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Lead poisoning of ducks in the lower Fraser valley of British Columbia : a chemical study Malysheff, Andrew 1951

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LEAD POISONING OP DUCKS IN THE LOWER FRASER VALLEY OF BRITISH COLUMBIA: A CHEMICAL STUDY by ANDREW MALYSHEFF A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in the Department of Zoology We accept.-'this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS. •Members of the Department of Zoology THE UNIVERSITY Of BRITISH COLUMBIA August,1951-ABSTRACT Macroscopic examinations of ducks in a hunter k i l l sample for the presence of ingested lead shot pellets in the gizzards indicated that a slight increase in active lead poi-soning since 1 9 ^ 7 , had taken-place among mallards (Anas platy-rhynchos) and that a more severe Increase had occurred in pintails (Anas acuta). Instances of proventrlculii stuffed with food occurred only in conjunction with a diet of oats or wild seed. Quantitative analysis of hone and liver ash by means of a specially designed dithizone-chloroform method was carried out to determine lead contents and their signi-ficance. Analysis shoiired that a considerable number of ducks of both species, though proportionately morehmallards than pintails were survivors of past contamination by lead and that, under normal conditions of weather and availability of food and perhaps other factors, the death t o l l attributable to plumbism should, not equal the active leading percentages obtained by gizzard examination. Pintails appeared to suffer more, both in terms of actual incidence of leading as well as their apparent inability to offer as much resistance as mal-lards, to the effects of lead. Though there appeared, in mallards, to be no significant difference in the probability of ingesting lead shot and the survival expectancies between the sexes in juvenile ducks, a lower survival of adult females was recorded. A lower survival was also recorded In juvenile mallards as compared totthe adults. Adverse weather condi-tions, probably expressing themselves as an effect on the availability and quality of food, appeared to give lead poi-soning an opportunity to exert greater influence. Although the current lead deposition taking place in any one year appears to be responsible for marked increases in the i n c i -dence of lead poisoning, evidence Indicated that lead shot is also available to ducks either on the nesting grounds or on the hunting grounds from the deposition of previous years. i CONTENTS I. INTRODUCTION 1 II. STATEMENT OF PROBLEM 3 III. PREVIOUS INVESTIGATIONS 5 A. Lead poisoning in waterfowl . 5 B„ Physiological action of lead ... 11 IV. DEFINITION OF STUDY AREA 15 V, PROCEDURES, METHODS AND MATERIALS 16 A. Collection and preparation of specimens 16 B„ Chemical analysis 20 VI.. DISCUSSION OF" PROCEDURES AND METHODS 2k-VII. RESULTS 2& A. Macroscopic examination of the gizzard for active or current leading 22 B. Chemical analysis of bone and liver for lead 30 VIII. DISCUSSION 36 IX. CONCLUSIONS 52 X. SUMMARY , 55 XI. SUGGESTIONS FOR FUTURE INVESTIGATIONS 59 XII. ACKNOWLEDGEMENTS 62 XIII. PREPARATION OF REAGENTS $7 i i LIST OF TABLES AND FIGURES Table I. Incidence of lead shot in hunter k i l l sample..63 II. Results of chemical analysis for lead in mallards 64 III. Results of chemical.-analysis for lead in -Din-t a i l s 6g IV. Results of chemical analysis for lead in miscellaneous species 70 V. Total incidence of lead poisoning (active. and previous cases) 72 VI. Distribution, of affected, mallards and pintails of the hunter k i l l . . . . .73 VII. Previous leading of mallards in relation to sex and age 74 VIII. Previous leading of mallards in relation to sex and age taken together ....... ,74 IX. Number of mallards affected .in relation to time of year .75 X. Results of chemical analysis on 11 mallards found sick and unable to f l y or dead ,7b XI. Observations of gizzard conditions of pin-t a i l s in relation to extent of active leading and bone and liver lead contents . . . . 77 .XII. Observations of gizzard conditions of mallards in relation to extent of active leading and bone and liver lead contents .....79 XIII. Observations of gizzard conditions of 11 mallards taken sick or dead in January 1950 in relation to extent of active leading and bone and. liver contents of lead ell XIV. Chemical analysis for copper, iron and zinc...$3 Figure 1. Map of the study area. 8" 6 LEAD POISONING OF DUCKS IN THE LOWER FRASER VALLEY OF BRITISH COLUMBIA: A CHEMICAL STUDY BY A. MALYSHEFF I. INTRODUCTION Lead poisoning of waterfowl as a result of ingesting loose lead shot found on the hunting grounds has been found to he a major destructive Influence. As a decimating factor i t arises from and complements increasing hunting pressure from a greater number of guns and gunners, ease of modern transportation and the congestion of hunters at choice hunt-ing spots where tons of lead in the form of loose shot are accumulating, often remaining readily available to ducks and other waterfowl. As with many other decimating factors, l i t t l e , atten-tion was at f i r s t given to lead poisoning or plumbism in game waterfowl. Early references went l i t t l e further, in most instances, than to record i t s occurrence. As the growing extent of the sickness became apparent along with the decline .in the numbers of ducks caused by the advance of c i v i l i z a t i o n into nesting areas, larger human populations and increased hunting pressures, the trouble became recognized as an addi-tional hazard to duck populations. More references on the subject of lead poisoning began 2 to appear in conservationist and w i l f - l l f e management l i t e r a -ture and authorities began to cast about for possible ways of removing this danger to an already seriously threatened game type. Emphasis in the study of lead poisoning gradually began to shift from the mere recording of occurrences to experiments in the correlation of the effects of the trouble with duck diet, development of non-toxic shot, the use of fluoroscopy to sutyd systemic and gizzard lead and the application, of chemical analysis to a study of lead contents of the various organs of the bodies of waterfowl. 3 II. STATEMENT OF THE PROBLEM In September of 1949 the author was assigned to an investigation into lead poisoning to further the study of the condition generally and to obtain more detailed data on i t s extent in ducks on the Pacific Flyway and the Lower Fraser River Valley of British Columbia in particular. The work was to consist primarily of a quantitative chemical analysis of various organs of ducks in a hunter-kill sample to determine the lead contents and their significance. On the basis of work by Adler (l) with geese and experimental knowledge of the action of lead in the mammalian body appearing In litera - v ture, i t was7 apparent that the most significant information would be obtained from treatment of what appeared to be key organs In plumblsm cases, namely the l i v e r and bones, parti-cularly those of the leg.; It was anticipated that the determination of the lead contents of these two organs would serve to indicate: 1. Difference in lead contents between healthy and lead poisoned ducks. 2. Possibility of survival which would be indicated by the absence of ,lead pellets in the gizzard in conjunction with high lead contents of the bone and low or almost normal lead contents of the l i v e r . 3. Possible extent of survival. 4. Total incidence of lead poisoning, past and present. 4 5. Some idea of the course of the poisoning, resist-ence of the various species, different rates of Incidence and survival between sexes and ducks of different ages. 6. Incidence of the disease at various times during the hunting season and any change in the severity with change in weather conditions and availability of food. 5 III. PREVIOUS INVESTIGATIONS A. Lead Poisoning In Waterfowl. The poisoning of game fowl by spent lead shot had been noted as early as the 1870*s. Phillips and Lincoln (2b) mentioned the occurrence of poisoning in waterfowl in 1874 and the poisoning of pheasants was described In 1876 by Calvert (8) and in 1882 by Holland ( 17) . Reports by Grinnell (lb) and Hough (18) in 1894, discussed the effects of lead poisoning on wildfowl. G-rinnell (lb) f i r s t began to make detailed descrip-tions of the condition in 1901. In 1908 (Bowles (7) noted the the incidence of lead poisoning on the Nisqually Flats, a large marsh in the Puget Sound area. In the same year McAtee (22) reported that canvas-backs were being affected by the disease In the Lake Surprise area in Texas. Some of the f i r s t experimental work on the subject of lead poisoning in waterfowl was carried out by wetmore (37) In 1919. In 1916 he had made note of the disease among mal-lards, pintails, canvasbacks, whistling swans and marbled god-wit at the northern end of the Great Salt Lake in Utah. He drew up the f i r s t complete symptomology of the disease, noting in particular: 1. paralysis and wing-droop; not always symmetrical loss of function. This he attributed to the b loss of function of the nerves supplying the pectoral muscles. 2e emaciation, particularly of the pectoral muscles. 3. thin, watery, green feces. 4 . bright eyes. 5. weakness and heart spasms which were often the direct causes of death. 6. good appetites. Post mortem examination showed that: 1. the flesh of the bird was pale. 2. blood was slow to coagulate. 3. heart stopped in systole. 4 . in chronic cases the pericardium was usually dis-tended with a watery lymph. 5. proventriculus and oesophagus were greatly dis-tended with food due to the paralysis of the giz-zard muscles. 6. intestines were irritated and discolored. 7. pads of the gizzard were corroded and sloughed off easily. 2. ga l l bladder was f u l l and sometimes distended. 9. bile was dark green. (Anticipating a trend which would be increasingly f o l -lowed in the future Wetmore began experimentation in the ef-fects of the disease.. He attempted to establish a lethal dosage by treating wild mallards, taken when young, with varying amounts of lead shot. He found that six pellets of No.6 shot were always fatal hut that even one pellet would sometimes result in death. He stated that plumbism is a dangerous and usually fatal malady and proved that the lethal agent in lead shot was the lead i t s e l f and not the impurities or any of the necessary constituents of lead shot. He quoted Cole to the effect that lead administered, to male domestic fowl had a powerful effect on v i r i l i t y . Many eggs were i n f e r t i l e and there was a higher percentage of deaths of embryos. Fairly detailed observations and investigations into the problem of lead poisoning were made by Shillinger and Cottam (29) in 1937o Symptoms described by them agreed with those reported earlier by we'tmore. They found that a lethal dose could vary from a single No.5 shot to many and that the losses were most prominent In winter. They compiled reports of the incidence of the disease from many parts of the United States such as North Carolina, Pamlico Sound of Virginia, Delaware Bay in New Jersey, in northern Ohio and Boyd Lake, Colorado. In the Chesapeake Bay region they found that two out of twenty ducks were carrying lead and that two more showed evidence of lead poisoning. Pirnie (27) In 1935* had.repor-ted that lead took the greatest t o l l over a l l diseases In the Great Lakes. Cottam (11) in 1939 reported an amazing extent of ingestion of shot by lesser scaups at Lake Puskeway, Marquette, Wisconsin in the spring hunting season of 1909? 76o5$ of the ducks were dosed with from 1 to 5$ shot per individual. With reference to British Columbia, Munro (24), in 1925, described the lead poisoning of several swans on Vancouver Island. Pathological reports made subsequently-paralleled the observations noted by Wetmore. In 1943, Munro (25), writing generally on the waterfowl of British Columbia, stated that lead poisoning was a restrictive fac-tor of unknown proportions, seldom occurring in the Interior of the Province but common in the Lower Fraser Valley where hunting was more concentrated. He reported that twenty out of ninety (13.3$) of the mallards shot contained lead pellets. He suggested also, that some evidence existed that mallards may develop a resistence to the effects of lead. Citing the example of two mallards that contained very worn-down shot but which were apparently normal and showed no evidence of the a f f l i c t i o n . Further studies in the Fraser Valley were conducted by Tener (33) i n 1948 who found active lead poisoning in 16.1$ of mallards and 5$ in pintails in a hunter k i l l sample. Tener further found that no widgeon or green-winged teal contained lead. Observing the small size of grit used by these two species Tener suggested that those ducks which ingest lead shot pick up the pellets as grit rather than food. 9 With plumbism assuming greater and greater importance, more workers began to follow the example of Wetmore and the amount of experimental work on the subject increased. In 1945 Cheatum and Benson (lO) studied the effects of lead poisoning on the reproduction of mallard drakes. Cottam (12) had suggested that s t e r i l i t y was an after-effect in those ducks which had survived the ravages of the disease. Weller (36) working with guinea pigs in 1915 con-cluded that chronic lead poisoning had a definite blastoph-thoric effect which could best be demonstrated on male germ plasm resulting in s t e r i l i t y , reduction In birth weight and increase in the number of deaths of young in their f i r s t week. That i t was the germ plasm already undergoing maturation