MONSTROSITIES PRODUCED BY THE INJECTION OF SELENIUM SALTS INTO HENS' EGGS1 KURT W. FRANKE, ALVIN L. MOXON, W. E. POLEP AND W. C. TULLY Chick monsters were consistently produced in eggs from localities where low hatchability had been observed and where toxic foodstuffs were produced (Franke and Tully, '35). I n order to prove that the foodstuff, and not the quality of the stock, was the causative factor, chicks of known stock were fed toxic grain produced in the localities where low hatchability and toxic foodstuffs are common (Tully and Franke, '35 ; Franke and Tully, '36). Following the clue provided by Robinson ( '33) who demonstrated the presence of selenium in a sample of toxic wheat, selenium salts were injected into normal eggs, thereby proving that selenium was the causative agent in the production of monsters. The methods employed were as follows :Eggs were candled to outline the air cells. The area over the air cell was swabbed with an iodine solution, and the selenium solution was injected by puncturing the shell with a hypodermic needle. The punctures were then closed by applying discs of thin paper dipped into egg white. Various concentrations of selenium were used, both in the form of sodium selenite and sodium selenate. I n estimating the concentration, it was assumed that the average egg white plus yolk weighed 50 gm. 'Published with the permission of the director of the South Dakota Agricultural Experiment Station as communication no. 19 from the Department of Experiment Station Chemistry and is Part XI1 of L ' A New Toxicant Occurring Xaturally in Certain Samples of Plant Foodstuffs." 15 16 KURT W. FRAXKE AND OTHERS The eggs were incubated according to the usual procedure. Some lots were opened and examined on the fourteenth day; others were allowed to incubate the full time. The eggs not hatching were opened on the twenty-second day. Some of the embryos were normal while others sliowed extreme deformities. OBSERVATIONS The 14-day-old embryos in figure 1 are from eggs injected with 0.6 parts per million of selenium as sodium selenate. A illustrates a chick with the upper beak slightly shortened and feet undeveloped; B has a still shorter upper beak; G has both eyes and upper beak missing; not only illustrates the underdeveloped, spindly body but also an encephalocelic condition; E shows still less body development, with eyes, beak and legs missing ; F has normal eye, beak and leg formations, but it is ectopic. A and E are edemic, but this condition also occurs in embryos that are otherwise normal. Malformations of the upper beak, following injection of 0.6 parts per million of selenium are best shown in figure 2. A illustrates a control head; B shows the upper beak slightly shortened, and it will also be noticed that it is not fully developed; C and n show the increasingly severe types of snppression with eyes also missing. E , figure 2, sliows the lack of eyes and upper beak when 0.1 parts per million of selenium was injected as sodiiim selenite, and F illustrates the same condition when only 0.01 parts per million was injected. Because of the detectioH of vanadium in a sample of toxic grain (Byers, '34), vanadium as sodium metavanadate and selenium as sodium selenate, were injected in the quantities : 0.1 to 0.6, 0.2 to 0.6 and 0.3 to 0.6 parts per million. The edemic condition was apparently greatly increased in the embryos obtained from these eggs. The greatest number of abnormalities were observed in the embryos obtained from eggs injected with selenium in concentrations between 0.6 and 0.8 parts per million either in the form of sodium seIenite o r sodium selenate. Above these eoiiccntrat i oils dcvelo pinen t was ci tlicr (111 tire1y prcvcti te(1, o r il high mortality occwrred in ttic embryos which had started to develop. T n conwntrations bclow 0.6 parts per inillion a grcatei. peiwmtage of normal appwring emhryos w.vei*ct pro- duced. ( Fig. 1 A , upper beak slightly short. Fcet rniusing. 13, upper bzak stubby. , eyes and upper beak missing. D,eyes ;ind uppc'~ beak iiiissing. Ericep!iiiloccalcB. 