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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 i n the Department of Zoology  We accept.-'this t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree o f MASTER OF ARTS.  •Members of the Department of Zoology THE UNIVERSITY Of BRITISH COLUMBIA August,1951-  ABSTRACT Macroscopic examinations of ducks i n a hunter k i l l sample for the presence of ingested lead shot p e l l e t s in the gizzards indicated that a slight increase soning since 1 9 ^ 7 ,  i n active lead p o i -  had taken-place among mallards (Anas platy-  rhynchos) and that a more severe Increase had occurred i n p i n t a i l s (Anas acuta).  Instances of p r o v e n t r l c u l i i stuffed  with food occurred only i n conjunction or wild seed.  Quantitative  with a diet of oats  analysis of hone and l i v e r  by means of a s p e c i a l l y designed dithizone-chloroform was  ash method  carried out to determine lead contents and t h e i r s i g n i -  ficance.  Analysis shoiired that a considerable  of both species, though proportionately  number of ducks  morehmallards than  p i n t a i l s were survivors of past contamination by lead  and  that, under normal conditions of weather and a v a i l a b i l i t y 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.  P i n t a i l s appeared to suffer  more, both i n terms of actual incidence  of leading as well as  t h e i r apparent i n a b i l i t y to o f f e r as much resistance as mallards, to the effects of lead.  Though there appeared, in  mallards, to be no s i g n i f i c a n t difference i n the p r o b a b i l i t y 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 a v a i l a b i l i t y and quality of food, appeared to give lead p o i 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  inci-  dence of lead poisoning, evidence Indicated that lead shot i s also available to ducks either on the nesting grounds or on the hunting grounds from the deposition of previous  years.  i  CONTENTS I. II. III.  IV. V,  VI.. VII.  INTRODUCTION  1  STATEMENT OF PROBLEM  3  PREVIOUS INVESTIGATIONS  5  A.  Lead poisoning  i n waterfowl  B„  Physiological action of lead  .  5 ... 11  DEFINITION OF STUDY AREA  15  PROCEDURES, METHODS AND MATERIALS A. Collection and preparation of specimens B„ Chemical analysis  16 16 20  DISCUSSION OF" PROCEDURES AND METHODS  2&  RESULTS A. Macroscopic examination of the gizzard for active or current leading B. Chemical analysis of bone and l i v e r f o r lead  ,  XI. SUGGESTIONS FOR FUTURE INVESTIGATIONS  XIII. PREPARATION OF REAGENTS  30  52  IX. CONCLUSIONS  XII. ACKNOWLEDGEMENTS  22  36  VIII. DISCUSSION  X. SUMMARY  2k-  55 59 62 $7  ii  LIST OF TABLES AND FIGURES Table  I. II. III. IV. V. VI.  sample..63  Results of chemical analysis for lead i n mallards  64  Results of chemical.-analysis for lead in -Dintails 6g Results of chemical analysis for lead i n miscellaneous species  70  Total incidence of lead poisoning (active. and previous cases)  72  Distribution, of affected, mallards and p i n t a i l s of the hunter k i l l .....73  VII.  Previous leading of mallards in r e l a t i o n to sex and age  74  VIII.  Previous leading of mallards i n r e l a t i o n to sex and age taken together .......  ,74  Number of mallards affected .in r e l a t i o n to time of year  .75  Results of chemical analysis on 11 mallards found sick and unable to f l y or dead  ,7b  IX. X. XI.  Observations of gizzard conditions of pint a i l s i n r e l a t i o n to extent of active leading and bone and l i v e r lead contents ....77  .XII.  Observations of gizzard conditions of mallards in r e l a t i o n to extent of active leading and bone and l i v e r lead contents .....79  XIII.  Observations of gizzard conditions of 11 mallards taken sick or dead in January 1950 in r e l a t i o n to extent of active leading and bone and. l i v e r contents of lead  XIV. Figure  Incidence of lead shot i n hunter k i l l  1.  ell  Chemical analysis f o r copper, iron and zinc...$3 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 hunting  spots where tons of lead i n 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 , attent i o n was at f i r s t given to lead poisoning or plumbism i n game waterfowl.  Early references went l i t t l e further, i n  most instances, than to record i t s occurrence.  As the growing  extent of the sickness became apparent along with the decline . i n 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 addit i o n a l hazard to duck populations. More references on the subject of lead poisoning began  2  to appear i n 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 t h i s danger to an already seriously threatened game type. Emphasis i n the study of lead poisoning gradually began to s h i f t 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  investigation into lead poisoning  assigned to an  to further the study of  the condition generally and to obtain more detailed data on i t s extent i n ducks on the P a c i f i c Flyway and the Lower Fraser River Valley of B r i t i s h Columbia i n p a r t i c u l a r .  The work was  to consist primarily of a quantitative chemical analysis of various organs of ducks in a h u n t e r - k i l l sample to determine the lead contents and t h e i r significance. work by Adler ( l )  On the basis of  with geese and experimental knowledge of  the action of lead i n the mammalian body appearing In l i t e r a - v ture, i t was apparent that the most s i g n i f i c a n t information 7  would be obtained from treatment of what appeared to be  key  organs In plumblsm cases, namely the l i v e r and bones, p a r t i c u l a r l y those of the leg.; It was  anticipated that the determination of the lead  contents of these two organs would serve to indicate: 1.  Difference i n lead contents between healthy  and  lead poisoned ducks. 2.  P o s s i b i l i t y of survival which would be indicated by the absence of ,lead p e l l e t s i n the gizzard i n conjunction  with high lead contents of the bone  and low or almost normal lead contents of the liver. 3.  Possible extent of s u r v i v a l .  4.  Total incidence of lead poisoning, present.  past  and  4 5.  Some idea of the course of the poisoning, r e s i s t ence of the various species, different rates of Incidence and survival between sexes and ducks of d i f f e r e n t ages.  6.  Incidence of the disease at various times during the hunting season and any change i n the severity with change i n weather conditions and of food.  availability  5 III.  A.  PREVIOUS INVESTIGATIONS  Lead Poisoning In Waterfowl. The poisoning of game fowl by spent lead shot had  been noted as early as the 1870*s.  P h i l l i p s and Lincoln (2b)  mentioned the occurrence of poisoning in waterfowl i n 1874 and the poisoning of pheasants was described In 1876 Calvert (8)  and i n 1882 by Holland ( 1 7 ) .  