and not the germinal epithelium that suffered was indicated by the fact that reproductive powers recovered after cessation of lead dosing* However, Cheatum and Benson found that no effect on f e r t i l i t y could be ascribed to lead poisoning on the basis of their data nor did there seem to be any impair-ment of hatchabllity or any Increase in mortality In the f i r s t seven weeks of l i f e . Adler (l) mentions Magath as being one of the f i r s t to chemically analyse the organs of waterfowl for lead. Following the example of Magath chemical analysis of the or-gans of waterfowl poisoned by lead was carried out by Adler (l) in 1944. He found that the amount of lead in the bones of lead poisoned Canada geese showed no correlation with the 10 amount of lead being carried in the gizzard and concluded that the increased lead content of the bone is more a measure of the length of exposure than of dosage. The amount of lead in the l i v e r bore closer relation to the amount of lead in the gizzard. He suggested that the li v e r was the best organ to use for the diagnosis of lead poisoning since, i f lead i s being absorbed from the alimentary tract i t w i l l be found in that organ while analysis of the bones at the same time would indicate whether or not the poisoning i s chronic. Jordan and Belrose (2l) reported in 1950 on some interesting work carried out. to determine the influence of diet on the course and effects of lead poisoning and on the development of shot that would be non-toxic and otherwise harmless to game waterfowl. They concluded that the nature of the diet was a more Important variable than the dosage of lead and that ducks on a diet of green, leafy material were less susceptibel to the disease than those on a seed diet. They further stated that 60% to g0$ of mallard drakes carry-ing one shot pellet in nature were l i k e l y to succumb i f they depended on a diet of seeds. They also confirmed the obser-vation of others, including Wetmore, that game farm mallards or ducks which had been kept for a considerable period-in captivity were less susceptible to lead. The latter fact was also brought out in a study by the I l l i n o i s Natural History Survey (19) In August 1 9 5 0 « This, along with other observations led to the suggestion 11 that there might be a correlation between the amount of food, taken and the expression of lead poisoning symptoms. It was also suggested in the same report that lead must be picked up for reasons other than Its similarity to g r i t . B. Physiological Action of Lead Since?the lead poisoning of humans in mines, and from water pipes, food containers, insecticides, paints and so on is not uncommon and has great c l i n i c a l importance, much work has been carried out on the physiological action of lead in the mammalian body. Many or most of the observations made appear to be equally true for the avian organism. Monier-willlams (23) writing in 1949 described lead as one of the most toxic of trace elements, made even more dangerous by Its cumulative action. According to this author, the f u l l toxic effect is created In the systemic blood where a characteristic anaemia is caused as result of the destruc-tion of erythrocytes by the deposition of insoluble lead phosphates on their surface. Much work on plumblsm was done by Aub, Fairhall, Minot and Reznikoff (4). In 1926 they stated that muscles, kidneys and l i v e r were the organs most readily damaged by lead while the bones and the alimentary tract were comparatively resist-ent. Best and Taylor (5) stated that, In lead poisoning, lead is deposited in the bones as a tertiary lead phosphate 12 (Pb^ (POy)a. ) displacing calcium from a tricalcium phosphate (Ca^ (POy)^ ). They regarded this deposition of lead in the bones as a device protecting the body from the toxic effects of the metal. A paper which is perhaps most pertinent to the present study was written in 1935 t>y Aub ( 2 ) . He states that, while lead is stored in the bone as a very insoluble tertiary lead phosphate, i t is carried in the blood as a more soluble d i -lead phosphate. He goes on to describe a pattern of the distribution of lead once i t is absorbed. Distribution takes place through-out the viscera but mostly in the li v e r , although in a few days, i t gradually collects almost entirely in the bones. Here i t can do no damage as i t does when i t is being carried by the circulatory system. His contention is that the direction of the lead stream in the body is similar to the calcium stream. If calcium is being concentrated and deposited in the bone of the i n d i v i -dual, lead w i l l be deposited and i f decalcifying action of any kind is brought into play lead w i l l be liberated from the bone along with the calciuip. Monier-Williams (23) also states that lead deposited . in the bones is not necessarily inert. He reports further on the work of Aub to the effect that lead deposited in the bones is least soluble within the normal range of acidity of the organism and that variations above or below this degree 13 favor solution and elimination of lead. Shock, starvation or changes in diet may effect the mobilization of lead and i t s liberation from the bone into the blood stream. Best and Taylor (5) state "according to Aub et a l , the increase in hydrogen ion concentration acts by converting the insoluble tertiary lead phosphate into a soluble di-lead salt." They further l i s t parathormone as an agent of bone lead liberation and both they and Monier-Wllllams also l i s t ammonium chloride. Aub (2) also states that the calcium withdrawn from the bone for body needs is supplied by the highly vascular trabecular structure of the bone. If calcium i s being stored It is in this portion of the bone that i t w i l l be deposited. It i s also here that lead w i l l be stored In i t s greatest con-centration and from here i t w i l l be liberated under the i n -fluence of any decalcifying agent or condition. Steiman (32) in 1939> reported on the action of lead on phosphocreatine during muscular paralysis caused by lead poisoning. It was his suggestion that lead had no dele-terious effects on nerves but was definitely injurious to muscle. The interrelation of calcium, phosphorus and vitamin E> were reported on by Sobel et a l (30) in 193c>. They state that greater deposition of lead in the bone results from multiplying the concentrations of blood calcium and phos-phorus. Vitamin D is instrumental in raising their concen-centrations and therefore in increasing the deposition of Ill-lead in the bone. However, Vitamin D, in rats on a lead diet, also raised the lead concentration in the blood. In another paper in 1939, Sobel et al (31) stated that vitamin D always caused a deposition of lead in the bone. Somewhat similar work was carried out by Tompsett (3^) in 1939* H e found that the absorption of lead from the a l i -mentary tract was low with a high calcium diet and high with a low calcium diet. Unlike Sobel et a l , he found no marked influence of vitamin D on the absorption of lead from the a l i -mentary tract. In a further paper in 1939, Tompsett (35) reported that a low calcium intake in humans resulted in a rise in blood lead and a high calcium intake caused a f a l l . In mice, a transfer of lead from soft tissues to bone resulted from a high calcium intake while a diet which was low in calcium had the reverse effect. Monier-Willlams (23) and Blaxter and Cowie (6) in 19^6 both mention that a greater part of the excretion of absorbed lead is accomplished through the bile. Thus the l i v e r again appears as the organ that plays a primary role in lead poisoning and would be most lik e l y to show, through chemical analysis, the presence of abnormal amounts of blood lead particularly. 15 IV. DEFINITION OF THE STUDY AREA The portion of the Fraser River Valley of British Columbia from which the specimens were received extended from the town of Matsqui and i t s environs, down river includ-ln the Pitt area, gradually widening as the coast was reached to include Canoe Pass and Ladner on the south, Lulu Island and West Point. Grey in the centre and the nprth shore of Burrard Inlet on the north. By far the greatest number of specimens were contributed from the central and southern points on the broad coastal salt marsh area, from Canoe Pass and Lad.ner to the north arm of the Fraser River. 16 V. PROCEDURES, METHODS AND MATERIALS A. Collection and Preparation of Specimens The collecting of a sufficient number of specimens for this study could only be done by enlisting the interest and aid of those who hunted ducks in the study area. Fellow stu-dents of the Zoology Department of the University of British Columbia, hunting clubs and individual hunters were contacted by notices, letters and personal v i s i t s and advised of the forthcoming project. The notices and letters requested their assistance and gave directions to be followed upon agreement to participate in the collections. A l l necessary collecting materials were then d i s t r i -buted or made available to the interested persons. These con-sisted of: (a) wooden kegs, partially f i l l e d with 10$ formalde-hyde which were to be kept at hunting centres, club houses and so on. " (b) quart glass sealers, about 1/3 f u l l of 10$ for-maldehyde for issue to individual hunters. (c) bundles of cheese cloth cut Into pieces large enough to hold a single specimen consisting of the entrails and the legs of a duck. (d) tags with strings attached on which the informa-tion desired from the hunters was l i s t e d as 17 follows: ( i ) SPECIE3 ( i i ) SEX ( i i i ) AGE (iv) PLACE (where shot or obtained) (v) DATE (vi) ETC. (for other pertinent information) Collectors were directed to wrap the entrails and the legs of each individual duck in a piece of the cheese cloth, tie securely with the strings provided, f i l l in the attached tag and deposit in the formaldehyde. A regular pick-up of specimens from the various collecting points throughout the valley was maintained during the hunting periods. When the actual preparation of the specimens for chemi-cal analysis commenced, wrapped specimens were taken at random from their kegs and bottles and the information appearing on the tag was entered into a log book beside a permanent cata-logue number. Throughout the entire procedure, even to the identification of a specimen in the f i n a l Tables, the specimen or portion of i t , retained this number. After logging the specimen the cloth was unwrapped and the entrails briefly examined. The gizzard was then cut open and i t s contents flushed into a white porcelain plate with a weak jet of water from a wash bottle. The condition of the gizzard and i t s lining was noted and recorded. The gizzard contents were then examined for the presence of lead shot. The incidence of lead shot, the number and condition of the shot, i f any, the type of food matter, the condition of the proventriculus and gizzard were a l l entered into the log book. 18 Following this, the liver of the specimen was separa-ted, finely chopped up and placed into an evaporating dish bearing the appropriate catalogue number. Both tarsi-metat-arsi of each specimen were skinned out and also placed in a properly numbered evaporating dish. When sufficiently dry, both bones and livers were ground into a powder with a mortar and pestle and placed in separate wide-mouthed via l s . The specimens were then sub-jected, to further drying in an electric oven for two days at 100° C temperature. Important during the grinding of the bone and liver samples was to thoroughly wash the mortar and pestle with soap and hot water between specimens. The washing was followed by rinsing with lead-free d i s t i l l e d water. When the samples were thoroughly dried the vials were corked and stored to await ashing. Due to the limited number of crucibles, the limited use possible of the one electric furnace large enough for the, purpose of this study, and the length of time necessary to accomplish ashing, i t was neces-sary to carry out the next step of the procedure in lots com-' posed of the bones and livers of about one dozen specimens. After having been thoroughly dried the bone sample and live r sample of each specimen were transferred to separate previously weighed and permanently numbered s i l i c a crucibles. The crucibles and their contents were always immediately placed into a dessicator in which the crucibles were already stored prior to their use. The crucibles and their contents 1 9 were then weighed. The crucible number, the weight of the crucible, the weight of the crucible plus i t s tissue were entered in order on separate "lot sheets" of the log book. The lot number, the crucible number and the nature and weight of the tissue contained were also entered under the proper catalogue numbers. The entire lot of crucibles was then placed into an electric furnace for the ashing of their contents at 550°C, a temperature that would accomplish the process with no danger of v o l a t i l i z i n g any of the lead that might be present in the sample. The ashing required from twenty-four to thirty-four hours per l o t . On removal from the furnace the hot crucibles contain-ing the ash were immediately placed into a dessicator for cool-ing. when cool, the crucibles and their contents were again weighed, the weight of the crucible was subtracted and the weight of the ash recorded on the "lot sheets". The weight of ash per gram of dry tissue was then cal-culated according to the formula: |[ = A where w » weight of ash sample, d » weight of the dry t i s -sue ashed, A = ash per gram of dry tissue. The figure for the ash per gram of dry tissue was then. entered into the last column of the "lot sheets". For