73, abnormal devcloprririit of e q w , u p p ~beak a d Icgs; IT, retol)ic 0.6 AtJout 0.46 in Sire. schiiuui i(8 sutliunl sc*k~latc~. ~.~.III. Traces of arsenic were fouiid to 1)c present in tlie soil on u-liiclithe toxic fooclstufls arc produced (Byers, '34). Therefore, undcr the same techic* of injection as rcleiiium, arsenic. 18 XTJRT W . F I t A N K l ? A N D OTHERS was i n j e c t d in a concenti*ation of 0.1 parts per. million or more ws sodium arstmite. This caused a high mortality in the tlevelopiiig cmhryo or prcventetl development of tlic ~ g g . Fig. 2 i f , noriiiul bead. 13, upper beak uiider-dPvcloped. C , upprr beak stnhby. 11, errs and upper Iwak missing. 23, G xiid 1). 0.6 p.p.rri. xoleninm as sndinm wleimte. Fourteen-tlsy-olt1 embryos. Blagnifiratioii x 1.6. E , 0.1 p.p.rn. ~elrmumas sodium selenitt,. k‘, 0.01 p.p.1n. st~leninm:IS sodiuni wlcnitc.. E and E’, 22-day old emhrpos. 0.8 in sim, i V i tli cwiicciitratioiis of 0.01 part p r million, a liigli pereentagt. of normals were producctl. ‘I’hc only type of abnormality pi*oducecl by wi*stJnic injections was that of tho cctopic conditioii. TABLE 1 A summary of the results obtained by the injection of selenium, arsenk, lead, and fluorine ions Sodium selenite Selenium 0.9 0.8 0.7 0.6 0.5 0.1 0.02 0.01 PRO.cent per cent per cent 24.4 53.8 34.3 20.0 31.0 26.0 50.0 8.3 2.3 3.8 9.4 20.0 56.5 25.0 0 41.7 73.3 42.3 56.3 4.8 0 5 16 4 4 12 131 78 64 Sodium selenate Selenium 1.0 0.8 0.7 0.6 0.5 21 15 11 26 40 18 36 76.2 13.3 27.3 7.7 17.5 22.2 11.1 19.0 86.7 18.2 57.7 5.0 27.8 0 34.6 77.5 50.0 88.9 0.05 6 7 0.01 15 83.3 14.3 0 0 14.3' 6.7' 16.7 71.4 93.3 0.4 0.1 54.5 Arsenio 0.1 Lead acetate Lead 1.0 0.8 0.6 Fluorine 1.0 0.8 0.6 0.4 15 16 16 15 20 18 17 87.5 33.3 15.0 11.1 5.6 0 0 22.22 0 86.7 85.0 66.7 88.8 20 KURT W. FBAKKE AN D OTHERS Fluorine has also been detected in well waters in these areas (unpublished data), and it was injected into eggs as sodium fluoride. It appears to be much less toxic than selenium, and the only abnormality produced was the ecotopic condition. Since the literature mentions lead as the the only element which has produced monsters (Fere, 1893; Hammett and Wallace, ,28) it was injected as lead acetate. Like fluorine, it seems to be less toxic than selenium, but it was similar to arsenic and fluorine in that it produced only ectopia as the abnormality. A summary of these various injections is tabulated in table 1. DISCFUSSION I n a former paper, a review was made of the different factors which might produce abnormal developrncnt (Franke and Tully, '35). Among these was that of chemical treatment to which Stockard ( '20, ,2l) referred as follows: The production of abnormal development and malformation of organs by treating eggs with strange chemical materials is brought about in a similar manner to the abnormalities following stoppinp The part or organ developing at the most rapid rate is inhibited more decidedly by treatment than are the less rapidly developing parts, and is, therefore, most affected or modified in its development. Child ( ? Z S ) summarizes a series of investigations showing that when inhibiting agents such as KCN, HCI, CuSO,, C,H,OH, and others, are applied just after fertilization, the highest susceptibility is at the apical region, causing microcephalia and inhibiting more or less the posterior growing regions. If, however, the exposure to an inhibiting agent is at a late stage after the head region has undergone part of its development and the posterior part has attained a high susceptibility, megacephalia and the inhibition of the posterior region will occur. I n his paper he states: To concentrations or intensities of agents which are above the limit of tolerance but not immediately lethal for the form or stage concerned, the most susceptible regions arc, in general, the most active physiologically, and susceptibility decreases with such activity along the axis. EFFECT OF SE O N EMBRYO DEVELOPMEKT 21 It is of interest to note that the injections with arsenic, lead, and