G r i n n e l l (lb)  and Hough (18)  i n 1894,  by  Reports by discussed the effects  of lead poisoning on wildfowl. G-rinnell (lb) f i r s t began to make detailed descriptions of the condition i n 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 i n Texas. Some of the f i r s t experimental work on the subject of lead poisoning i n waterfowl was carried out by wetmore In 1919.  In 1916  (37)  he had made note of the disease among mal-  lards, p i n t a i l s , canvasbacks, whistling swans and marbled godwit at the northern end of the Great Salt Lake i n Utah. He drew up the f i r s t complete symptomology of the disease, noting i n p a r t i c u l a r : 1.  p a r a l y s i s and wing-droop; not always loss of function.  symmetrical  This he attributed to the  b  loss of function of the nerves supplying the pectoral muscles. 2  e  emaciation, p a r t i c u l a r l y of the pectoral muscles.  3.  thin, watery, green feces.  4.  bright eyes.  5.  weakness and heart spasms which were often the d i r e c t causes of death.  6.  good appetites.  Post mortem examination showed that: 1.  the f l e s h of the b i r d was pale.  2.  blood was  3.  heart stopped i n systole.  4.  i n chronic cases the pericardium was usually d i s -  slow to coagulate.  tended with a watery lymph. 5.  proventriculus and oesophagus were greatly d i s tended with food due to the paralysis of the g i z zard muscles.  6.  intestines were i r r i t a t e d and discolored.  7.  pads of the gizzard were corroded and off  sloughed  easily.  2.  g a l l bladder was f u l l and sometimes distended.  9.  b i l e was dark green.  (Anticipating a trend which would be increasingly f o l lowed i n the future Wetmore began experimentation i n the effects of the disease..  He attempted to establish a l e t h a l  dosage by treating wild mallards, taken when young, with varying amounts of lead shot.  He found that six p e l l e t s of  No.6  shot were always f a t a l hut that even one p e l l e t would  sometimes result i n death.  He stated that plumbism i s a  dangerous and usually f a t a l malady and proved that the l e t h a l agent i n 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 . eggs were i n f e r t i l e and there was a higher percentage  Many of  deaths of embryos. F a i r l y detailed observations and investigations into the problem of lead poisoning were made by S h i l l i n g e r and Cottam (29)  i n 1937o  Symptoms described by them agreed with  those reported e a r l i e r by we'tmore. dose could vary from a single No.5  They found that a l e t h a l shot to many and that the  losses were most prominent In winter. of  They compiled reports  the incidence of the disease from many parts of the United  States such as North Carolina, Pamlico Sound of V i r g i n i a , Delaware Bay i n 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. ted  Pirnie (27)  In 1935*  had.repor-  that lead took the greatest t o l l over a l l diseases In the  Great Lakes. Cottam (11)  i n 1939 reported an amazing extent of  ingestion of shot by lesser scaups at Lake Puskeway,  Marquette, Wisconsin i n the spring hunting season of 1909?  76o5$  of the ducks were dosed with from  1  to  5$  shot per  individual. With reference to B r i t i s h Columbia, Munro (24), i n 1925, described the lead poisoning of several swans on Vancouver Island.  Pathological reports made subsequently-  p a r a l l e l e d the observations  noted by Wetmore.  In 1943,  Munro (25), writing generally on the waterfowl of B r i t i s h Columbia, stated that lead poisoning was a r e s t r i c t i v e factor of unknown proportions,  seldom occurring i n the Interior  of the Province but common i n the Lower Fraser Valley where hunting was more concentrated. of ninety  (13.3$)  He reported that twenty out  of the mallards  shot contained lead p e l l e t s .  He suggested also, that some evidence existed that may develop a resistence to the effects of lead. example of two mallards  mallards C i t i n g the  that contained very worn-down  shot  but which were apparently normal and showed no evidence of the a f f l i c t i o n . Further studies i n the Fraser Valley were conducted by Tener (33)  i  16.1$ of mallards  n  1948 who found active lead poisoning i n and 5$ i n p i n t a i l s i n a hunter k i l l sample.  Tener further found that no widgeon or green-winged t e a l contained lead.  Observing the small size of g r i t used  by these two species Tener suggested that those ducks which ingest lead shot pick up the p e l l e t s as g r i t 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 Cottam (12)  had suggested that s t e r i l i t y  drakes.  was an a f t e r -  effect i n those ducks which had survived the ravages of the disease.  Weller (36)  working with guinea pigs i n 1915  con-  cluded that chronic lead poisoning had a d e f i n i t e blastophthoric effect which could best be demonstrated on male germ plasm r e s u l t i n g i n s t e r i l i t y ,  reduction In b i r t h weight and  increase i n the number of deaths of young i n their f i r s t week. That i t was  the germ plasm already undergoing maturation  not the germinal epithelium that suffered was  and  indicated by  the fact that reproductive powers recovered a f t e r 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 h a t c h a b l l i t y or any Increase i n mortality In the f i r s t seven weeks of l i f e . Adler ( l ) to  mentions Magath as being one of the f i r s t  chemically analyse the organs of waterfowl for lead.  Following the example of Magath chemical analysis of the organs of waterfowl poisoned by lead was (l)  i n 1944.  carried out by Adler  He found that the amount of lead i n the bones  of lead poisoned  Canada geese showed no correlation with the  10  amount of lead being carried i n the gizzard and concluded that the increased lead content of the bone i s more a measure of the length of exposure than of dosage.  The amount of lead  i n the l i v e r bore closer r e l a t i o n to the amount of lead i n the gizzard.  He suggested that the l i v e r was the best organ  to use f o r 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 i n 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 i n 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 d i e t . They further stated that 60% to g0$ of mallard drakes carrying one shot p e l l e t i n 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 f o r a considerable period-in c a p t i v i t y were less susceptible to lead. The l a t t e r fact was also brought out i n a study by the I l l i n o i s Natural History Survey (19)  In August  1950«  This, along with other observations l e d to the suggestion  11 that there might be a correlation between the amount of food, taken and the expression of lead poisoning symptoms. also suggested  i n the same report that lead  It was  must be picked  up for reasons other than Its s i m i l a r i t y to g r i t .  B.  Physiological Action of Lead Since?the lead poisoning of humans i n mines, and from  water pipes, food containers, insecticides, paints and so on i s 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 i n the mammalian body.  