quick reference the weight of the ash sample and the figure represent-ing the ash per gram of dry tissue were also recorded under the 20 catalogue number along with the other information already there. On completion of this step for each lot, the contents of the crucibles were transferred by means of folded slips of paper, a separate slip being used for each sample, to small glass vials. These were corked, labelled with catalogue num-ber and the letter B or L to denote the tissue and set aside to await chemical analysis. B. Chemical Analysis A portion of ash, generally about 40 mgs. of bone or 20 mgs. of l i v e r , was placed in a small beaker, ^ to 1 cc. of 3^  hydrochloric acid and , 2 gm. of hydroxylamine hydrochloride were .added and the mixture, heated gently, was evaporated near dry-ness. A few ccs. of water were then added and the heating was continued to enable as much of the residue as possible to go into solution. The solution was then removed from the hot plate and made up to 5 ccs. in volume in a graduated mixing cylinder. When a portion of the solution was to be used for the deter-mination of other metals, the volume of the solution was made up to 10 ccs. Five ccs. were removed by pipetting, transfer-red to a test tube and labelled with catalogue number and tissue letter. To the solution in the mixing cylinder 5 ccs. of 25$ ammonium citrate buffer was then added to bring the pH to 21 between 9»5 and 10. This was followed by the addition of 2.5 ccs. of 10$ potassium cyanide solution and the pH was checked with litmus paper and a pH chart. An Incorrect pH could be adjusted with a small addition of the buffer solution. A dithlzone-chloroform solution containing 30 mgms. of dithizone per l i t r e of chloroform was then added, .1 or .2 ccs. at a time and accompanied with very vigorous shaking, from a burette lubricated with equal volumes of glue of gly-cerine. This was continued until the color of the chloroform layer at the bottom of the mixing cylinder became bluish grey, neither purplish nor green. The volume of reagent required to bring about this point was then recorded. Though the grey color indicates the end point i t does not mean that a l l the dithizone added has reacted with the metal as i t would in carbon tetrachloride and acid solutions. Some of the dithizone w i l l have been lost to the alkaline water layer with the result that the dithizone remaining in the chloro-form and therefore the grey color of the end point, w i l l be somewhat diluted. To determine the extent of this dilution a portion of the chloroform layer was transferred by pipette to a small test tube and i t s color compared to a series of chloro-form dilutions previously prepared from the color standard. The extent of the dilution was recorded to be used in the calcu-lations as the coefficient of dilution. After treatment by the coefficient of dilution the amount of dithizoneu.used in titration indicates the amount of lead present in the portion of the bone or liv e r sample subjected to 22 analysis. For comparison with other specimens the lead con-tent per gram of the dry tissue had to be calculated. This was done according to the formula: A x 1, L w where A =» ash per gram of dry tissue, w = weight of the ash sample analysed, 1 - lead content of the ash sample analysed i n gammas, L = lead per gram of dry tissue in parts per million. Analyses of several selected specimens for copper, zinc, iron and manganese were carried out by Miss R. Irish of the Geology Department of the University of British Columbia. Each preserved 5 aliquot portion of some of the samples was transferred from the test tube to a mixing cy-linders The pH of the solution was adjusted to between 2 and three with a drop or two of 3N hydrochloric acid. Copper was then extracted with a solution made up of 60 mgs. of dithizone per l i t r e of carbon tetrachloride. The end point was in d i -cated by the blue-grey color of the tetrachloride layer. A small portion of the water layer, usually 5 ccs. and now free of copper, was transferred by a pipette to a mixing cylinder and the volume was made' up to 5 ccs. The pH was adjusted to between 5°5 and 6 with an acetate buffer to pre-vent the reaction of the lead. Dithizone was again used to estimate zinc, the end point again being characterized by a dark blue-grey. Separate small aliquots of the original water layer were then removed for the iron and manganese determinations. 23 The aliquot for iron had to be diluted, in many cases, 50 to 100 times. More hydroxylamine was added to ensure that a l l the iron was reduced. The solution was made slightly acid and bipyridyl was' added. This reagent with iron gives a red color, the Intensity of which i s matched with a set of standards. To the diluted aliquot set aside for the manganese deter-mination formaldoxene and sodium hydroxide were added. A brown color signified the end point and was matched against a set of standards. 24 VI. DISCUSSION OF PROCEDURES AND METHODS t The only sample of duck specimens that i t was possible to obtain was one consisting of a hunter k i l l . This i s pro-bably as close to a random sample as i t i s possible to obtain without some very elaborate c o l l e c t i n g technique which might eventually prove less f a u l t l e s s than o r i g i n a l l y anticipated. It i s s t i l l necessary however, to determine whether lead poisoning a f f e c t s f l i g h t to an extent s u f f i c i e n t to make d i s -eased ducks more prone to shooting than healthy individuals. For the present study however, since previous i n v e s t i -gations into the lead poisoning of waterfowl have been l a r g e l y l i m i t e d to macroscopic gizzard examinations of ducks obtained by a hunter k i l l , v a l i d comparisons can s t i l l be made. Monier-Williams ( 2 3 ) stated that lead i s retained more by plate bone than tubular, in the ends of the bones mores than in the shafts and i n the femur and t i b i a more than i n r i b s and vertebrae. Aub ( 2 ) too, writes that lead concen-trates i n the trabeculae scattered through the marrow, p a r t i -c u l a r l y at the epiphysis. T a rgo-metatarsi were chosen as the bone tissue to be analysed In the present study primarily because i t would be the portion of the specimen most readily obtained from the c o l l e c t o r s 8 It was f e l t however, that i n the the l i g h t of Monier-Williams 1 statement, t h i s part of the skeleton x«mld be quite suitable for the purpose. 25 Due to the uneven distribution of retained lead that takes place in the bone, the whole of the bone was ground up and thoroughly mixed in the preparations of the samples for chemical analysis. Throughout this part of the work as in the later chemi-cal analysis, the greatest care had to be observed to avoid the contamination of the samples by lead, of samples by the residues of other samples and so on. Some d i f f i c u l t y was experienced in developing a method of analysis for lead which was suited to the conditions imposed by this study. That no simple but well substantiated method of quantitative analysis exists for small quantities of this metal i s attested to b y the continuing stream of new papers on this subject. Those methods which are in use for c l i n i c a l , food technology and other purposes were found to be, without exception, either Inadequate for the purposes of this study or far too lengthy for the number of determinations involved and often requiring equipment, laboratory f a c i l i t i e s and techniques which i t was not possible to obtain. It was necessary, therefore, to develop some method specifically for this study, one which would be rapid, reason-ably accurate and capable of being performed with the equipment f a c i l i t i e s available. Many approaches and t r i a l s were attempted with the help of Dr. R. Delavault and Miss R. Irish of the Geology Department to whom the problem was referred. The methods f i r s t considered "were based on the precipitation of lead as a sulphide. 26 However,the constant interference of copper and undissolved ash residue, particularly in the liver samples, forced aban-donment of this approach in favor of the use of dithizone (diphenylthiocarbazone), an organic reagent widely used for the microdetermination of various metals. According to Felgl (14), dithizone reacts with numer-ous metallic ions as well as lead to form inner complex com-pounds which are soluble In organic liquids. However, under proper conditions, dithizone w i l l be specific for lead. It forms a bright red inner complex compound with lead salts in neutral to alkaline and cyanide solutions. H H H N—h—C 6H-r N—N—C6H«-S = C / S=C NPb/e ^ N=N—C6RV XN==N—C6H0-Dithlzone Sandell (28) regarded the pH of 9«5 - 10 as being the most suitable for driving the reaction P b + % DZH *DzPb H* to the right. Since neither dithizone nor i t s heavy metal complexes are soluble in water, chloroform was used as the solvent. Carbon tetrachloride, used as the solvent for dithizone in the analysis for copper and other metals, was rejected for lead analysis since, at the high pH at which dithizone i s most specific for lead, carbon tetrachloride w i l l share too much of the dithizone with the alkaline aqueous layer. The di l u -tion of dithizone caused by the smaller loss of the reagent 27 to the aqueous layer from a chloroform solution could more easily be determined by comparison of the grey end point with several dilutions of the standard. It was expected that copper, Iron, zinc and perhaps man-ganese would be present, particularly in the liver samples. Since these metals could hinder the determination of lead by dithizone, i t was necessary to create conditions under which this interference would be eliminated. Preliminary t r i a l s showed that the dithizone was being badly oxidized by the large quantities of ferric iron in the livers. To overcome this effect hydroxylamlne hydrochloride was added during the I n i t i a l hydrochloric acid digestion of the sample. This reduced the iron prior to the treatment of the sample with dithizone. Fe++* +• NH£OH > Fe^ + |N2 "-»- H.,0 +• H + Any small amount of oxidation of dithizone which was st ' i l l found to take place merely accentuated the diluting effect of the loss of dithizone to the aqueous layer and was compensated for when the extent of dilution was determined. The divalent iron resulting from the hydroxylamlne treat-ment, the copper, zinc, and any other metals which could inter-fere with the lead determination were, through the use of potassium cyanide, transformed into complex salts, insoluble in the dithizone solution. Any effects they may have created by behaving similarly to lead were thus overcome. 2g VII. RESULTS A. Macroscopic Examination of the Gizzard for Active or  Current Leading The total hunter k i l l sample consisted of 15^ ducks of thirteen species. Mallards (Anas platyrhynchos) and pintails (Anas acuta) totalled 11^ Individuals, the remainder being made up of widgeon (Anas amerlcana), gadwall (Anas strepera). shoveller (Anas clypeata), lesser scaup (Aythya a f f i n i s ) , greater scaup (Aythya marila), Barrow's golden eye (Buce-phala lslandlca), bufflehead (Bucephala albeola). scoters (genus Melanltta), red-breasted merganser (Mergus serator), and ring-necked duck (Aythya c o l l a r i s ) . Of the 79 mallards in the sample, 13 were found to con-tain lead shot inthelr gizzards, that i s , were actively leaded, with individual gizzards dosed with from 1 to 27 pellets. The total number of pellets removed from the mallards was 62 which gave a figure of U-.77 for the average number of shot per duck. In the case of pintails, g of 35 contained a total of 26 shot, an average of 3 « 2 5 per duck. Individual gizzards contained from 1 to 5 shot. The only other species containing any shot were lesser scaup, in which 1 of the 6 individuals was found with 1 pellet, and greater scaup in which 1 of the H- taken was dosed with 2 pellets. In both cases the pellets were of f u l l size and did 29 not show any evidence of having been carried for very long. None of the remaining1 thirty specimens of either sur-face or diving ducks was found to be actively leaded. The mallards and pintails were thus shown to be the most affected by lead poisoning, the mallards being 16.45$ actively leaded and the pintails 22.86$ actively leaded. (Table I). In several of the active lead poisoning cases, the giz-zards were found to show signs of the condition as described by many previous investigators. Discoloration was noted in several instances in both the mallards and the pintails; more frequently, the gizzard pads and lining were found to be loose and corroded. It was observed however, that the giz-zards sometimes showed signs of damage of the type ascribed to lead though no lead could be found in the gizzard contents. On the other hand also, not in a l l cases of active lead poison-ing did the gizzards show evidence of having suffered any deleterious effects. One of the symptoms often mentioned in the literature and regarded as an almost certain manifestation in cases of lead poisoning, namely, a proventriculus stuffed with food mat-ter, appeared very infrequently among the individuals of the sample. It occurred in only one p i n t a i l containing five pel-lets, one mallard containing one well-ground pellet and two mallards which were captured after the hunting season and which were in very poor physical condition. The latter two 30 ducks contained 21 and 12 pellets. It was noted that when the proventrlculus of lead poisoned duck contained food matter, such food matter was of a seed nature, oats in three cases and wild seed in the case of the hunter k i l l mallard. The gizzards were often f u l l of green leafy and animal food matter and i t was further noted that very large amounts of grit frequently appeared in active lead poisoning cases. Gizzards f u l l of food however, were not limited.just to in d i v i -duals that were suffering from active leading. B» Chemical Analysis of Bone and Liver for Lead A circumstance which imposed great d i f f i c u l t i e s and existed only as a serious disadvantage to the study was the fact that not a l l the specimens were complete as to the leg bones, livers, gizzards and information on sex, age, date and place of shooting and so on^ Many of the aspects of the work were so hampered by the lack of a sufficient number of com-plete specimens or properly identified specimens that only bare trends and general indications could be obtained. The tarso-metatarsi and the livers of 4-9 mallards of the 66 which were found to be free of gizzard lead were sub-jected, to chemical analysis to determine their lead contents. It was found that the content of lead could vary from less than two parts per million in the bone of non-leaded individuals to 137 parts per million in the bone of actively leaded specimens. The liver lead varied from [l ppm in non-leaded mallards to 44 ppm in the livers of actively poisoned individuals. 