total of 850 millimoles of sodium in the solution. In a solution containing 950 millimoles of sodium there was a sharp drop in heart rate from 290 beats a minute to 175 a minute, and the rate decreased proportionately in - - V ' h f / / '/ y 1^ Ÿ tiÇ5 9 1 j 37^ _ .V A 7 x : r / > M -, r 7 r; { CV y / / \ ■ 1 / /y\ r // 1 Ac: u t Ù _ 0 ( / / V .S ' )G j . j - p < 7 F i V O Ü MiKjrft . X l*i y u [S ' V 1 _ .. % A ( ? />Via '£ § K I ' . t c it k I- " M ■ I t ;c u r" 4 V /t ) k b > O J P % A \| o V .. , C j b i) \f 0 V s . L T » J : u fL , t k a ? o r b ( j 3 "" ' — ■ • ■ " ■ - " ■ 1 P _ _ P p _ V : .A \J 1 o < V . t , L p . f § S i X) t) i 0 v | K l : 1 f ■ _ o g j 0 - 1 0 0 q 0 ■ 1 m : - 0 o g E a » s. Ê t^- T :M 1 * o 0 'L o f i. \ 0 '' i o' / X 1 , Ô — . , „ ( 4 .. - D 1 . 1 si n J 5 , i^ T O J V M J Ü L c j 0 S ( r y 1 / b -s " , 0 s \t ' V k i s . N . 1 < ' X X o * '< ! 0 o P Aj g 0 1 8 , 0 ,y i: ' 0 J k V r n V t; f : ' r 1 ' < o • f - 't - f 4 - I r - < r / r< A f/ v <u £ /f 1 ? 7 " / &7i < • y n 7 Oe d o J S ’ " ■ ' 1 'd 1 p ' . Cjzp« > A :^<5 r i v < ^ ► / p fikf, ■ f J J 4 /ÿr7 /wA ÎT f- r î ? J - A ? r ] /t r k r? '. I / / / / / S / Ü C j/ f d 4 - / r/< C - C-4// r- % ! C | fj e /f7"V // Y _ 38 hypotonie solutions of sodium from N-200 millimoles down to N-400 millimoles (leaving only 50 millimoles of sodium). The rates in natural sea water and artificial sea water were almost the same. The presence of lithium of a toxic concentration in hypotonic or isosmotic solutions de creased the heart rate. Sodium counteracted this effect and as more sodium was added in the presence of a toxic concentration of lithium, the heart beat was gradually brought up to its normal rate. In a solution containing 50 millimoles of lithium and from which 100 millimoles of sodium was removed the heart rate was 140 a minute, but in a solution containing 50 millimoles of lithium in which an additional 200 millimoles of sodium was added, the heart rate reached the average of 250 per minute. Since if 200 millimoles of sodium were removed in the absence of lithium the heart rate was 240 per minute, it appeared that lithium did in effect decrease the heart rate. In solutions containing calcium or magnesium counter acting the effects of lithium, the heart rates were always less than normal, but not as low as when calcium and magnesium were omitted. Calcium and magnesium ions appeared therefore, to counteract the retarding effect of lithium on heart rate. The presence of non-electrolytes in low sodium 39 solutions did not appear to substantially increase the heart rate even though the solutions were isosmotic. Concerning the range of the heart beat rates of animals in a given solution there was a spread of about plus or minus 20 beats per minute. In a separate test with 30 animals the rates of heart beat in animals 2.0 millimeteis in length averaged 10 to 20 more per minute than animals 2.5 millimeters in length, and those 3.0 millimeters in length were 5 to 15 beats per minute slower than the 2.5 millimeter animals. In addition to this study, additional experiments were performed in which the heart beat rates of single isolated animals were studied under varying conditions and for periods of time. (Tables IX, X, and XI) Table IX indicates that the heart of the animal placed in distilled water stopped beating in two and one-half hours. This did not indicate death however, since the heart resumed its beat after the animal was placed in artificial sea water. In distilled water the heart rate appeared to slow down in a linear manner. Animals placed in toxic solutions of lithium containing the normal con centrations of other ions except the'" sodium, had a de crease in heart beat rate to 55 beats a minute at 6° centigrade which increased to 220 beats at 24^ in one hour. m fSC 41 Statistically this experiment is of little value but is included for interest. Table X was a time study over a period of days in which the heart beat rate was observed. It can be seen that as the animals were placed in toxic solutions of lithium, the heart rates steadily decreased until death occurred. The table also shows the rates