Many or most of the observations made  appear to be equally true f o r the avian organism. Monier-willlams  (23)  writing i n 1949 described lead  as one of the most toxic of trace elements, made even more dangerous by Its cumulative action.  According to t h i s author,  the f u l l toxic effect i s created In the systemic blood where a c h a r a c t e r i s t i c anaemia i s caused as result of the destruction of erythrocytes by the deposition of insoluble lead phosphates on t h e i r surface. Much work on plumblsm was done by Aub, and Reznikoff (4).  In 1926  F a i r h a l l , Minot  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 i s deposited i n the bones as a t e r t i a r y lead phosphate  12  (Pb^ (POy)a. ) displacing calcium from a tricalcium phosphate (Ca^ (POy)^ ).  They regarded this deposition of lead i n the  bones as a device protecting the body from the toxic effects of the metal. A paper which i s perhaps most pertinent to the present study was  written i n 1935  t>y Aub  (2).  He  states that, while  lead i s stored i n the bone as a very insoluble t e r t i a r y lead phosphate, i t i s carried i n the blood as a more soluble d i lead phosphate. He goes on to describe a pattern of the d i s t r i b u t i o n of lead once i t i s absorbed.  D i s t r i b u t i o n takes place through-  out the v i s c e r a but mostly i n the l i v e r , although i n a few days, i t gradually c o l l e c t s almost e n t i r e l y i n the bones. Here i t can do no damage as i t does when i t i s being carried by the c i r c u l a t o r y system. His contention  i s that the d i r e c t i o n of the lead stream  in the body i s similar to the calcium being concentrated and deposited dual, lead w i l l be deposited  stream.  I f calcium i s  i n the bone of the  indivi-  and i f d e c a l c i f y i n g action of  any kind i s brought into play lead w i l l be l i b e r a t e d from the bone along with the calciuip. Monier-Williams (23)  also states that lead  . i n the bones i s not necessarily inert. on the work of Aub  deposited  He reports further  to the effect that lead deposited  i n the  bones i s least soluble within the normal range of a c i d i t y of the organism and that variations above or below t h i s degree  13  favor solution and elimination of lead.  Shock, starvation or  changes i n diet may effect the mobilization of lead and i t s l i b e r a t i o n from the bone into the blood stream. Best and Taylor (5)  state "according to Aub et a l ,  the increase i n hydrogen ion concentration  acts by converting  the insoluble t e r t i a r y lead phosphate into a soluble di-lead salt."  They further l i s t parathormone as an agent of bone  lead l i b e r a t i o n 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 i s supplied by the highly trabecular structure of the bone.  vascular  I f calcium i s being stored  It i s i n 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 l i b e r a t e d under the i n fluence of any decalcifying agent or condition. Steiman (32)  i n 1939> reported on the action of  lead on phosphocreatine during muscular paralysis caused by lead poisoning.  I t was his suggestion that lead had no dele-  terious effects on nerves but was d e f i n i t e l y injurious to muscle. The  i n t e r r e l a t i o n of calcium, phosphorus and vitamin  E> were reported on by Sobel et a l (30)  i n 193c>.  They state  that greater deposition of lead i n the bone r e s u l t s from multiplying the concentrations of blood calcium and phosphorus.  Vitamin D i s instrumental  i n r a i s i n g t h e i r concen-  centrations and therefore i n increasing the deposition of  Illlead i n the bone.  However, Vitamin D, i n rats on a lead  d i e t , also raised the lead concentration i n the blood. another paper i n 1939,  Sobel et a l (31)  In  stated that vitamin  D always caused a deposition of lead i n the bone. (3^)  Somewhat similar work was carried out by Tompsett i n 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 d i e t .  Unlike Sobel et a l , he found no marked  influence of vitamin D on the absorption of lead from the a l i mentary t r a c t . In a further paper i n 1939,  Tompsett (35)  reported that  a low calcium intake i n humans resulted in a r i s e i n 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 i n calcium had the reverse e f f e c t . Monier-Willlams 19^6  (23)  and Blaxter and Cowie (6)  in  both mention that a greater part of the excretion of  absorbed lead i s accomplished  through the b i l e .  Thus the l i v e r  again appears as the organ that plays a primary role i n lead poisoning and would be most l i k 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 B r i t i s h Columbia from which the specimens were received extended from the town of Matsqui and i t s environs, down r i v e r includln the P i t t area, gradually widening as the coast was reached to include Canoe Pass and Ladner on the south, Lulu Island and West Point. Grey i n the centre and the nprth shore of Burrard Inlet on the north.  By f a r 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. A.  PROCEDURES, METHODS AND MATERIALS  Collection and Preparation of Specimens The c o l l e c t i n g of a s u f f i c i e n t number of specimens  for  this study could only be done by e n l i s t i n g the interest and  aid  of those who hunted ducks i n the study area.  Fellow stu-  dents of the Zoology Department of the University of B r i t i s h Columbia, hunting clubs and individual hunters were contacted by notices, l e t t e r s and personal v i s i t s and advised of the forthcoming project.  The notices and l e t t e r s requested their  assistance and gave directions to be followed upon agreement to participate i n the c o l l e c t i o n s . A l l necessary c o l l e c t i n g materials were then buted or made available to the interested persons.  distriThese con-  sisted of: (a)  wooden kegs, p a r t i a l l y f i l l e d with 10$ formaldehyde 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$ f o r maldehyde f o r issue to individual hunters.  (c)  bundles of cheese cloth cut Into pieces large enough to hold a single specimen consisting of the e n t r a i l s and the legs of a duck.  (d) tags with strings attached on which the information desired from the hunters was l i s t e d as  17  follows: ( i ) SPECIE3 SEX ( i i i ) AGE (iv) PLACE (where shot or obtained) (v) DATE (vi) ETC. (for other pertinent information) (ii)  Collectors were directed to wrap the e n t r a i l s and the legs of each individual duck i n a piece of the cheese cloth, t i e securely with the strings provided, f i l l i n the attached tag and deposit i n the formaldehyde.  A regular pick-up of  specimens from the various c o l l e c t i n g points throughout  the  v a l l e y was maintained during the hunting periods. When the actual preparation of the specimens for chemic a l 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 catalogue number.  