31 It was also discovered however, that Individuals which were actively leaded could have lead contents of bone and liv e r which were no higher than in apparently healthy speci-mens while, at the same time, the bone lead content of some of the currently non-leaded specimens could be as high as 195 ppm with l i v e r contents as high as 23 ppm. (Table II). These abnormally high lead contents of bone, liver or both, of specimens which were not actively leaded, were ac-cepted as satisfactory evidence that the individuals in ques-tion had previously suffered from lead poisoning, had elimi-nated or assimilated the ingested shot and had survived the sickness. Out of the 49 mallards subjected to analysis, 18 appeared to be such survivors of previous leading, that i s , 36.7$. The status of 8 more out of the 49 was in doubt. The application of the percentage of 36e7 to the figure gave an answer of 2.93 which was added to the original IS to give a total for previous leadings of 20*93* In order to treat the entire mallard sample as a unit i t was necessary to adjust the figure of 20.93 out of 49 to 28.19 out of the original 66 lead-free mallards. This figure, added to the 13 actively leaded individuals out of the sample of 79» indicated that a total of 52.14$ of the hunter k i l l mallards were either suf-fering or had suffered from lead poisoning, (Table V). The bone lead contents of the 8 actively leaded pin-t a i l s analysed varied from 11 ppm to 151 ppm. Liver contents varied from 2 ppm to 53 PP m» The bone Lead contents of the non-leaded pintails were found to vary from an apparently 32 healthy [2 ppm to 70 ppm while l i v e r contents ranged from £l ppm to 12 ppm, (Table III). Of the lb lead-free pintails that were analyzed, three, or 1B% were thought to have been previously leaded. With one doubtful case treated by this percentage and added to the three definite cases of survival, a figure of 3*18 was obtained. The figure of 3*18 o u t of 16 was adjusted to 5.36 out of the entire 27 pintails which had been found, to be free of ingested lead. The overall percentage of pintails that were currently leaded and those that had survived previous leading was 3 $ » 1 7 $ (Table V). The percentage of previously leaded lesser scaup and greater scaup x«;ere not calculated due to the inadequate num-ber of specimens of those species. With respect to the remaining species and individuals that were chemically analyzed, four widgeon had bone lead con-tents varying from JjL to 4- ppm and liver lead contents a l l [2 ppm. One green-xvinged teal had [ l ppm of lead In both t i s -sues and a shoveller contained [2. ppm in the bone and 4- ppm of lead in the l i v e r . Of the four lesser scaup chemically analyzed only one was currently leaded with one uneroded pel-l e t . Both the bone and the liver content of lead was [2 ppm. Of the remaining three one had a bone lead content of 9 ppm while the other two gave figures of 73 PP™ and 77 ppm. The latter two specimens appeared to be survivors of the disease. Their liver lead contents were [ l ppm and 3 PPn* respectively. The bone lead content of the one actively leaded greater scaup 33 was f o u n d t o be 4 ppm and o f t h e n o n - l e a d e d one, £2 ppm. T h e i r l i v e r l e a d c o n t e n t s were b o t h / l ppm. The a c t i v e l y l e a d e d g r e a t e r s c a u p was d o s e d w i t h two q u i t e . u n e r o d e d p e l l e t s . One B a r r o w ' s g o l d e n e ye, one b u f f l e h e a d , one w h i t e - w i n g e d s c o t e r and two r e d - b r e a s t e d m e r g a n s e r s h a d bone l e a d c o n t e n t s v a r y i n g f r o m 4 ppm t o 9 ppm and l i v e r l e a d c o n t e n t s v a r y i n g f r o m £ l ppm t o 5 ppm» ( T a b l e I V ) . An a t t e m p t was made t o o b t a i n some i n d i c a t i o n o f t h e i n c i d e n c e o f b o t h p r e s e n t a n d p r e v i o u s l e a d i n g i n t h e main g e n e r a l a r e a s o f h u n t i n g . The l a c k o f s u f f i c i e n t numbers o f s p e c i m e n s made i t i m p o s s i b l e t o a c h i e v e any v e r y d e f i n i t e r e -s u l t . However, i t d i d a p p e a r t h a t t h e Canoe P a s s and L a d n e r a r e a s were r e g i o n s o f g r e a t e s t i n c i d e n c e o f t h e d i s e a s e f o r m a l l a r d s and t h a t p i n t a i l s s u f f e r e d t h e i r g r e a t e s t p e r c e n t a g e o f l e a d i n g s i n t h e N o r t h Arm o f t h e F r a s e r R i v e r , ( T a b l e V I ) . O n l y t h e g r e a t e r number o f m a l l a r d s p e c i m e n s made i t p o s s i b l e t o a t t e m p t c o r r e l a t i o n s o f age and s e x w i t h t h e i n c i -d e nce o f p r e v i o u s l e a d i n g s , and time o f y e a r w i t h b o t h p r e s e n t a n d p r e v i o u s s i c k n e s s . I t was f o u n d t h a t m a l e s showed t h e h i g h e s t f i g u r e f o r s u r v i v a l a f t e r l e a d i n g , namely 50.38$ o f t h e t o t a l o f 26 w h i c h h a d b e en s u f f i c i e n t l y i d e n t i f i e d . Of t h e 23 f e m a l e m a l l a r d s , i d e n t i f i e d a s s u c h i n t h e h u n t e r k i l l sample, 34.06$ were t h o u g h t t o have been s u r v i v o r s o f p r e v i o u s l e a d i n g . A d u l t s showed g r e a t e r s u r v i v a l a f t e r l e a d i n g t h a n j u v e n i l e s , t h e f i g u r e s b e i n g 48.78$ and 30 .0$ r e s p e c t i v e l y , ( T a b l e V I I ) . 3^ Smaller numbers of mallards were Identified as to both sex and age together and calculations suffered accordingly. However, i t was indicated that 59«>17$ of the non-leaded male adults were survivors of past leadings while the figure for female adults stood at 35 .01$ , for juvenile males at 29.53$ and juvenile females at 30 .37$ , (Table VIII). The incidence of current leading in mallards and the evidence of previous poisoning was determined for each of the three months over which the two periods of the 19^9 hunting season extended. A figure of 1*4-. 31$ active leading was re-corded for the month of October. In November there was a slight rise to 16.66$ which remained almost steady into the month of December for which a percentage of 16.0 was obtained. The figures obtained for previous leadings followed a very different pattern. For the month of October 37.3^$ of the^p lead-free mallards were recorded as being survivors of lead poisoning. During the month of November this figure had fallen to 33*59$ hut in December i t increased suddenly to 56.36$, (Table IX). Chemical analysis was carried out on 11 mallards which were not included in the hunter k i l l sample. They consisted of Individuals found dead or too sick to escape capture. A l l were taken in the last days of December, 19^9 and the early days of the month of January, 1950. While six of these mallards were found to be free of lead shot, the remaining five were very heavily leaded with from 12 to 35 pellets per individual. The bone lead contents in the leaded specimens 35 ranged from 44 ppm to a high of 228 ppm in the specimen carry-ing the 35 pellets. Liver lead contents were equally high, varying from 82 ppm to 176 ppm, the highest figure again appearing in the most heavily leaded of the ducks. Two of the non-leaded individuals of this group had hone and l i v e r lead contents of 96 and 14 ppm and 7 and 11 ppm. The remain-ing four specimens had insignificant quantities of [2. to 3 ppm in the case of bone tissue and £l to [2 in the case of livers with the exception of one individual which showed 14 ppm of lead in the li v e r , (Table X). Correlating the extent of gizzard damage with both the number of lead pellets carried in the gizzard and the lead contents of bone and l i v e r showed that there was very l i t t l e relationship with either* Rather, there seemed to be more of an association of gizzard damage with the actual presence of pellets which had undergone erosion, indicating that they had been carried for some period of time, (Tables XI to XIII). The analysis for copper, iron and zinc in representa-tives of various species and conditions within species con-tributed very l i t t l e of direct interest to this study. Further work along these lines, however, would be of value, (Table XIV). 36 VIII. DISCUSSION Macroscopic examination of the gizzard contents of ducks in a hunter k i l l sample showed that 16.45$ of the mall-ards and 22.86$ of the pintails were carrying Ingested lead. Previous figures for leading in mallards in the Lower Fraser Valley included those of Munro ( 2 5 ) , 13 .3$ in 1935 and Tener ( 3 3 ) , 16.1$ in 1947. At the same time Tener obtained a figure of 5$ active leading in pintails in the same area. The figure of 16.45$ leading In mallards substantiates Tener 1s 1948 result and Indicates the growing seriousness of the disease over the years as a greater number of hunters are progressively confined to smaller shooting areas. The figure of 22.86$ leading for pintails obtained in this study, however, is a tremendous increase over Tener Ts 5$ i n 1947. It is an increase out of a l l proportion with that taking place in the case of mallards and leads to the suggestion that the high figure for 1949 may have been due to some spe-c i a l circumstances which might well be made the subject of future studies, (Table I). None of the ducks except the scaup, in the group refer-red to as miscellaneous species, was found to contain lead. This fact would tend to add confirmation to the suggestion made by Tener (34) that lead pellets are picked up as grit since those ducks which u t i l i z e a sand-like grit do not appear 37 to suffer contamination. Widgeon and green-winged teal are the prime examples of this group and shovellers could perhaps be added though Tener did note the leading of a shoveller In one instance. The observation of Pirnle (27) that lead poi-soning appears to be more serious in areas of gravel deficien-cy, adds support to the thought that lead is picked up as grit, (Table IV). On the other hand Elder (13) mentions the leading of blue-winged teal and the Progress Report of the I l l i n o i s Natural History. Survey (19) t e l l s of shot incidence in shovel-ler, green-winged teal, and widgeon, with a f a i r l y consider-able percentage in the latter. The report makes the strong suggestion that lead is picked up for reasons other than i t s similarity to gr i t . S t i l l , the figures for the extent of lea,ding were, in the case of shovellers, green-winged teal, 2 .15$; widgeon, 4.84$ as compared to 7 « & 2 $ in mallards, 9.77% in pintails and 13*37$ in redheads. The users of sand-like grit appeared considerably less prone to leading and their contamination in that locality might have been due to some aspect of the observation mentioned by Pirnie. Conclusive proof is s t i l l lacking and the entire ques-tion is regarded, by the present writer, as being s t i l l open. Contrary to its frequent mention as an almost invariable symptom of lead poisoning, a proventriculus stuffed with food matter was rarely found. In the four instances when i t could be said that the proventriculus was stuffed or contained food, 3* the food matter was of a seed nature, usually oats and once, wild seed. In each case the lead pellets were s t i l l present in the gizzard. This fact appears to add further evidence to the thesis that diet plays an important role in the course and the effects of the malady. Jordan and Belrose (21) suggested that ducks on a seed diet succumbed more readily to lead poisoning than those on a green, leafy diet. It may be there-fore, that the effect of lead poisoning on seed eating ind i -viduals is sufficiently sudden and severe to result in paraly-sis of the gizzard muscles, the piling up of food in the pro-ventriculus and the retention of lead in the gizzard. It is suggested that paralysis of the gizzard muscles, which has also been frequently mentioned as one of the effects of lead contamination, cannot be an invariable, immediate or complete result of lead poisoning. The results of the study indicate that victims of the disease often either pass off the lead or completely grind and assimilate i t or both. Both would require some action of the gizzard muscles and the i n -testinal tract. It was often found that the gizzards of ducks in the hunter k i l l sample were stuffed with green plant food sometimes mixed with quantities of animal matter, particularly in pintails, consisting of shells and parts of crayfish. How-ever, a stuffed gizzard