of heart beats in animals in hypertonic and hypotonic solutions (with respect to sodium); how they lived for days at either high or low rates of heart beat. Table XI illustrated the prolonged effect of lithium. After living for 15 days in a solution containing a toxic concentration of lithium,, the animals failed to recover on being placed in a natural medium. The heart beat seemed to be on the way to a normal rate when the heart failed. It was decided to observe the interaction of tempera ture with toxic concentrations of lithium for Limnoria. As the temperature was reduced to a minimum of 5° centi grade the death rate in 14 days fell from 88 per cent to 60 per cent. Increasing the temperature probably had the reverse effect although in this experiment the effects of the increased temperature and lithium by themselves could not be shown, (see Table Xll) Lithium appeared to have a prolonged effect. Animals m av. - f S C . . i W « u c - s j — ' W\rYO f.>9TOAV 1 m U s , 4 ^ ^ ^ x î _ ^ e $ p 4 7 p g r : / ! _ -zk-Æ /%/! 1 1 L^rciISi<_<:^ 45 exposed 19 days to toxic solutions did not recover com pletely when returned to natural sea water. In fact the death rate continued as before. (Table XIII) The animals could not become conditioned to the effect of lithium. Table XIV illustrates that when animals were placed in solutions containing a toxic concentration of lithium, and managed to live for 15 days, and when these animals were transferred into a slightly more toxic con centration, the death rate continued as if these animals had been placed directly into the latter solution from natural sea water. The animals could not become conditioned to sub-toxic concentrations of lithium, as is evidenced by Table XV. The death rate continued at the same rate as if the animals had been placed directly into the more toxic solution from natural sea water, rather than being brought gradually into it. PH changes in representative solutions are shown in Table XIV. It will be seen that the pH levels off at the end of two days to a value comparable to that of natural sea water. r - J - T ç l /& % C àr lO j r_ ^ ? - 2 r a i m z : < d : ^ L l H/^7T£Æ - / f / e i Æ . Sà ^ ■ rz/Oj^ i^ d t ’ Aj üL'^Z^fu'._ s S z p S ® A ^ ^ ^ o j R Æ t k X - WÆZ> Î D 3 L - ^ Ï - A 4 — - :^6g_Sâ&2L^2Z= — ----------------- Ï r î i a ^ O i :<4^4X ~]^izrlik'immzs:i-i :i'$£zivi£75£$ /D l CHAPTER IV DISCUSSION Prom results obtained from preliminary studies it was known that if a certain concentration of lithium was sub stituted for a like concentration of sodium a very high death rate would occur. The actual mechanism that caused the death could only be conjectured until further experi mentation was undertaken. For example, it was not known whether the animals were dying from effects due to a low ered sodium concentration, a hypotonic situâtion or from toxic lithium effects. Therefore, a fairly large mass of data was obtained which indicated that the animals were dying primarily from toxic effects of lithium, and only when a considerable concentration of sodium was removed, did lowered sodium play an important role in the death of the organism. Since the use of non-electrolytes in main taining osmotic pressure failed to lower the death rate, the factor of hypotonicity appeared a minor one. The studies indicated that several cations were antagonistic to lithium. These were magnesium, sodium and calcium. It was established that an Increased concentration of these ions quite decisively antagonised the toxic effects of lithium. Using a given toxic level of lithium which resulted in an 88 per cent death rate in 14 days, sodium decreased this 51 rate to 28 per cent, magnesium to 20 per cent, and calcium to 76 per cent. The antagonisms of sodium and magnesium are therefore quite striking. The heart rates of animals subjected to various modi fications of ionic concentrations were correlated with the findings and it was shown that increased lithium and low ered sodium depressed the heart rate. Hearts beat faster in hypertonic sodium solutions than in hypotonic solutions and increased sodium counteracted the effect of lithium in