Throughout the entire procedure, even to the  i d e n t i f i c a t i o n of a specimen i n 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 e n t r a i l s b r i e f l y 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 l i n i n g 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 t h i s , the l i v e r of the specimen was separated, f i n e l y chopped up and placed into an evaporating dish bearing the appropriate catalogue number.  Both tarsi-metat-  a r s i of each specimen were skinned out and also placed i n a properly numbered evaporating dish. When s u f f i c i e n t l y dry, both bones and l i v e r s were ground into a powder with a mortar and pestle and placed i n separate wide-mouthed v i a l s .  The specimens were then sub-  jected, to further drying i n an e l e c t r i c oven for two days at 100° C temperature. Important during the grinding of the bone and l i v e r samples was to thoroughly wash the mortar and pestle with soap and hot water between specimens.  The washing was followed by  r i n s i n g with lead-free d i s t i l l e d water. When the samples were thoroughly dried the v i a l s were corked and stored to await ashing.  Due to the l i m i t e d number  of crucibles, the limited use possible of the one e l e c t r i c furnace large enough for the, purpose of this study, and the length of time necessary to accomplish ashing, i t was necessary to carry out the next step of the procedure i n l o t s com-' posed of the bones and l i v e r s of about one dozen specimens. After having been thoroughly dried the bone sample and l i v e r sample of each specimen were transferred to separate previously weighed and permanently numbered s i l i c a crucibles. The crucibles and t h e i r contents were always immediately placed into a dessicator i n which the crucibles were already stored p r i o r to t h e i r use.  The crucibles and their contents  19  were then weighed.  The crucible number, the weight of the  crucible, the weight of the crucible plus i t s tissue were entered i n order on separate " l o t sheets" of the l o g book. The l o t number, the crucible number and the nature and weight of the tissue contained were also entered under the proper catalogue numbers. The entire l o t of crucibles was then placed into an e l e c t r i c furnace f o r 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 i n the sample.  The ashing required from twenty-four to thirty-four  hours per l o t . On removal from the furnace the hot crucibles ing the ash were immediately placed into a dessicator  containfor cool-  ing. when cool, the crucibles and t h e i r contents were again weighed, the weight of the crucible was subtracted and the weight of the ash recorded on the " l o t sheets". The weight of ash per gram of dry tissue was then c a l culated according to the formula: where  |[ = A w » weight of ash sample, d » weight of the dry t i s sue ashed, A = ash per gram of dry tissue.  The figure f o r the ash per gram of dry tissue was then. entered into the last column of the " l o t sheets".  For quick  reference the weight of the ash sample and the figure representing 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 l o t , the contents of the crucibles were transferred by means of folded s l i p s of paper, a separate s l i p being used for each sample, to small glass v i a l s .  These were corked, l a b e l l e d with catalogue num-  ber and the l e t t e r 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 i n 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 dryness.  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. i n volume i n a graduated mixing cylinder. When a portion of the solution was to be used f o r the determination 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 l a b e l l e d with catalogue number and tissue letter. To the solution in the mixing cylinder 5 ccs. of 2 5 $ ammonium c i t r a t e 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 glycerine.  This was continued u n t i l the color of the chloroform  layer at the bottom of the mixing cylinder became b l u i s h 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 i n carbon tetrachloride and acid solutions.  Some of  the dithizone w i l l have been l o s t to the alkaline water layer with the r e s u l t that the dithizone remaining  i n the chloro-  form and therefore the grey color of the end point, w i l l be somewhat d i l u t e d .  To determine the extent of t h i s d i l u t i o n 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 chloroform d i l u t i o n s previously prepared from the color standard. The  extent of the d i l u t i o n was recorded to be used i n the calcu-  l a t i o n s as the c o e f f i c i e n t of d i l u t i o n . After treatment by the c o e f f i c i e n t of d i l u t i o n the amount of dithizoneu.used i n t i t r a t i o n indicates the amount of lead present i n the portion of the bone or l i v 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. was  done according  This  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 i n parts per m i l l i o n . Analyses of several selected specimens for copper, zinc, iron and manganese were carried out by Miss R. I r i s h of the Geology Department of the University of B r i t i s h Columbia.  Each preserved 5  aliquot portion of some of  the samples was transferred from the test tube to a mixing cylinders  The pH of the solution was adjusted to between 2 and  three with a drop or two of 3  N  hydrochloric acid.  Copper was  then extracted with a solution made up of 60 mgs. of dithizone per l i t r e of carbon t e t r a c h l o r i d e .  The end point was i n 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 prevent 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 o r i g i n a l water layer were then removed for the iron and manganese determinations.  23  The aliquot for iron had to be diluted, i n many cases, 50 to 100 times.  More hydroxylamine  was added to ensure that a l l  the iron was reduced.  The solution was made s l i g h t l y acid and  b i p y r i d y l was' added.  This reagent with iron gives a red color,  the Intensity of which i s matched with a set of standards. To the d i l u t e d aliquot set aside for the manganese determination formaldoxene and sodium hydroxide were added.  A  brown color s i g n i f i e d the end point and was matched against a set of standards.  24  VI.  DISCUSSION OF PROCEDURES AND  METHODS  t The  only  to o b t a i n was  sample of duck specimens that i t was  one  c o n s i s t i n g of a hunter k i l l .  possible  T h i s i s pro-  b a b l y as c l o s e to a random sample as i t i s p o s s i b l e to without some v e r y e l a b o r a t e  obtain  c o l l e c t i n g technique which might  e v e n t u a l l y prove l e s s f a u l t l e s s than o r i g i n a l l y a n t i c i p a t e d . It poisoning  is s t i l l  necessary however, to determine whether l e a d  affects flight  to an extent  eased ducks more prone to shooting  s u f f i c i e n t to make d i s -  than h e a l t h y i n d i v i d u a l s .  