also often occurred in completely unaffected individuals. Whether the lead played" any role in the retention of food in the leaded gizzards was a question which did not lend i t s e l f to determination within the scope of this study. It is thought though, the food matter having 39 always appeared fresh and green, that the shooting of the ducks had taken place Immediately after feeding. The study of lead poisoning of waterfowl through chemical analysis was based primarily on the conclusions of Aub (2) to the effect that lead, after absorption from the alimentary tract, concentrated at f i r s t in the soft tissues, particularly in the liver and. then Ms gradually .transferred to the bone. Liver would naturally be the f i r s t organ affec-ted since i t l i e s directly in the path of and absorbs the blood stream fiom the Intestines, the hepatic-portal system, Blaxter and Cowie (6) further, showed that the excretion of lead from the body is carried out through the bile so that, for two reasons, the liver would- be expected to show a high lead content immediately after contamination, throughout the active poisoning and later, during heavy mobilization of lead from the bones. Again according to Aub, in concentrating in the tra-beculae of the bone, the lead stream parallels the calcium stream to which i t behaves similarly throughout. The lead is deposited in the bone as an insoluble tertiary lead phos-phate and while thus storedcan do the organism no further injury. However, the action of any decalcifying agent or condition such as starvation, shock or change in the acidity of the body, which w i l l cause the release of calcium from the bone, w i l l also result in the liberation of lead, as a d i -lead phosphate, Into the blood where i t can exert i t s f u l l toxic effect. That bones and livers can be regarded 4-0 and used as key centres in the study of lead poisoning through chemical analysis, is confirmed by the work of Adler (l) on Canada geese. He found that the bones of his actively leaded specimens ranged from 67 ppm. to 93 ppm and showed no corre-lation with the number of lead pellets being carried in the'g gizzard. There was a greater degree of correlation between the actual dosage and the l i v e r lead content which varied from 9 ppm In the least leaded individual to 27 ppm in the most heavily poisoned specimen,, The bone lead contents of the two controls which were later discovered to have been pre-viously leaded were 3 ° PP m and 3^ PP m while the liver lead contents were 0 and 1 ppm. Similar results were obtained in the present study with the bone and l i v e r contents of poisoned ducks appearing of the same order as those of Adler's geese. It was also found, however, more so in mallards than in pintails, that many ducks carrying lead had,almost normal bone and li v e r lead contents while many non-leaded ducks had lead contents of bone and l i v e r equal to and higher than those in which the gizzards were found to contain lead shot pellets, (Tables II and III). The former appeared to be individuals only recently con-taminated. The latter, without doubt, were individuals that had either eliminated or completely eroded and assimilated the lead and survived whatever deleterious effects had had time to develop. That actual elimination of the pellets occurs exclusively or, at least, more frequently than erosion and assimilation, is indicated by the number of non-leaded 41 juvenile ducks which have only slightly higher than normal lead contents of hone and li v e r . A juvenile duck would pro-bably not have had time in i t s short existence to d.evelop a severe case of plumbism, to store lead to the extent shown by the results to be possible and then to eliminate i t to just higher than what appear to be normal, healthy levels. The statement of Adler (l) to the effect that bone lead content is more a measure of the length of exposure than of dosage of lead appears to be confirmed by the fact that often, much higher bone lead contents are associated with fewer pellets. In one instance two mallards, No.'s 45 and 60, (Table II), each were found to contain one pellet. One showed no indication of lead deposition in the bone while the other had 62 ppm of lead in the bone and 15 ppm in the li v e r , indicating an advanced state of plumbism. Similarly, mallard No. 34 with six pellets, had 44 ppm of lead in the To) bone and. Jl ppm In the l i v e r while mallard. No. 76 with three pellets and No. 105 with four pellets had 127 ppm and 129 ppm of lead in the bone and 41 ppm and 5 P P m * n "the livers. The pellets carried by the latter two specimens were eroded and worn. On the other hand, mallard No. 123, (Table II), con-taining 27 very eroded pellets and having a gizzard, showing a great extent of injury, had bone and liver contents of only lOg and S ppm. Other factors besides the length of exposure or dosage are clearly indicated. The widely varying amounts of bone and liver lead k2 sometimes made i t d i f f i c u l t to determine or decide, in non-leaded ducks, whether they had or had not been previously leaded. A gradation of lead contents upwards from [2 ppm in the bones and ]_\ ppm in the livers existed and with i t , a question as to where the dividing line f e l l between completely healthy individuals and those which had suffered leading in the past but had almost entirely eliminated any lead that may have been stored in the bones, (Table II). It was noted, however, that a great number of mallards and pintails which were not carrying lead, had bone lead con-tents from about 3 PP m to [2 ppm and liver lead oontents from 2 to less. These would appear to be normal healthy quanti-ties, particularly when considered together with evidence from the species which did not seem to be subject to lead poisoning, namely widgeon, green-winged teal and shoveller, (Table IV). In these species bone could have a lead content of as much as 4- ppm while l i v e r was 2 or below. It was con-sidered therefore, that individuals in which the bone lead content was found to be 5 ppm °r over must have suffered contamination by lead. However, since the method of analysis was not very definite for quantities of lead under 2 ppm i t was considered best to treat those individuals having bone lead contents from 5 Ppm to 7 ppm as probable cases of pre-vious lead poisoning. A content of lead in bone of 8> ppm was regarded as definite evidence of previous sickness itfhile bone lead contents of H- ppm and 3 PP m were accepted as the 43 upper limits of a normal healthy state unless accompanied by li v e r contents of more than 2 ppm. Whenever the latter con-dition occurred in conjunction with a bone lead content of less than 8> ppm, the individual was also recorded as a pro-bable survivor of past contamination by lead. Wetmore (37) had stated that plumbism in waterfowl is a dangerous and nearly always fatal malady. Munro (25) , on the basis of his observations.in mallards, had concluded though, that mallards at least may build up a resistence to the disease. The present study shows that mallards may over-come sometimes very heavy or prolonged dosages and, when In the wild, can occasionally survive even the dosages considered by Wetmore to be always fatal, six No.6 shot. The total percentage of mallards in the hunter k i l l affected by lead poisoning, that i s , actively or currently, leaded and.previously leaded was found to be 52.14%. About 36$ of the hunter k i l l mallards were survivors of previous leading. These figures show that, while the total Incidence of the disease is considerably higher than gizzard examina-tions w i l l disclose, the balance of the higher figure is made up of survivors of the disease who can possibly live out a normal l i f e span. Consequently i t can be stated that, under the normal conditions of good diet and suitable weather, the seriousness of the disease from the point of view of the numbers involved, w i l l never be as great as gizzard examina-tions may lead observers to believe since a certain survival "rate can be expected. kk Lead poisoning appears to have more serious consequen-ces among the pintails which, though suffering a heavier i n -cidence of leading, do not seem able to offer the same resistence to i t as the mallards. Though the total percent-age of pintails affected is 3&»17 as compared to 52Blk% in the mallards, only approximately 15.0 7° of the total p i n t a i l hunter k i l l are survivors while the incidence of active lead-ing is 22.86% compared to the mallard 16 .45$. In short, more become leaded while fewer survive, (Table V), In both species, under normal conditions of weather, diet and so on, a similar pattern in the development and course of the disease makes i t s e l f evident through the figures representing the lead contents of bone and livers. Immediately after contamination the rise in l i v e r lead is followed quickly by the deposition of the metal in the bone. The liver content appears to reach, in most instances a maxi-mum in mallards of between 15 and 20 ppm although higher con-tents were recorded, usually in correlation with higher than average number of pellets. In pintails, higher liver con-tents were recorded; up to 53 and 63 ppm with only average dosage of three or four pellets. After elimination of the lead pellets the l i v e r lead drops sharply to normal or just above normal. The level of l i v e r lead then depends on the rate of elimination from the body of bone lead which in most cases can be expected to pro-cede gradually. The rate of elimination of the bone lead - may be expected to decrease or perhaps temporarily, cease K5 entirely as the hone content becomes smaller. Mallard No .6l (Table II), with a bone lead ;content of 29 ppm and a liver lead content of £l ppm may Illustrate such a condition. I n some specimens, such as mallard No.9 (Table II), an adult with a bone lead content, of 4- ppm and. a l i v e r content of K ppm, i t is d i f f i c u l t to say whether the case is one of almost completed elimination of lead from the bones after exposure or rather, the occurrence of the death of the duck immediately after the elimination, from the gizzard, of a pellet which had not been retained long enough to make the disease more apparent to analysis. That the latter situation can take place i s illustrated by the case of mallard No.97 (Table II), in which the l i v e r lead content is 19 ppm and the bone lead content is 11 ppm, indicating an i n i t i a l progress of the disease, elimination of the pellet or pellets and the subsequent death of the duck shortly after elimination. The bone lead had just begun i t s rise when the lead xiras elimi-nated. The duck was then shot before the liver lead could f a l l to more normal levels. Pintail No,59 (Table.Ill), also shows such a state before elimination and death. It was s t i l l carrying one pellet and had a bone lead content of 11 ppm and a liver lead content of 12 ppm. Mentioned earlier was the apparent tendency of pin t a i l livers to suffer higher lead contents than mallard livers, even with fexver pellets. It seems l i k e l y that the loxver sur-vival of pintails from the effects of plumbism may find at .least part of Its explanation in this fact. It x<ms noted that in the leaded individuals of the eleven mallards found sick or dead at the end of December 19^9, a period of adverse weather conditions, the li v e r lead contents were very high, reaching levels four and five times greater than the liver lead contents of the leaded ducks of the hunter k i l l sample. It is true that the dosages in this sample xvere heavier than the average dosages in the hunter k i l l but at the same time, the pellets were not eroded to any great extent. In mallard No.123, (Table II), of the hunter k i l l , carrying 27 pellets nearly a l l of which were ground to small f l a t discs, the bone and liver contents of lead were only lok and $ ppm as com-pared to 73 and 113 ppm of mallard No. 132, (Table 10), which contained 23 pellets. The poor physical condition of the latter specimen due to malnutrition and perhaps other factors, apparently contributed to the exaggerated effects of the poi-soning. The entire pattern of high liver lead contents indi -cates some accentuated condition which resulted in the very abnormal amounts of laad in that organ. This could be a mal-function of the liver i t s e l f which prevented the prompt trans-fer of lead to other tissues. More li k e l y however, i t is due to starvation which has been often quoted as a decalcifying agent. Mobilization of calcium would be accompanied by the release of lead from the bone and the inabi l i t y to deposit more. In addition, low calcium levels in the Blood would also result in a greater absorption of calcium, and with i t lead, from the alimentary tract. Both would cause greater concen-trations of lead in the blood, and result in a higher content 47 of lead in the l i v e r . With respect to pintails, i t might be added, that i f l i v e r or blood damage is more easily accomplished by lead in that species, the higher incidence of lead poisoning apparent-ly existing may have been due to the adverse effect of the disease on their flight and escape a b i l i t i e s . As mentioned earlier, i t s t i l l remains to be shown whether fl i g h t , while s t i l l possible for a poisoned duck, is nevertheless affected to the extent that It is more prone to shooting than Is a healthy Individual. It would remain necessary then, in order to explain the very high leading figure for 1949, to determine whether any factor existed to make the effects of the sickness more severe in the- pintails alone during that year. Shillinger and Cottam (29) mentioned that lead poison-ing seems to have