slowing the rate, and the sodium concentration could be altered to bring it back to the normal rate. Magnesium and calcium counteracted the lithium effect but hearts did not increase in beat to the normal value. It was indicated that as f i u i animal was dying from the toxic effects of lithium its heart beat rate was steadily decreasing to zero, shortly after .which time death occurred. Animals were not able to adjust or condition themselves to the effect of lithium and continued to die at the ex- pec tèd rate when shifted from one solution into another. They also appeared unable to recover from the effects of lithium on being placed in a normal environment after a long subjection to lithium, and the death rate continued as before. Decreased temperature evidently lowered the animals * 52 metabolic rates to such a degree that the effect of lithium was greatly lessened or merely delayed, (from 88 to 60 per cent death rate). It was known previously that the animal could survive in strongly hypotonic solutions and this study indicated an even greater ability to adjust to extremely hypertonic solutions• It was found that for several days after the solutions were prepared, whether the animals were in them or not the pH did not stabilize at the normal natural sea water level (8.1-8.3) for several days. This was not considered a sig nificant factor in introducing error since tests starting animals before and after this stabilizing gave almost identical results. It is believed that more work should be done in studying the phenonemon of pH stabilization in light of the consistently long time it took to occur. This study has its value in laying the foundation work for further experimentation on the actual ionic mechanisms, with perhaps specific studies on isolated nerve and heart tissues in respect to effects caused by cation shifting. There is no real way to understand just what the actual mechanisms were in this study until further work is done. The heart of Limnoria is believed to be neurogeni- cally controlled (Prosser 1950) and it may be that these 53 ionic effects are intimately connected with the interrup tions of the nerve impulse in nerve and muscle tissue# It may be that lithium is displacing the sodium, especially so in lowered sodium concentrations and thus interfering with the action potential# It may be that a sufficient concen tration of sodium, magnesium, and calcium somehow blocks this action# All this is purely conjectural in nature and it is not the purpose of this study to indicate the specific ionic mechanisms. CHAPTER V SUMMARY To summarize the results it was shown that: (1) Limnoria was much more able to accommodate to a hypertonic concentration of sodium, but at a high level of concentration the death rate increased markedly. (2) When 50 millimoles of lithium was substituted for 50 millimoles of sodium in an artificial sea water solution, 88 per cent of the animals died in 14 days. 75 millimoles of lithium was extremely toxic but the animal could fairly well tolerate 25 millimoles. Thus the threshold toxic level was bound by narrow limits. (3) The deaths of Limnoria were mainly due to lithium toxicity rather than low sodium concentration or hypotoni- 'city. Moderate hypotonicity was a less important factor than the loss of sodium and the presence of lithium. (4) Magnesium, sodium and calcium were shown to be antagonistic (in that order) to lithium. The antagonisms of sodium and magnesium were quite striking. (5) Increased lithium and lowered sodium depressed the heart rate. Hearts beat faster in hypertonic sodium solutions and increased sodium counteracted the effect of lithium in slowing the heart beat rate. Magnesium and calcium counteracted the lithium effect to a lesser degree. 55 As an animal was dying sfrom the toxic effects of lithium its heart beat rate was steadily decreasing to zero. (6) Decreased temperature evidently lowered the animal’s metabolic rate to such a degree that the effect of lithium was greatly lessened or delayed. (7) Animals were not able to adjust or condition themselves to the effect of lithium. They appeared unable to recover from the effects of lithium on being placed in a normal environment after a long subjection to lithium. LITERATURE CITED A. BOOKS Cushny, A. R., "Pharmacology and Therapeutics." Lea and Pebiger, Philadelphia, 1940. 