For the present study however, s i n c e p r e v i o u s g a t i o n s i n t o the l e a d p o i s o n i n g  of waterfowl have been l a r g e l y  l i m i t e d to macroscopic g i z z a r d examinations of ducks by a hunter k i l l ,  v a l i d comparisons can  Monier-Williams ( 2 3 )  in  s h a f t s and  r i b s and v e r t e b r a e .  t r a t e s i n the c u l a r l y at the  Aub  trabeculae  obtained  s t i l l be made.  s t a t e d that l e a d i s r e t a i n e d  more by p l a t e bone than t u b u l a r , mores than i n the  investi-  i n the  ends of the bones  i n the femur and (2)  too, w r i t e s  t i b i a more than that l e a d concen-  s c a t t e r e d through the marrow, p a r t i -  epiphysis.  T g o - m e t a t a r s i were chosen as a r  the  bone t i s s u e to be analysed In the present study p r i m a r i l y because i t would be obtained the  the  from the  the p o r t i o n o f the  collectors  8  I t was  l i g h t of M o n i e r - W i l l i a m s  s k e l e t o n x«mld be  quite  1  specimen most r e a d i l y f e l t however, that i n  statement, t h i s p a r t of  s u i t a b l e f o r the  purpose.  the  25 Due to the uneven d i s t r i b u t i o n of retained lead that takes place i n the bone, the whole of the bone was ground up and thoroughly mixed i n the preparations of the samples f o r chemical analysis. Throughout this part of the work as in the l a t e r chemical 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 i n developing a method  of analysis f o r lead which was suited to the conditions imposed by t h i s 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 t h i s subject.  Those methods which are i n use f o r 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 t h i s study  or f a r 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 s p e c i f i c a l l y for t h i s study, one which would be rapid, reasonably 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. I r i s h of the Geology Department to whom the problem was referred.  The methods f i r s t considered  "were based on the p r e c i p i t a t i o n of lead as a sulphide.  26  However,the constant interference of copper and undissolved ash residue, p a r t i c u l a r l y i n the l i v e r samples, forced abandonment of this approach i n favor of the use of dithizone (diphenylthiocarbazone), the microdetermination According  an organic reagent widely used f o r  of various metals.  to F e l g l (14), dithizone reacts with numer-  ous metallic ions as well as lead to form inner complex compounds which are soluble In organic l i q u i d s .  However, under  proper conditions, dithizone w i l l be s p e c i f i c for lead. I t forms a bright red inner complex compound with lead salts i n neutral to alkaline and cyanide solutions. H H N—h—C H-r 6  S=C  /  H N—N—C H«S=C Pb/e N==N—C H 6  N  ^ N=N—C RV Dithlzone 6  X  6  0  Sandell (28) regarded the pH of 9«5 - 10 as being the most suitable for driving the reaction Pb % +  DH Z  *DzPb  H*  to the right. Since neither dithizone nor i t s heavy metal complexes are soluble i n water, chloroform was used as the solvent. Carbon tetrachloride, used as the solvent for dithizone i n the analysis for copper and other metals, was rejected for lead analysis since, at the high pH at which dithizone i s most s p e c i f i c for lead, carbon tetrachloride w i l l share too much of the dithizone with the alkaline aqueous layer.  The d i l u -  tion of dithizone caused by the smaller loss of the reagent  27 to the aqueous layer from a chloroform solution could more e a s i l y be determined by comparison of the grey end point with several d i l u t i o n s of the standard. It was expected that copper, Iron, zinc and perhaps manganese would be present, p a r t i c u l a r l y in the l i v e r  samples.  Since these metals could hinder the determination of lead by dithizone, i t was necessary to create conditions under which t h i s interference would be eliminated. Preliminary t r i a l s showed that the dithizone was being badly oxidized by the large quantities of f e r r i c iron i n the l i v e r s .  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 p r i o r to the treatment of the sample with dithizone. Fe * +• ++  NH OH £  > Fe^  +  |N  2  "-»-  H.,0  +•  H  +  Any small amount of oxidation of dithizone which was s t ' i l l found to take place merely accentuated the d i l u t i n g effect of the loss of dithizone to the aqueous layer and was compensated for when the extent of d i l u t i o n was  determined.  The divalent iron r e s u l t i n g from the hydroxylamlne  treat-  ment, the copper, zinc, and any other metals which could i n t e r 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 s i m i l a r l y to lead were thus overcome.  2g  VII.  A.  RESULTS  Macroscopic Examination of the Gizzard f o r Active or Current Leading The t o t a l hunter k i l l sample consisted of 15^ ducks of  thirteen species.  Mallards (Anas platyrhynchos)  and p i n t a i l s  (Anas acuta) t o t a l l e d 11^ Individuals, the remainder being made up of widgeon (Anas amerlcana), shoveller (Anas clypeata),  gadwall (Anas strepera).  lesser scaup (Aythya a f f i n i s ) ,  greater scaup (Aythya marila), Barrow's golden eye (Bucephala l s l a n d l c a ) , 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 i n the sample, 13 were found to cont a i n lead shot i n t h e l r gizzards, that i s , were a c t i v e l y leaded, with i n d i v i d u a l gizzards dosed with from 1 to 27 p e l l e t s . The t o t a l number of p e l l e t s removed from the mallards was 62 which gave a figure of U-.77 f o r the average number of shot per duck. In the case of p i n t a i l s , g of 35 contained a t o t a l of 26 shot, an average of 3 « 5 per duck. 2  Individual gizzards contained  from 1 to 5 shot. The only other species containing any shot were lesser scaup, i n which 1 of the 6 individuals was found with 1 p e l l e t , and greater scaup i n which 1 of the H- taken was dosed with 2 pellets.  In both cases the p e l l e t s were of f u l l size and d i d  29  not show any evidence of having been carried for very long. None of the remaining t h i r t y specimens of either sur1  face or diving ducks was found to be a c t i v e l y leaded. The mallards and p i n t a i l s were thus shown to be the most affected by lead poisoning, the mallards being 16.45$ a c t i v e l y leaded and the p i n t a i l s 22.86$ a c t i v e l y leaded. (Table I ) . In several of the active lead poisoning cases, the g i z zards were found to show signs of the condition as described by many previous investigators.  Discoloration was noted i n  several instances i n both the mallards and the p i n t a i l s ; more frequently, the gizzard pads and l i n i n g were found to be loose and corroded.  