more severe results in \ •••••winters. It can be expected that any adverse period resulting in malnutrition w i l l exaggerate the severity of the condition and result in losses more closely corresponding to the leading figures obtained by gizzard examination. Of the species termed "miscellaneous" only the scaups appeared to be subject to lead poisoning, one lesser and one greater scaup being actively leaded and tiro lesser scaup apparently having been previously leaded, (Table IV). The latter.two showed bone lead contents of 73 and 77 ppm. Liver contents were low, £l in one case and 3 PP**1 in the other. One lesser scaup had lead contents of 9 ppm in the bone and 4 ppm in the l i v e r and may have been another survivor. It 4-g was d i f f i c u l t to determine however, whether these contents should be regarded as an indication of previous leading or looked upon as normal contents for the tissues of diving ducks. It was noted that such species as golden eye and scoter showed bone lead contents of 9 ppm while buffleheads and mergansers had consistent bone lead contents of 3 ppm. Insufficient numbers of specimens makes this section of l i t t l e value for definite conclusions,, However, i t can be expected that animal-eating diving ducks, feeding in possibly lead-bearing estuary waters, may carry more lead in their bones than pond ducks. Chapman (9) states that molluscs and crus-taceae found in the mouths of industrial rivers may accumu-late lead. This could find i t s way into the systems of ducks feeding on such animal matter and at the same time, the high calcium level to be expected in the blood of such ducks would fa c i l i t a t e the deposition of lead in i t s harmless state in the bone. The lower figure for previous leading in Juveniles, (Table VII), probably indicates lower rate of occurrence of the condition rather than lower survival after leading. This does not mean that juveniles are less l i k e l y to pick up lead. On the contrary, though numbers and identifications were i n -adequate for definite conclusions, out of the actively leaded ducks in the samples obtained for this study, the majority were juveniles. Out of the seven mallards carrying lead and identified as to age, tv/o were definitely identified as juveniles and another three were identified, as mot probably ^9 Juveniles. Out of the eight pintails leaded, five were Juveniles. Elder (13) , analysing the situation among blue-winged teal, mallards and pintails found that the figure for active leading was always higher among the Juveniles. How-ever, i t is s t i l l logical to anticipate that the figures for previous leading would be lower for juveniles since there would have been less time in the l i f e of juveniles for many such cases to develop. Adult ducks on the other hand, may carry lead residue in their bones for a period of years and thus swell the numbers of those individuals entered as sur-vivors. A definite comparison of the survival expectancy of juveniles and adults would constitute a valuable and interest-ing study. The results reported in Table VIII suggest that there is no difference in the incidence of lead poisoning between sexes and that the survival rates of male and female juve-niles are equal. The lower figure for the survival of ad.ult females however, does indicate a higher death rate among females from the effects of lead poisoning. The August 1950 Report of the I l l i n o i s Natural History Survey (19) states that males are less susceptible than females except during nesting. Then females become less susceptible due to the greater amount of food consumed at that time. It is sug-gested by the present writer however, that the nesting period may well be one of additional danger to the female, rather than one of increased resistence. As body calcium is mobi-lized for use in egg production the general calcium level 50 in the body should decrease. The mass of evidence to be found in the literature suggests that this fact would be calculated to increase blood lead, both through accelerated absorption of any lead s t i l l carried in the gizzard and the mobilization of inert lead carried in the bones. Increase in the blood lead at a time when the female is undergoing the stress of reproduction may play a part in the greater t o l l of female ducks apparently taken by plumbism. It is significant that this difference in death rate between sexes does not take place t i l l after the female is of an age to have experienced i t s f i r s t nesting. There would appear to be a definite increase in the i n -cidence of lead poisoning as the shooting season progresses. The figures for active leading do not change markedly, the percentage of leaded mallards in October being 14-.31$, in November 16 .66$ and in December 16 .00$ . The percentage of survivors of previous leading, which had remained in the thirties throughout October and November, rose to 56$ in December, (Table IX). In order that such a figure should re-sult a considerable increase in the Incidence of leading during the season must have taken place. This suggests that ducks suffer most from the current deposition of lead on the hunting grounds. To obtain a figure of 14-.31$ for October however, indicates that some lead must be present on the nesting grounds or present and available on the hunting grounds from previous years of shooting or both. The rise of survivors percentage to 5b.36$ with only 51 a small Increase in active leading figures suggests that the duration of any individual case of leading could not be very long, that in most cases the pellets are not held for more than perhaps a week or two. This would bear out many of the observations made in Table II where bone and liver contents indicate the beginning and then the sudden halting of lead poisoning. There is some correlation between the number of shot present in the gizzard and bone and l i v e r lead contents, with visible gizzard damage. If lead is s t i l l present in the giz-zard of ducks with a high bone and l i v e r content of lead, indicating that the pellet or pellets have been present for some time, the gizzard almost invariably shows great injury in the form of corroded and loose linings which slough off with ease. On the other hand, in those individuals which appear to have suffered a heavy dosage as shown by high bone lead contents, but which were currently free of lead, the gizzard damage did not appear to be extensive. The indica-tion is that damage is due to the actual presence of a heavy dosage of lead or lead which has been held for a considerable length of time, and that very quick recovery takes place after the elimination of the lead. Macroscopic examination of gizzard linings could not be used as an index of previous leadings. 52 IX. CONCLUSIONS 1. There has been an increase in the incidence of. lead poi-soning in mallards and pintails in southern British Columbia in the last twenty years. 2 . The total incidence of lead poisoning is considerably higher than the macroscopic examinations of the gizzard.s for lead pellets have previously indicated. 3. The fact that so many ducks have apparently passed off the pellets they had ingested and appear to be healthy survivors suggests that, under normal circumstances of good health,weather and proper diet, the effects of the disease are not as serious as had been previously thought. A considerable survival rate can usually be anticipated and the percentage of ducks succumbing to the effects of Ingested lead w i l l normally be less than the percentage found to be contaminated with lead shot. 4 . In the wild, ducks,, particularly mallards, appear to be able to withstand the effects of sometimes very heavy or prolonged dosages, even greater than that described by Wetmore as "always fatal", namely six No.,6 shot. 5. Plumbism w i l l exert i t s greatest influence and w i l l do it s greatest damage during period.s of adverse conditions such as heavy snowfalls and resulting famine. Presumably decalcifying agents and conditions making themselves f e l t at such times result in an increase of lead in the blood where i t can exert i t s f u l l toxic effects. 6. Further evidence, though very sparse, was obtained to indicate that diet has an effect on absorption of lead from the alimentary tract, deposition of lead in the bone, amount of lead concentrating in the liver, in.short, plays an important role In the entire course and effects of lead poisoning. 7. Pintails appear to suffer much more than mallards from lead poisoning, both from the point of view of the actual incidence but particularly from the effects of the inges-ted lead. They do not seem to have the same resistance to these effects as mallards. 3. Adult female ducks appear to be less l i k e l y to survive the effects of lead poisoning than adult males. 9. There appears to be no difference in the probability of ingesting lead between the sexes nor of the rate of sur-vival in the Juveniles. 10. Juveniles appear to be more prone to Ingesting lead shot than adults. 11. Lead shot remains available to ducks on the hunting grounds from the previous year or years or may be avail-able on the summer range. Fresh lead deposition during the hunting season does, however, result in an increase in the incidence of poisoning. 12. The period of actual retention of lead pellets in the gizzard appears, in most cases, to be considerably less 54 than one month in duration. 55 X. SUMMARY In the f a l l of 1949 the author was assigned to carry out further researches into the lead poisoning of waterfowl in the Lower Fraser River Valley of British Columbia. The major and most significant portion of the work was to consist of the application of chemical analysis to the study of the con-dition. Previous workers had shown that the key organs in plumb-ism cases are the livers, which, without delay, reflect the amount of lead appearing in the blood stream of the indi v i -dual, and the bones into which blood lea.d may be transferred under suitable circumstances and where i t i s , temporarily at least, rendered harmless to the organism. It was therefore decided to carry out chemical analysis on the bones and livers of ducks in a hunter k i l l sample, to determine lead contents and their possible significance„ The hunter k i l l sample consisted of 154 ducks of 13 species \ and included 79 mallatds, 35 pintails and 40 individuals of the remaining 11 species. A further sample submitted for study consisted of 11 mallards which were picked up towards the end of December 1949 and in the early days of January 1950• They were a l l either alread.y dead or in too poor physical con-dition to escape capture. Existing methods of quantitative microanalysis for lead 56 were found to be unsuitable and a new method was developed specifically for this study. It was based on the use of dithizone (diphenylthiocarbazone) with chloroform used as the solvent. A macroscopic examination of the gizzards of the ducks in the hunter k i l l sample disclosed that an increase in the incidence of lead poisoning was taking place in the study area. A startling increase in leading was noted among pin-t a i l s which were found, to be 22.36$ actively leaded as com-pared to their 5$ leading In 194-7 as reported by Tener. Among mallards, Munro had reported 130$ leading in 1935> Tener reported 16.1$ in 194-7 and the present study showed 16.4-5$ leading in 194-1. The results of the chemical analysis showed that many of the mallards and pintails shot out of the air by hunters were survivors of previous cases of lead poisoning. The total incidence of the sickness was thus seen to be much higher than gizzard examinations have shown in previous studies. This very fact, however, indicates that a certain rate of survival, dependent perhaps on weather, availability of food and other factors, can be anticipated and the death t o l l attributable to plumbism should not normally equal the per-centage of ducks found to be actively leaded. In the wild, mallards at least, appear able to withstand the effects of very heavy dosages, sometimes even greater than the six No.6 shot, considered by Wetmore to be always fatal. The possibility of survival after leading appeared to be 57 considerably higher in mallards than in pintails.who do not seem able to offer the same resistence to the effects of lead as do the mallards. This fact, considered together with the higher Incidence of leading in this species indicates that plumbism constitutes a far greater hazard for pintails than for mallards and that l i t t l e of the optimism that may "be fe l t over the survival expectancies of the latter can be extended to the former. As in the case with many other hazards lead poisoning may exert i t s greatest influence during periods of severe weather, lack of suitable foods and other adversities. Most adverse conditions would probably express themselves as dele-terious effects on diet which is seen to be an important fac-tor in the course and effects of plumbism. In Juvenile ducks there appears to be no difference between the sexes in the probability of ingestion of lead shot and survival after leading. Adult females however, appear to be less l i k e l y to survive plumbism than adult males. Though the incidence of poisoning was increased by the new deposition of lead on the hunting grounds, the duck popu-lations were found to be already suffering from leading before the hunting season had commenced or gotten well under way. This early leading must jfchen have occurred on the nesting grounds or on the hunting grounds from the shot s t i l l present and available from previous years of shooting. Comparison of active and. previous leading figures throughout the three months of the hunting period, indicated that lead pellets 53 in any one contamination are carried for periods consider-ably less than one month in duration. 