852 pp. Harrow, Benjamin, "Textbook of Biochemistry." W. B. Saunders Co., Philadelphia, 1950. 609 pp. Heilbrunn, L. V., "General Physiology.” W. B. Saunders Co. Philadelphia, 1943. 748 pp. Hober, Rudolf, "Physical Chemistry of Cells and Tissues." The Blakiston Co., Philadelphia, 1945. 676 pp. Menzies, R. J., "Limnoria." A Dissertation, U. S. C., 1951a. 538 pp. Prosser, C. Ladd, David W. Bishop, Prank A. Brown, Theodore L. John and Verner J. Wulff, "Comparative Animal Physiology.” W. B. Saunders Co., Philadelphia, 1950. 888 pp. Rogers, C. G., "Textbook of Comparative Physiology." McGraw-Hill, New York, 1938. 715 pp. Sollmann, T., "A Manual of Pharmacology." W. B. Saunders Co., Philadelphia, 1948. 1132 pp. Sverdrup, H. U., M. W. Johnson, and R. H. Fleming, "The Oceans, Their Physics, Chemistry and General Biology." Prentice-Hall, Inc., New York, 1946. 1087 pp. 57 B. PERIODICAL LITERATURE Corcoran, A* C., R. D. Taylor and I. H. Page, "Lithium poisoning from the use of salt substitutes." J. A. M. A. 139; 685-687, 1949. Poulks, James G., "Renal excretion of cation in the dog during infusion of isotonic solutions of lithium chloride." Am. J. Physiol. 168: 642-649, 1952. Poulks, James G., Gilbert H. Mudge, and Alfred Gilman, "Effect of lithium on the renal excretion of Potassium. Fed. Proc. 9: 41-42, 1950. Gallego, A., and R. Lorente de No, "On the effect of sever al monovalent ions upon frog nerve." Jour. Cell, and Corap. Physiol. 29: 189-206, 1947. Gallego, A., and R. Lorente de No, "On the effect of ammonium and lithium ions upon frog nerve deprived of sodium." Jour. Gen. Physiol. 35: 227-244, 1951. Hall, Thomas S., "The mode of action of lithium salts in amphibian development." Jour. Exp. Zool. 89: 1-34, 1942. Hanlon, L. W., M. Romaine, P . J. Gilroy, and J. E. Deitrick, "Lithium chloride as a substitute for sodium chloride in the diet." J. A. M. A. 139: 688-692, 1949. Lindahl, Eric, "Einige Bemerkungen zu der Arbeit Lithium und Echinoderm Exogastrulation." Protoplasma 36: 58 558-570, 1942. Lyman, John, and R. H. Fleming, "Composition of sea water." Jour. Marine Res. 3: 134-146, 1940. MacLeod, John, R. C. Swan, and O. A. Aitken, "Lithium: Its effect on human spermatozoa, rat testicular tissue and upon rats in vivo." Am. Jour. Physiol. 157: 177- 183, 1949. Masson, 0., "Toxicity of lithium chloride in rats." J. A. M. A. 139: 685-687, 1949. Menzies, Robert J., "A new species of Limnoria (Crustacea: Isopoda) from Southern California." So. Calif. Acad. Sci. 50 (L): 86-88, 1951b. Overton, E., "Beitraege zur allgemeinen Muskel und Nerven- physiologie." Arch. ges. Physiol. 92: 346-361, 1902. Radomski, J. L., "The toxic effects, excretion and distri bution of lithium chloride. " Jour. Pharm. and Exp. Therapeutics 100: 429-444, 1950. Rulon, 0., "Modifications of sand-dollar development by exposure to lithium chloride and sodium thiocyanate before and after fertilization." Physiol. Zool. 19: 58-86, 1946. Rulon, 0., "The control of reconstitutional development in planarians with sodium thiocyanate and lithium chloride. " Physiol. Zool. 21: 231-237, 1948. 59 Runnstrom, J., "Analysis of the effect of lithium on sea- urchin development." Biol. Bull. 68: 379-384, 1935. Stern, R. L., "Lithium chloride poisoning." J. A. M. A. 139: 710-712, 1949. Talbott, J. H., "Use of lithium salt as a substitute for sodium chloride." Arch. Int. Med. 85: 1-10, 1950. Talso, P. J., and R. W. Clarke, "Excretion and distribution of lithium in the dog." Am. J. Physiol. 166: 202- 208, 1951. University of Southern California Lfbrarv

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Creator Hinshaw, Lerner B. (author)

Core Title Some effects of partial substitutions of lithium for environmental sodium on Limnoria species

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Degree Master of Arts

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c39-277784

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