It was observed however, that the g i z -  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 i n a l l cases of active lead poisoning did the gizzards show evidence of having suffered any deleterious e f f e c t s . One of the symptoms often mentioned in the l i t e r a t u r e and regarded as an almost certain manifestation i n cases of lead poisoning, namely, a proventriculus stuffed with food matter, appeared very infrequently among the individuals of the sample.  I t occurred i n only one p i n t a i l containing five p e l -  l e t s , one mallard containing one well-ground p e l l e t and  two  mallards which were captured after the hunting season and which were i n very poor physical condition. The l a t t e r two  30  ducks contained 21 and 12 p e l l e t s .  I t was noted that when the  proventrlculus of lead poisoned duck contained food matter, such food matter was of a seed nature, oats i n three cases and wild seed i n 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 g r i t frequently appeared i n active lead poisoning cases. Gizzards f u l l of food however, were not limited.just to i n d i v i duals that were suffering from active leading.  B  »  Chemical Analysis of Bone and Liver f o r 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 l e g bones, l i v e r s , 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 s u f f i c i e n t number of complete specimens or properly i d e n t i f i e d specimens that only bare trends and general indications could be obtained. The tarso-metatarsi and the l i v e r s of 4-9 mallards of the 66 which were found to be free of gizzard lead were subjected, to chemical analysis to determine their lead contents. It was found that the content of lead could vary from less than two parts per m i l l i o n i n the bone of non-leaded individuals to 137 parts per m i l l i o n i n the bone of a c t i v e l y leaded specimens. The l i v e r lead varied from [l  ppm i n non-leaded mallards to  44 ppm i n the l i v e r s of a c t i v e l y poisoned individuals.  31 It was also discovered however, that Individuals which were a c t i v e l y leaded could have lead contents of bone and l i v e r which were no higher than i n apparently healthy specimens while, at the same time, the bone lead content of some specimens could be as high as 195  of the currently non-leaded  ppm with l i v e r contents as high as 23 ppm. (Table I I ) . These abnormally high lead contents of bone, l i v e r or both, of specimens which were not a c t i v e l y leaded, were accepted as satisfactory evidence that the individuals i n question had previously suffered from lead poisoning, had e l i m i 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$.  The status of  8  more out of the  application of the percentage of  36e7  49 was  i n doubt.  The  to the figure gave an  answer of 2.93 which was added to the o r i g i n a l IS to give a t o t a l for previous leadings of  20*93*  In order to treat the  entire mallard sample as a unit i t was necessary to adjust the figure of  20.93  lead-free mallards.  out of  49  to  28.19 out  of the o r i g i n a l  66  This figure, added to the 13 actively  leaded individuals out of the sample of 79» indicated that a t o t a l of 52.14$ of the hunter k i l l mallards were either suffering or had suffered from lead poisoning, (Table V). The bone lead contents of the 8 a c t i v e l y leaded p i n t a i l s analysed varied from 11 ppm to 151 ppm. varied from 2 ppm to 53 PP » m  Liver contents  The bone Lead contents of the  non-leaded p i n t a i l s were found to vary from an apparently  32  healthy [2 ppm ppm  to 12 ppm,  to 70 ppm while l i v e r contents ranged from  £l  (Table I I I ) .  Of the l b lead-free p i n t a i l s 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 d e f i n i t e 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 p i n t a i l s which had been found, to be free of ingested lead.  The o v e r a l l percentage of p i n t a i l s that were currently  leaded and those that had survived previous leading was  3$»17$  (Table V). The percentage of previously leaded lesser scaup and greater scaup x«;ere not calculated due to the inadequate number of specimens of those species. With respect to the remaining species and individuals that were chemically analyzed, four widgeon had bone lead contents varying from JjL to 4- ppm [2 ppm.  and l i v e r lead contents a l l  One green-xvinged t e a l had [ l ppm of lead In both t i s -  sues and a shoveller contained [2. ppm of lead in the l i v e r .  i n the bone and 4- ppm  Of the four lesser scaup chemically  analyzed only one was currently leaded with one uneroded p e l let.  Both the bone and the l i v e r 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  l a t t e r two specimens appeared to be survivors of the disease. Their l i v e r lead contents were [ l ppm  and 3 PPn* respectively.  The bone lead content of the one a c t i v e l y leaded greater scaup  33 was  found  liver  lead  greater  was d o s e d  golden  4 ppm  and o f the non-leaded  were b o t h / with  lead  incidence  of both  present  areas  areas  of  and t h a t  i n the North  Only possible dence and  of greatest pintails  the greater  to attempt  of previous  previous It  that  had  sufficiently  showed figures  leading,  as such  t o have greater being  that  any very  definite r e and Ladner  o f the disease f o r  suffered their  number  numbers o f  t h e Canoe P a s s  incidence  of mallard  and time  of the  greatest River,  percentage (Table V I ) .  specimens  made i t  o f year  with  the i n c i -  both  present  sickness.  after  thought  £l  i n t h e main  of sufficient  Arm o f t h e F r a s e r  leadings,  was f o u n d  identified  varying  from  c o r r e l a t i o n s o f age and sex w i t h  survival been  leading  to achieve  i t d i d appear  regions  leadings  The l a c k  i t impossible  However,  mallards  varying  One  scoter  contents  some i n d i c a t i o n  and previous  of hunting.  made  were  pellets.  (Table I V ) . w a s made t o o b t a i n  sult.  lead  Their  leaded  one w h i t e - w i n g e d  contents  attempt  specimens  ppm.  The a c t i v e l y  m e r g a n s e r s h a d bone  An  general  o n e , £2  two q u i t e . u n e r o d e d  t o 9 ppm a n d l i v e r  t o 5 ppm»  l ppm.  e y e , one b u f f l e h e a d ,  two r e d - b r e a s t e d  from ppm  contents  scaup  Barrow's and  t o b e 4 ppm  males  showed  the highest  namely  50.38$  o f the t o t a l  identified.  i n the hunter  been  survivors  survival  after  O f t h e 23 k i l l  sample,  of previous leading  than  figuref o r o f 26  female 34.06$  leading.  mallards, were Adults  j u v e n i l e s , the  48.78$ and 30.0$ r e s p e c t i v e l y , ( T a b l e  which  VII).  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 f o r female adults stood at 3 5 . 0 1 $ , f o r juvenile males at 2 9 . 5 3 $ and juvenile females at 3 0 . 3 7 $ ,  (Table VIII).  The incidence of current leading i n mallards and the evidence of previous poisoning was determined  f o r 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 r e -  corded f o r the month of October.  