59 XI. SUGGESTIONS FOR FUTURE INVESTIGATIONS The present study, being the f i r s t of i t s kind and suf-fering from the lack of essential information which might be more carefully specified and aquired in future work, is one that could be usefully progressed further. Various controlled experiments would be of great value in confirming and ampli-fying some of the trends and observations making themselves apparent during the present investigation. For instance, the different survival expectancies bet-ween pintails and mallards, the fate of lead pellets, the duration of contamination and extent of erosion taking place in that time are several aspedts of the work which would lend themselves to controlled investigation. If a hunter k i l l sample can be obtained from persons ful l y qualified to make invariably accurate identifications of sex and age other detailed work w i l l be possible. In order to determine, for example, just where the greatest damage is done in the entire population of any one species, the actual number of pellets carried by males and females, adults and juveniles and so on can be counted and the figures for the incidence of lead poisoning generally could be broken down to adult male and females and juvenile males and females. The results can then be considered against other evidence obtained on the ingestion and survival rates of the different sexes 60 and ages. An interesting and useful study is suggested by the knowledge that lead in the body follows a path similar to the calcium stream It appears li k e l y that when calcium i s with-drawn from the bones of the female for use in egg production, lead may also be liberated from the bone. S t i l l following the calcium stream, this lead may be deposited, with calcium, in the egg shells. It is thus conceivable that chemical analy-sis of egg shells collected on the nesting grounds may serve as another index to the Incidence of lead poisoning. A project which lends i t s e l f to organization into a three-year schedule is suggested. A student, desiring to carry through the entire project could begin, in the f a l l of his graduate year, by inducing plumbism in domestic and captive wild mallards. These ducks could then be mated in various combinations and the influence on mating, reproduction and the survival of nesting females determined. The results could constitute the basis of the student's BA thesis. In the late spring of the same year or during the student's f i r s t post-graduate summer or portion of i t , the egg shells obtained from the f i r s t part of the experiment could be tested for the presence of lead. Should the test prove positive the student can make his study of the literature and write the preliminary protion of his MA thesis during his f i r s t post-graduate session. A collection of egg shells from the nesting grounds, which could have been initiated during the previous summer, 61 A l l survey parties entering the f i e l d can be instructed to make collections of duck eggs complete with f u l l data on species, location, eggs per nest and so on. Chemical analysis of the egg shells can then be carried out during the f a l l and winter of the student's second post-graduate session and the entire project could be completed by the spring or summer of that year. 62 XII. ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. W.A. Clemens, the Head of the Department of Zoology, for permission to carry out the present study and to Dr. I.McT. Cowan for sug-' gesting and. directing the investigation. I am particularly indebted to Dr. R. Delavault and Miss Ruth Irish of the Department of Geology, for their assist-ance with the chemical aspects of the project and to Dr. W.S. Hoar and Dr. W.M. Cameron for the use of laboratory space and f a c i l i t i e s . I also wish to extend my thanks to my wife and to V. Vishnlakoff for the many long hours spent with me in preparation and analysis of specimens. For their criticisms, advice and assistance I am grate-f u l to Mr. G.J. Spencer and other members of the Department of Zoology and to J.S. Tener, E. Taylor, D. Robinson and other fellow students. My appreciation i s extended to the many students of the Zoology Department as well as to the hunters of the study area for the contribution of specimens and to the British Columbia Game Commission for their financial support of the project. 63 Table I. Incidence of lead shot in hunter k i l l sample. Species Total No. of Ducks No. of Ducks with Lead Shot Percentage with Lead Shot Total No. of Shot Found Average No. of Shot per Duck Mallard 79 13 16.4-5 62 KU P i n t a i l 35 22.86 26 3.25 Widgeon 15 0 — — — Green-winged Teal 2 0 — — . — G-adwall 2 0 — — — Shoveller 1 0 — — — Lesser Scaup 6 1 — 1 — Greater Scaup 4- 1 — 2 — Barrow1s Golden eye 2 0 — — — Bufflehead 2 0 — — — Scoters 3 0 — — — Red-breasted Merganser 2" 0 — — — Ring-necked Duck 1 0 — — — TOTALS 15^ 23 1^.93 91 Table II. Results of Chemical Analysis for Lead in Mallards ( Cata-logue No. Sex Age Month when Shot Place No. of shot in Lead in Parts/Million of drv tissue G-izzard Bone Liver 1 2 ? Ad. Oct. Ladner 0 5 2 2 3 Ad. Oct. Nicomen Island 0 1 2 Z i 3 5 cri- Ad. Oct. Nicomen Island 0 i z 3 4 g er7 Ju. Oct. North Vancouver 0 21 3 5 9 cT Ad. Oct. N. Arm Fraser 0 4 4 6 12 a" Ju. Oct. Matsqui 0 2 Z i 7 13 a" Ju. Oct. Canoe Pass 0 Z2 Z i g l b Ju. Oct. Canoe Pass 0 b 1 9 23 % Ad. . Oct. Canoe Pass 0 7 2 10 24 a" Ju. Oct. Canoe Pass 0 195 15. n 2g ¥ Ju. Oct. Canoe Pass g 187 44 12 31 Ju. Oct. Ladner 0 Z2 . .4 13 34 Ju. Oct. Canoe Pass b 44 31 14 3b ? Ju. Oct. Canoe Pass 0 Z2 Z i 15 45 1 Z2 Z i l b 5b 9 Ju. Oct. " Nicomen Island. 0 Z2 Z i Table II. Cont'd. Results of Chemical Analysis for Lead In Mallards. Cata-logue . No. Sex Age Month when Shot Place No. of shot in G-lzzard Lead in Pg of dr\ irts/Mllllon r tissue Bone . Liver 17 57 ? Ju. Oct. Nicomen Island 0 Z2 Zi 13 60 Ad. Dec. Canoe Pass 1 62 15 19 6i ? Ju. Dec. Canoe Pass 0 12 Zi 20 65 ? Ju. Dec. Canoe Pass 0 2 Zi 21 63 ¥ Ju. Nov. — 0 3 3 22 71 Ad. Dec. Ladner 0 7 Zi 23 72 Ju. Nov. North Arm Fraser 0 Z2 2 2* 73 ? Ju. Nov. North Arm Fraser 0 2 Zi 25 76 — Nov. North Arm Fraser 1 127 41 26 73 Ju. Nov. North Arm Fraser 0 % 3 27 9^ ? -- Nov. Ladner 0 %> 2 23 96 ? -- Nov. Ladner 0 Z2 Zi 29 97 ? — Dec. Ladner 0 11 19 30 99 ? — Dec. Ladner 0 13 Zi 31 100 — Nov. Ladner 0 62 5 Table II. Cont'd. Results of Chemical Analysis for Lead In Mallards -Cata-logue No. Sex Age Month when shot Place No. of shot in Gizzard Lead in Parts/Million of drv tissue Bone Liver 32 101 ? — Dec. Ladner 0 Z2 Zi 33 102 — Nov. Ladner 0 Z2 Zi 3^  104 ? — Dec. Ladner 1 3 6 35 105 — Dec. Ladner 4 129 5 36 106 — Oct. Ladner 0 4 Zi 37 107 — Nov. Ladner 0 3 Zi 33 103 — Nov! Ladner 0 io4 39 109 ? ' — Oct. Ladner 0 3 Zi 40 110 — Oct. Ladner 0 ±2 . - Z i 4i 111 ? — Nov. Ladner 0 12 Zi 42 114 a* — Oct. Ladner 0 102 Zi 43 116 cT — Oct. Pitt River 0 Z2 Zi 44 119 ? Ad. Dec. Pitt River 0 Z2 Zi 45 120 Ad. Dec. Pitt River - 2 Zi 46 121 Ad. Dec. Pitt River 0 /2 Zi cr Table II. Cont'd. Results of Chemical Analysis for Lead in Mallards. Cata-logue No. Sex Age Month when shot Place No. of shot in G-i z zard Lead in Parts/Million of drv tissue Bone Liver 47 122 ? —— Oct. Westham Island 0 Z2 Zi 48 123 Ad. Oct. Westham Island 21. 104 g 49 124 or* Ju. Nov. North Arm Fraser 0 13 Zi 50 137 Ad. Dec. Pitt River 0 Zi 51 138 ? Ju. Dec. Pitt River 0 6o io 52 140 Ad. Dec. Pitt River 0 11 1 53 142 — Dec. Ladner 0 22 2 64 143 Ad. Dec. Pitt River 0 6 Zi 55 144 ? Ad. Dec. Pitt River 0 41 4 56 145 ? Ju. Dec. Pitt River 0 7 2 57 146 Ad. Nov. Pitt River 0 Z2 Z2 Table III. Results of Chemical Analysis for Lead in Pintails Cata-logue No. Sex Age Month when shot Place No. of shot in Lead in Parts/Million of dry tissue G-lzzard Bone Liver 1 14 Ju. u c t . Canoe Pass 0 4 Zi 2 15 ? Ad. Oct, North Aym Fraser 2 151 5 3 • 17 ? Ju. Oct. North Arm Fraser 5'" 49 22 4 19 Ju. Oct. Canoe Pass 1. 120 53 5 20 <yn Ad. °ct. North Arm Fraser 0 45 -6 26 ? Ju. Oct. North Arm Fraser 0 Z2 Zi 7 27 ? Ju. Oct. North Arm Fraser 0 1 Zi 8 38 ? Ju. Oct. Canoe Pass 4 53 63 9 40 . ? Ju. Oct. Canoe Pass 0 Z2 Z2 10 41 ? Ju. Oct. Canoe Pass 0 3 2 n 44 ? Ju. Oct. ^adner 0 Zo 12 12 46 ? Ju. Oct. North Arm Fraser 0 4 Zi 13 49 ? Ju. Oct. Canoe Pass 0 Z2 Zi 14 54 ? Ju. Oct. Ladner 84 34 » 15 55 Ju. Oct. Canoe Pass 0 Z2 Z2 Table III. Cont'd. Results of Chemical Analysis for Lead in Pintails. Cata-logue No. Sex Age Month when shot Place No. of shot in Gizzard Lead in Parts/Million of drv tissue Bone Liver 16 59 ' ? Ju. Oct. Canoe Pass 1 11 12 17 63 9 Ad. Dec. North Arm Fraser 1 19 2 12 75 9 Ju. Nov. North Arm Fraser 0 Z2 Z i 19 79 9 Ad. — Lulu Island 0 5 Z i 20 95 cf — Nov. Ladner 0 44 Z i 21 93 cf — Dec. Ladner 0 Z2 Z i 22 112 — — Ladner 0 Z2 Z i 23 141 ¥ — — Pitt River 0 4 Z i Table IV. Results of Chemical Analysis for.Lead in Miscellaneous Species. Cata-logue No. Species Sex Age Month when Place No.of shot in G-izzard Lead in PPM of dry tissue shot Bone Liver 1 13 Widgeon ? Ju. Oct. N. Arm.Fraser 0 Z2 2 21 Widgeon cf Ju. Oct. N. Arm Fraser 0 4 Z2 3 30 Widgeon Ju. Nov. N. Arm Fraser 0 2 Zi 4 33 Widgeon £ Ju. Oct. Canoe Pass 0 3 Zi 5 42 Widgeon Ju. Oct. N. Arm Fraser 0 4 Zi 6 4g G-reen-winged teal 9-Ju. Oct. N. Arm Fraser 0 Zi Zi 7 35 Shoveller ? Ju. Oct. N. Arm Fraser 0 Z2 4 g 11 Lesser Scaup Ad. Nov. North Vanoauver 0 22 Zi 9 25 Lesser Scaup ? Ju. . °ct. North Vancouver 0 Zi Zi 10 66 Lesser Scaup — Dec. N. Arm Fraser 0 9 4 n 67 Lesser Scaup % — Dec. N. Arm Fraser 0 ZZ 1 12 77 Lesser Scaup Ju. Nov. N. Arm Fraser 1 - Z2 Table IV. Cont'd. Results of Chemical Analysis for Lead in Miscellaneous Species. Cata-logue No. Species Sex Age Month When Place No. of shot in Gizzard Lead"in PPM' of dry <3Ufi Shot Bone Liver 13 14 22 64 G-reater Scaup Greater Scaup 9 9 Ad. Nov. Dec. North Vancouver N. Arm Fraser 0 2 12 4 Z i Z i 15 74 Barrow's Golden Eye Ju. Nov. N, Arm Fraser 0 9 Z i 16 50 Buffle-head Ad. Oct. North Vancouver 0 3 2 17 33 w . w . Scoter Ad. °ct. Canoe Pass 0 9 Z i 13 34 Red-Breasted merganser ? Ju. Dec. Lulu Island 0 3 5 19 35 Red-breasted merganser Ju. Oct. N. Arm Fraser 0 3 Z i 72 Table V. Total incidence of lead poisoning (active and previous cases). Species 1 2 3 4 5 b 7 . 8 Mallard 79 13 bb 49 <T 18 24. 24 37*24 47.14 Doubtful cases-S 18x8 ~$^"~2.93; added. 20.93 28.19 41.19 52.14 Pi n t a i l 35 8 27 lb 3 . 5.0b 13.06 37.31 Doubtful cases-1 ^g- = .18;added. 3*18 5-36 13.3b 33.17 Lesser Scaup 6 1 5 4 3 — — — Greater Scaup 4 1 3 1 0 - - -Totals 124 23 101 70 - - -1. Total number of ducks in sample. 2. Number of ducks actively leaded. 3. Number of ducks not actively leaded. 4. Number of lead-free ducks chemically analyzed. 5. Number of ducks invwhich previous lead poisoning is indicated by high lead contents of bone and liver, b. Number of previously leaded ducks raised to the total of lead-free ducks. (5x3.. ) ( 4 * b ) 7. Total number of ducks actively or previously leaded (2 + b). . 8. Percentage of ducks affected. Table VI. Distribution of AffectedMallards and Pintails of the Hunter K i l l . Area Mallards Pintails 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Canoe Pass 12 . 3 9 .7 (,2.7- 3-5 54.26 12 3. 9 5 0 0 25.00 Ladne r 27 3 2k 12 2.7 11.6 54.00 5 1. 4 4 l 1 40.00 Lulu Island. 2 \ 5 0 — — 37-5 3 0 3 1 .12 .54 ^ 12.00 Matsqui 1 0 1 1 — -- — - - - - - - -N.Arm Fraser 6 l 5 5 1.4- 1*7 39.45 11 2 5 3.2 56.36 Nlcomen Is. 4- f 0 4 4 2 2 50.00 - - - - - - -N.Vancouver 1 0 1 1 1 - 1 Pitt River 12 0 12 11 4.7 5.1 43.64 2 . 1 1 l Surrey 3 1 2 0 - - -Westham Is. 2 1 1 0 - - -Unknown 3 1 2 1 - - - 1 Totals 79 13 66 49 35 2 27 16 1. 2. I: 5. No. of ducks taken in area. 6. No. of ducks actively leaded. No. of ducks not actively leaded. No. of ducks chemically analysed. 1. No. of ducks apparently previously leaded. No. of ducks apparently previously leaded raised to total no. of ducks not actively leaded. Percentage of ducks affected in area <6 * 2 x 100> ( 1 x 1 0 U ) 7h. Table VII. Previous leading of mallards in relation to the sex and age (using onlylthose individuals which have been sufficiently identified and chemical-Ly analyzed). Male Female Juvenile Adult Total Identified 26 23 17 l 4 Previous Leading 13.101 7.335 . 5.101 6.5 Percent survivors of previous leading 50.3&1 34.06 30.00 48.73 Table VIII. Previous leading in mallards in relation to sex and age taken together (using only those individuals which have been sufficiently identified and chemically analyzed). Adult Male Adult Female Juvenile Male Juvenile Female Total Identified 3 6 3 9 Previous Leading 4.734 2.101 2.367 2.734 Percent sur-vivors of previous leading 59-17 35.01 29.53 30.37 75 Table IX. Number of mallards affected in relationtto time of year. Month 1 2 3 4 . 5 6 October 27 4 14.81$ 20 7.47 37.34 November 18 3 16.66$ 13 4.36 33.59 December 25 4 16.00$ 16 9.10 56.86 1. Number of ducks whose month of death was known. 2. Number of ducks currently leaded. 3 . Percentage of current leading. 4. Number of ducks chemically analyzed, excluding those currently leaded. 5. Number of ducks previously leaded. 6. Percentage of previous leading. Table X. Results of Chemical Analysis on 11 Mallards Found Sick and unable to Fly or Dead. Cata-logue No. Sex Age . Condition When Found Place No.of shot in gizzard Lead in Parts/Million of dry tissue Bone Liver 1 126 cf Ju. Dead Lulu Island 0 Z2 Z i 2 127 cf Ad. Dead Lulu Island 0 3 Z i . 