fluorine, seem to conform to the limit of tolerance. However, lesser concentrations do not produce the abnormalities that occur when selenium is injected, as only an ectopic condition occurred. Fere (1893) injected solutions of codeine, morphine and lead nitrate into the egg white and reported the occurrence of monsters. The amouIit of lead injected was equivalent to 31.2 and 52.6 parts per million, if the egg white and yolk totaled 50 gm. Hammett and Wallace ('28) stated that the lead ion retarded growth and that the differential development of the head and optic regions was markedly inhibited. When based on 50 gm. of egg white and yolk, 3 cc. of a 0.05 per cent lead ion solution (lead nitrate) would be equivalent to 5 parts per million. The higher concentrations of lead ions, or else the influence of nitrate ions, caused the monster formation that was not noticed with injections of 1.0 part per million, or less. Landauer ('29) found that lithium was more poisonous for the chick embryo than magnesium, but states that no malf ormations occurred under the influence of these elements. Based on 50 gm. of egg white and yolk he injected practically 7.14 and 28 parts per million of lithium as lithium chloride or bromide, and 14 to 28 and 56 parts per million of lithium as lithium sulphate. The magnesium ion was injected on the basis of 24, 49, and 97 parts per million as magnesium chloride, bromide or sulphate. In table 1, a great variation as to the percentage of abnormal, dead, and normal embryos can be noticed. It seems plausible that a number of factors may enter here, such as, stage of cleavage reached before injections were made, the small number of embryos obtained in some groups, and also the vitality inherent to the egg itself. Injections of selenium salts have been made up to the seventh day of incubation and, contrary to expectations based on the theory of susceptibility gradients, abnormal eye and 22 KURT W. FRANKE AXD OTHERS beak formations were produced as late as the seventh day, even though the apical regions had reached an advanced stage of development. SUMXARY The injection of selenium salts into the air ceIl of hen’s eggs before incubation produced monsters similar to those occurring naturally. Sublethal concentrations of arsenic, fluorine, and lead ions produced ectopic conditions, but no monstrosities such as produced by selenium. LITERATURE CITED EYERS,H. G. 1934 Selenium, vanadium, chromium and arsenic in one soil. Ind. Eng. Chem., News Ed., vol. 12, p. 122. CHILD, C. M. 1928 The physiological gradients. Protoplasma, vol. 5, pp. 447-476. FEILE, M. CII. 1893 Note snr I’influence des injections de liquides dans l’albumen sur l’incubation de l’oeuf de poule. Compt. rend soc. biol., vol. 45, pp. 787-789. FRANEE, K ~ W., T A ~ W. D G. TULLY 1935 A new toxicant occurring naturally in certain samples of plant foodstuffs. V. Low hatchability due t o deformities in chicks. Poultry Science, vol. 14, pp. 273-279. 1936 A new toxicant occurring naturally in certain aamples of plant foodstuffs. VII. Edonsters from eggs produced by hens fed toxic grains. Poultry Scienoe, vol. 15. HAMMETT,FEEDERICK S., AND VILMAL. WALLACE1928 Biologp of metals. VII. Influence of lead on the development of the chick embryo. J. Exp. Med., vol. 48, pp. 659-665. L A N D A WALTER ~, 1929 Experimental studies concerning the development of the chicken embryo. I. The toxic action of lithium and magnesium salts. Poultry Science, vol. 8, pp. 301-312. ROBINSON, W, 0. 1933 The determination of selenium i n wheat and soils. J. Assoc. Off. Agric. Chem., vol. 16, p. 423. STOCKARD, CHARLES R. 1920-1921 Developmental rate of structural expression : An experimental study of twins, double monsters and single def ormities, and the interaction among embryonic organs during their origin and development. Am. J. Auat., vol. 28, pp. 115-277 1935 A new toxicant occurring naturally TULLY,W. C., AND KURTW. FRANKE in certain samples of plant foodstuffs. VI. A study of the effect of affected grains on growing chicks. Poultry Science, vol. 14, pp. 280284.