In November there was a  s l i g h t r i s e to 16.66$ which remained almost steady into the month of December f o r which a percentage  of 16.0 was obtained.  The figures obtained f o r previous leadings followed a very d i f f e r e n t pattern.  For the month of October 3 7 . 3 ^ $ of  the^p  lead-free mallards were recorded as being survivors of lead poisoning.  During the month of November this figure had  f a l l e n to 33*59$ hut i n December i t increased suddenly to 56.36$, (Table IX). Chemical analysis was carried out on 11 mallards which were not included i n the hunter k i l l sample.  They consisted  of Individuals found dead or too sick to escape capture. A l l were taken i n the l a s t days of December, 19^9 and the early days of the month of January, 1 9 5 0 . While s i x of these mallards were found to be free of lead shot, the remaining f i v e were very heavily leaded with from 12 to 35 p e l l e t s per individual.  The bone lead contents i n the leaded specimens  35 ranged from 44 ppm to a high of 228 ppm i n the specimen carrying  the 35 p e l l e t s .  Liver lead contents were equally high,  varying from 82 ppm to 176 ppm, the highest figure again appearing i n the most heavily leaded of the ducks.  Two of  the non-leaded individuals of this group had hone and l i v e r lead contents of 96 and 14 ppm and 7 and 11 ppm. ing  The remain-  four specimens had i n s i g n i f i c a n t quantities of [2. to 3  ppm i n the case of bone tissue and £l  to [2 i n the case of  l i v e r s with the exception of one i n d i v i d u a l which showed 14 ppm of lead i n the l i v e r , (Table X). Correlating the extent of gizzard damage with both the number of lead p e l l e t s carried i n the gizzard and the lead contents of bone and l i v e r showed that there was very relationship with either* of  little  Rather, there seemed to be more  an association of gizzard damage with the actual presence  of p e l l e t s which had undergone erosion, indicating that they had been carried f o r some period of time, (Tables XI to XIII). The analysis f o r copper, iron and zinc i n representatives of various species and conditions within species contributed very l i t t l e of d i r e c t interest to t h i s study. Further work along these l i n e s , however, would be of value, (Table XIV).  36  VIII.  DISCUSSION  Macroscopic examination of the gizzard contents of ducks in a hunter k i l l sample showed that 16.45$ of the mallards and 2 2 . 8 6 $ of the p i n t a i l s were carrying Ingested lead. Previous figures for leading i n mallards Valley included those of Munro ( 2 5 ) , Tener ( 3 3 ) ,  16.1$  in 1947.  in the Lower Fraser  13.3$  i n 1935  and  At the same time Tener obtained  a figure of 5$ active leading i n p i n t a i l s i n the same area. The figure of 16.45$ leading In mallards Tener s 1948 1  substantiates  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. figure of 2 2 . 8 6 $ leading f o r p i n t a i l s obtained  i n t h i s study,  however, i s a tremendous increase over Tener s 5$ i T  The  n  1947.  I t i s an increase out of a l l proportion with that taking place i n the case of mallards and leads to the that the high figure for 1949  may  suggestion  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, i n the group r e f e r red to as miscellaneous species, was  found to contain lead.  This fact would tend to add confirmation to the made by Tener (34)  suggestion  that lead p e l l e t s are picked up as g r i t  since those ducks which u t i l i z e a sand-like g r i t do not appear  37  to  suffer contamination.  Widgeon and green-winged t e a l are  the prime examples of t h i s group and shovellers could perhaps be added though Tener d i d note the leading of a shoveller In one instance.  The observation of Pirnle (27) that lead p o i -  soning appears to be more serious i n areas of gravel d e f i c i e n cy, adds support to the thought that lead i s picked up as g r i t , (Table IV). On the other hand Elder (13) mentions the leading of blue-winged t e a l and the Progress Report of the I l l i n o i s Natural History. Survey (19) ler,  t e l l s of shot incidence i n shovel-  green-winged t e a l , and widgeon, with a f a i r l y consider-  able percentage i n the l a t t e r .  The report makes the strong  suggestion that lead i s picked up for reasons other than i t s s i m i l a r i t y to g r i t .  Still,  the figures for the extent of  lea,ding were, i n the case of shovellers,  green-winged  t e a l , 2 . 1 5 $ ; widgeon, 4.84$ as compared to 7 « & 2 $ i n mallards, 9.77% i n p i n t a i l s and 13*37$ i n redheads.  The users of sand-  l i k e g r i t appeared considerably less prone to leading and their contamination  i n that l o c a l i t y might have been due to  some aspect of the observation mentioned by P i r n i e . Conclusive proof i s s t i l l lacking and the entire quest i o n i s regarded, by the present writer, as being s t i l l open. Contrary to i t s frequent mention as an almost invariable symptom of lead poisoning, a proventriculus stuffed with food matter was r a r e l y found.  In the four instances when i t could  be said that the proventriculus was stuffed or contained  food,  3* the food matter was wild seed. in  of a seed nature, usually oats and once,  In each case the lead p e l l e t s were s t i l l present  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 r e a d i l y to lead poisoning  than those on a green, leafy diet.  fore, that the effect of lead poisoning  It may  be  on seed eating  thereindi-  viduals i s s u f f i c i e n t l y sudden and severe to result i n paralysis  of the gizzard muscles, the p i l i n g up of food i n the  pro-  ventriculus and the retention of lead i n the gizzard. It  i s 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 r e s u l t 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.  I t 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, p a r t i c u l a r l y in p i n t a i l s , consisting of shells and parts of c r a y f i s h .  How-  ever, a stuffed gizzard also often occurred in completely unaffected  individuals.  Whether the lead played" any role in  the retention of food i n the leaded gizzards was  a question  which did not lend i t s e l f to determination within the scope of t h i s 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 The  study of lead poisoning  chemical analysis was Aub  feeding.  of waterfowl through  based primarily on the conclusions  (2) to the effect that lead, after absorption  of  from the  alimentary t r a c t , concentrated at f i r s t in the soft tissues, p a r t i c u l a r l y in the l i v e r and. then Ms  gradually .transferred  to the bone. Liver would naturally be the f i r s t organ a f f e c ted since i t l i e s d i r e c t l y i n 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 i s carried out through the b i l e so that, for  two  reasons, the l i v e r would- be expected to show a high  lead content immediately after contamination, throughout the active poisoning  and l a t e r , during heavy mobilization of lead  from the bones. Again according to Aub,  i n concentrating  in the t r a -  beculae of the bone, the lead stream p a r a l l e l s the calcium stream to which i t behaves s i m i l a r l y throughout.  