3 12g cf ~ ~ • — Lulu Island 0 Z2 14.3 4 129 cf Ju. Sick Lulu Island 0 Z2 Z2 5 130 cf Ju. Dead Lulu Island 0 96.0 1 6 131 cf Ju. — Lulu Island 14 44 g2 7 •132 cf Ad. Sick Westham Is. 23 7g 113 g 133 cf Ad. Dead Westham Is. 21 42 164 9 134 Ju. — Westham Is. 35 22g 176 10 135 cf Ju.? — Westham Is. 12 133 i i 136 cf Ju.? -- Westham Is. 0 lit I i Table XI. Observations of gizzard conditions of pintails in relation to extent of active leading and bone and liver contents. Cata-logue No. No. of Shot in Gizzard Observations Lead in ppm of dry Bone Llver 15 2 * One pellet worn. Gizzard lining loose and discolored. 151 5 17 5 • Pellets various sizes and shapes. Giz-zard lining loose and flakey. Liver a light color. Gizzard f u l l of animal food. 49 22 19 .3 Pellets very worn. Gizzard sloughing and f u l l of plant food. 120 53 33 4 Some pellets worn. Gizzard lining loose and discolored. Gizzard distended with much animal and some plant food. 53 63 54 5 Gizzard lining loose and gizzard stuffed with green plant food. 34 34 59 1 Gizzard showing no effect. 11", 12 Table XI. Observations of gizzard conditions of pintails in relation to extent of CoA/r'a. active leading and bone and liver contents. Cata-logue No. No. of Shot in G-izzard Observations Lead in p t.1 S S pm of dry us Bone Liver 1 G-izzard discolored 19 2 117 5 Three pellets worn. Proventriculus and gizzard stuffed with oats. — — Table XII. Observations of gizzard conditions of mallards in relation to extent of active leading and bone and liver contents. Cata-logue No. No. of Shot in G-izzard Observations Lead in ppm of dry tissue Bone Liver 23 0 G-izzard showing slight effects associated with lead poisoning 7 2 24 0 Gizzard showing slight effects associated with lead poisoning 195 15 28 8 Gizzard lining loose and brown in color 187 44 34 6 Two'-pellets worn to discs. Gizzard lining loose and discolored. Gizzard f u l l of seeds and oats. 44 31 45 1 Gizzard not visibly affected. Z2 Z i 60 1 Gizzard not greatly affected. 62 15 76 3 One pellet very worn. Gizzard showing some sloughing of the lining. 127 41 78 0 Gizzard lining showing definite effects associated with lead poisoning. 34 3 104 1 Pellet worn. Gizzard discolored and con-tained, seeds in proventricular end 3 6 105 4 Pellets worn and small in size. Gizzard lining loose. 129 5 123 27 Nearly a l l worn small and f l a t . Gizzard lining loose; putrid odor. io4 8 Table XII. Cont'd. Observations of gizzard conditions of mallards in relation to extent of active leading and bone and liver contents. Cata-logue No. No. of Shot in Gizzard Observations Lead in ppm of dry tissue Bone Liver 154 2 Pellets worn f l a t . Gizzard f u l l of green plant food. — — 159 k Pellets worn. Gizzard f u l l of green plant food. -Table XIII. Observations of gizzard conditions of 11 mallards taken sick or dead in January 1950 in relation to extent of active leading and bone and liver contents of lead. Cata-logue N o . . No. of Shot in G-izzard Observations Lead in ppm of dry tissue B o n e L i v e r 126 0 Found dead. G-izzard undersized and empty with lining.firm and dark in color. Z2 Z i 127 0 Found dead. Emaciated. G-izzard undersized and. empty with lining firm and dark in color. 3 Z i 128 0 G-izzard empty and undersized with lining firm and dark In color. Z2 14.3 129 0 Emaciated. G-izzard lining sloughing slightly, almost empty and containing grit of a small size. Z2 Z2 130 0 • Found, dead. G-izzard very small and lining black in color. I 131 14 Pellets f u l l size. Gizzard lining loose. 44 82 132 23 Pellets f u l l size. G-izzard lining in poor condition. 78 113 Table XIII. Cont'd. Observations of gizzard conditions of 11 mallards taken sick or dead in January 1950 in relation to extent of active leading and bone and liver contents of lead. Cata-logue No. No. of Shot in Gizzard Observations Lead in ppm of dry tissue Bone Liver 133 21 Proventriculus stuffed with oats. 42 164 134 35 Gizzard lining loose. Gizzard f u l l of green plant food. 223 176 135 12 Proventriculus stuffed with oats. Gizzard containing oats and green plant food. 47 - - 133 136 0 Emaciated. * Gizzard empty. 11 i i Table XIV. Chemical analysis for Copper, Iron and Zinc. Cata-logue No. Description No. of Shot per Duck Lead Copper Iron Zinc Bone Liver Bone Liver Bone Liver Bone Liver 14 . Pintail, normal 0 4.1 Z i 41 142 Tfrabe 415 330 142 32 Pintail, leaded 4 53-0 63.0 44 121 Trace 6922 142 699 77 Lesser Scaup 1 Z2 Z2 - 64 - 2670 - 252 64 Greater Scaup 2 4.5 Z i - 33 - 430 - 21 50 Bufflehead 0 3 2 - 37 - 99 - 161 23 White winged Scoter 0 9 Z i - 57 .2674 - 201 42 Green-winged Teal 0 Z i Z i — 94 — 222 . — 142 35 Shoveller 0 Z2 4 - 34 - 2237 - 207 22L. Greater Scaup 0 Z2 Z i - 26 - 720 - 76 Table XIV. Cont'd. Chemical analysis for Copper, Iron and Zinc. Cata-logue No. Description No. of Shot per Duck Lead Co] Dper Iron Zinc Bone Liver Bone Liver Bone Liver Bone Liver 21L. Widgeon 0 Z2 Z2 _ 5S 490 _ 137 74 Barrow's Golden eye 0 9 Z i — 5b _ 2064 2g0 85 Red-breasted Merganser 0 3 5 - 28 - 1254 - 193 bb Lesser Scaup 0 77 3 - b4 - 2007 - 186 94 Mallard (survivor) 0 56 Z2 .37 — 37 927 -9b . Mallard _ . 0 Z2 Z i 79 - • 1780 - 1429 -97 Mallard (survivor) 0 11 1 9 - 36 •- 513 - 1824 -101 Mallard 0 Z2 Z i 81 - 1671 - 1393 102 Mallard 0 Z2 Z i 71 - 825 - 1800 -Table XIV. Cont'd. Chemical analysis for Copper, Ironrand Zinc. Cata-logue No. Description No. of Shot per Duck Lead Copper Iron Zinc Bone Liver Bone Liver Bone Liver Bone Liver 123 Mallard. (Leaded) 27 104 51 1400 114 127 Mallard, (Dead) 0 Z2 Z2 134 - . 1637 - 215 -86 Figure 1. S7 XIII. PREPARATION OF REAGENTS Preparation of the Dithizone Reagent. The dithizone reagent is prepared by simply dissolving 30 mgms of dithizone (DIPHENYLTHIOCARBAZONE) in 1 l i t r e of chloroform, the chloroform being added slowly due to an exo-thermic reaction taking place during the mixing. The pre-paration-- sould be carried out in the presence of an abundance of fresh a i r . Preparation of the Lead Standard. A small quantity was obtained of an already prepared lead standard consisting of 3^ HNO containing 2 mgms or -2000 ppm (gammas) of lead per cc. The small bottle contain-ing the standard was f i r s t shaken to ensure that no lead remained out of solution around the stopper and the dry end of the bottle and then 1 cc of the standard was transferred by pipette to the bottom of a 200 cc volumetric flask. Care was taken during the transfer, the pipette being f i r s t washed and rinsed in lead-free d i s t i l l e d water and then rinsed with the lead standard. Di s t i l l e d water was then used to bring up the volume of the standard up to 200 ccs. A standard was thus obtained containing 10 ppm (gammas) of lead :per cc. m Preparation of the Am'monium Citrate Buffer. D i s t i l l e d water was used to dissolve 250 gms of ammon-ium citrate and hring the volume of the solution to 100 ccs. Lead impruities in thehbuffer were extracted by shaking with a sufficient quantity of dithizone in a separating funnel. When the dithizone ceased to turn red on addition and shak-ing, but rather retained i t s green color, i t was allowed to settle and was then drawn off, leaving the lead-free ammon-ium citrate intthe funnel. Preparation of the Potassium Cyanide Solution. D i s t i l l e d water was used tocldissolve 10 gms of potas-sium cyanide and bring the volume of the solution to 100 ccs. Since the solution was found to contain practically no lead, extraction with dithizone was considered unnecessary. Preparation of the Color Standard. To 2 ccs of the diluted lead standard in the bottom of a 50 cc mixing cylinder, 2 ccs of 25$ ammonium citrate buf-fer and 1 cc of 10$ potassium cyanide solution wgre added. This was titrated with the dithizone, about .2 ccs at a time with the mixture being vigorously shaken between additions of the dithizone. The titration was continued t i l l a blue-grey color of the dithizone layer, which settles below the water layer, indicated the end point. The amount of the dithizone reagent required to reach the end point was recor-ded. 29 Seven ccs of dithizone were required for 2 ccs of the lead standard. Three decimal five ccs of dithizone were required for 1 cc of the lead standard, that i s , to react with 10 gammas of lead. One gamma of lead would therefore he indicated by .35 cc of dithizone. The dithizone-chloroform layer was then pipetted from the bottom of the mixing cylinder and transferred to the f i r s t of five small test tubes mounted on a rack which was open on both sides. Small amounts of this standard were then trans-ferred to each of the other test tubes in the rack and diluted with chloroform to twice, three times, four times and five times their original volumes. This color series was later used to determine the extent of dithizone loss to • the water layer and any loss of color of the dithizone com-plex due to reduction by ferric iron, particularly present in the liver samples. Throughout the preparation of the reagents and stan-dards i t was most improtant to guard against accidental con-tamination by lead. In the later chemical analysis of the samples i t was equally important to guard against such con-tamination as well as contamination of samples by residues of other samples.. A l l glassware was i n i t i a l l y treated with HCl, washed and then rinsed in d i s t i l l e d water. Pipettes were kept standing in d i s t i l l e d water and before use were rinsed in d i s t i l l e d water and then in the solution to be ~ measured or transferred. They were rinsed once more in dis-~ t i l l e d water after use. 90 Other precautions observed in working with such minute amounts of a metal to be analyzed included the shaking of bottles containing standard solutions to ensure that none of the lead had crystallized around the top of the bottle or under the stopper; avoiding the loss of any standardizing solution contained in a pipette by touching the sides of the vessel from which i t was being drawn or to which i t was being transferred; using s i l i c a crucibles in the ashing pro-cess. LITERATURE CITED 1. Adler, F, E. w. Chemical analysis of organs from lead-poisoned Canada geese. Jour, wildl. Mgt., 8 ( l ) . 8 3 - 8 5 . 1944. 2. Aub, Joseph G, The biochemical behaviour of lead in the body. Jour. Amer. Med. Assoc., 104(lj:37-90, 1935. 3 . cited from Monier-williams, G.w. 4. Aub, J. C., L. T. Fairhall, A. S. Minot and P. Reznikoff. cited from Monier-williams, G. w. 5. Best, Charles Herbert and Norman Buske Taylor. The physiological basis of medical practice. (A text in applied physiology) 5th Ed., Baltimore, 1950. Williams and Wilkins Co. 6. Blaxter, R. L. and A. T. Cowie. Nature 157, May 4, 1946, 588. 7. Bowles, J. H. Lead poisoning in ducks. The Auk, 25. 312-313. 1908. 8 . Calvert, J. Hindle. Cited from Jones, J. C. 9. Chapman, A. C. Cited from Monier-Willlams, G. W. Cheatum, E. L. and Dirck Benson. Effects of lead poison-ing on reproduction of mallard drakes. Jour. Wildl. Mgt., 9(l ) : 2 6 - 3 0 , 1945. 11. Cottam, Clarence C. Cited from Jones, J. C. 12. Cited from Cheatum, E. L. and Dirck Benson. 13. Elder, William H. Measurement of hunting pressure in water-fox?! by means of X-ray. Trans, of 15th Nor. Am. Wildl. Conf., 490-503. 14. Feigl, F. Qualitative analysis of spot tests. Third edition. 1946. Elsevier Publishing Company, Inc., New York - Amsterdam. 15. G-rinnell, G. B. Cited from Jones, J. C. 16. Cited from Shillinger, J. E. and Clarence C. Cottam. 10. 17. Holland, George. Cited from Jones, J. C. 18. Hough, E. Cited from Jones, J. C. 19. I l l i n o i s Natural History Survey. Progress Report, August, 1950. (Unpublished) Havana I l l i n o i s . 20. Jones, J. C. On the occurrence of lead shot in the stomachs of North American Cruiformes. Jour. Wildl. Mgt., 3(4)5353-357,1939. 21. Jordan, James S. and Frank C. Belrose. Shot alloys and lead poisoning in waterfowl. Trans, of 15th Nor. Am. Wildl. Conf., 155-170, 1950. 22. McAtee, W. L. The Auk, 25; 472, 1902. 23. Monler-Willlams, C. W. Trace Elements in Food. John Wiley and Sons, 1949, New York. 24-. Munro, J. A. Lead.poisoning in trumpeter swans. Canad. Field Nat., 39:160-162, 1925. 25. Munro, J. A. Studies of waterfowl in B. C.. Canad. Jour. Res., D 21, 223-260, 1943. 26. Phi l l i p s , John C, and Frederick C. Lincoln. Cited from Jordan, James S. and Frank C. Belrose. 27. Pirnie, D. M. Cited from Shillinger, J. E, and Clarence C. Cottam. 22, Sandell, E. B. Colorimetric determination of traces of metals. Interscience Publishers, 1950. New York, N. Y. 29. Shillinger, J. E. and Clarence C. Cottam. Trans, of 2nd North Am. Wildl. Conf. 392-403, 1937. The importance of lead poisoning in waterfowl. 30. Sobel, Alber E., Oscar Gawron and Benjamin Kramer. Influence of Vitamin D in experimental lead poisoning. Proc. Soc. Exp. Biol, and Med. 32(3):433-435,1933. 31. Sobel, Albert E., Irving B. Wexler, David D. Petrovsky and Benjamin Kramer. Influence of dietary calcium and phosphorus upon action of Vitamin D in experi-mental lead poisoning. Proc. Soc. Exp. Biol, and Med., 38(3):435-437, 1938. Steiman, S. E. The action of lead on the phosphocrea-tine in the muscular paralysis of lead poisoning. Amer. Jour. Physiol'., 12b ( 2 ) : 2 b l - 2 b 9 , 1939. Tener, John Simpson. An investigation of some of the members of the sub-family Anatinae in the Lower Fraser Valley of B. C, A thesis submitted in partial fulfilment of the requirements for the Degree of B.A. in the Department of Zoology, U.B.C., April, 194g. Tompsett, Sidney Lionel. The influence of certain constituents of the diet upon the absorption of lea.d from the alimentary tract. Further studies on the absorption, mobilization and excretion of lead. Brit. Jour. Exp. Path., 20(b):512-516, 1939. Weller, C. V. ' The.blastophthoric effect of chronic lead poisoning. Jour. Med, Res. Vol. 23 (New Series, Vol. 23), 271-293, 1915-Wetmore, Alexander. Lead poisoning in waterfowl. United States Dept. of Agriculture, Bulletin' No.793, J u l y 31, 1919. 

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