The  lead  i s deposited i n the bone as an insoluble t e r t i a r y lead phosphate and while thus storedcan do the organism no injury.  further  However, the action of any d e c a l c i f y i n g agent or  condition such as starvation, shock or change in the a c i d i t y of the body, which w i l l cause the release of calcium from the bone, w i l l also result in the l i b e r a t i o n of lead, as a d i lead phosphate, Into the blood where i t can exert i t s f u l l toxic e f f e c t . That bones and l i v e r s can be regarded  4-0  and used as key centres in the study of lead poisoning through chemical analysis, i s confirmed by the work of Adler ( l ) on Canada geese.  He found that the bones of his a c t i v e l y leaded  specimens ranged from 67 ppm.  to 93 ppm  and showed no corre-  l a t i o n with the number of lead p e l l e t s being c a r r i e d 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 i n d i v i d u a l to 27 ppm  most heavily poisoned specimen,,  i n the  The bone lead contents of  the two controls which were l a t e r discovered to have been previously leaded were 3 ° PP contents were 0 and 1  m  and 3^ PP  m  while the l i v e r lead  ppm.  Similar results were obtained i n 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.  I t was also found,  however, more so i n mallards than in p i n t a i l s , that many ducks carrying lead had,almost normal bone and l i 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 i n which the gizzards were found to contain lead shot p e l l e t s , (Tables I I and I I I ) . The former appeared to be individuals only recently contaminated.  The l a t t e r , 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 p e l l e t s  occurs exclusively or, at least, more frequently than erosion and assimilation, i s indicated by the number of non-leaded  41 juvenile ducks which have only s l i g h t l y higher than normal lead contents of hone and l i v e r .  A juvenile duck would pro-  bably not have had time i n 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 l e v e l s . The statement of Adler ( l ) to the effect that bone lead content i s 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 p e l l e t s . and 60, One  In one instance two mallards, No.'s  45  (Table I I ) , each were found to contain one p e l l e t .  showed no indication of lead deposition i n the bone while  the other had 62 ppm  of lead i n the bone and 15 ppm  l i v e r , indicating an advanced state of plumbism.  i n the  Similarly,  mallard No. 34 with six p e l l e t s , had 44 ppm of lead i n the To) bone and. Jl  ppm  In the l i v e r while mallard. No. 76 with three  p e l l e t s and No. 105 with four p e l l e t s had 127 ppm ppm  of lead in the bone and 41 ppm and 5 P P  m  *  n  and  129  "the l i v e r s .  The p e l l e t s carried by the l a t t e r two specimens were eroded and worn. On the other hand, mallard No. 123,  (Table I I ) , con-  taining 27 very eroded p e l l e t s and having a gizzard, showing a great extent of injury, had bone and l i v e r contents of only lOg and S ppm.  Other factors besides the length of  exposure or dosage are c l e a r l y indicated. The widely varying amounts of bone and l i v e r lead  k2 sometimes made i t d i f f i c u l t to determine or decide, i n nonleaded ducks, whether they had or had not been previously leaded.  A gradation of lead contents upwards from [2 ppm  the bones and ]_\ ppm question  in  i n the l i v e r s existed and with i t , a  as to where the dividing l i n e f e l l between completely  healthy individuals and those which had  suffered leading i n  the past but had almost e n t i r e l y eliminated any lead that  may  have been stored in the bones, (Table I I ) . It was  noted, however, that a great number of mallards  and p i n t a i l s which were not carrying lead, had bone lead contents from about 3 PP 2 to l e s s .  m  to [2 ppm  and l i v e r lead oontents from  These would appear to be normal healthy  t i e s , p a r t i c u l a r l y when considered  quanti-  together with evidence  from the species which did not seem to be subject to lead poisoning, (Table IV).  namely widgeon, green-winged teal and  shoveller,  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 i n which the bone lead content was  found to be 5 ppm  contamination by lead.  °r over must have suffered  However, since the method of analysis  was  not very d e f i n i t e for quantities of lead under 2 ppm i t  was  considered  best to treat those individuals having bone  lead contents from 5 Ppm vious lead poisoning. was  to 7 ppm  as probable cases of pre-  A content of lead i n bone of 8> ppm  regarded as d e f i n i t e evidence of previous sickness itfhile  bone lead contents of H- ppm  and 3 PP  m  were accepted as the  43 upper l i m i t s of a normal healthy state unless accompanied by l i v e r contents of more than 2 ppm.  Whenever the l a t t e r con-  d i t i o n occurred i n conjunction with a bone lead content of less than 8> ppm,  the i n d i v i d u a l was also recorded as a pro-  bable survivor of past contamination by lead. Wetmore (37)  had stated that plumbism i n waterfowl i s  a dangerous and nearly always f a t a l malady.  Munro ( 2 5 ) ,  the basis of h i s observations.in mallards, had  on  concluded  though, that mallards at least may b u i l d 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 f a t a l , six No.6 The t o t a l percentage  shot.  of mallards in the hunter  kill  affected by lead poisoning, that i s , a c t i v e l y 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. of  These figures show that, while the t o t a l Incidence  the disease i s considerably higher than gizzard examina-  tions w i l l disclose, the balance of the higher figure i s made up of survivors of the disease who out a normal l i f e span.  Consequently  can possibly l i v e  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 examinations may lead observers to believe since a certain survival "rate can be  expected.  kk Lead poisoning appears to have more serious consequences among the p i n t a i l s 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 t o t a l  age of p i n t a i l s affected i s 3&»17 as compared to the mallards, only approximately 15.0  percent-  52 lk% i n B  7° of the t o t a l p i n t a i l  hunter k i l l are survivors while the incidence of active leading i s  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 r i s e i n l i v e r lead i s followed quickly by the deposition of the metal i n the bone. The l i v e r content appears to reach, i n most instances a maximum  i n mallards of between 15 and 20 ppm  although higher con-  tents were recorded, usually i n correlation with higher than average number of p e l l e t s .  In p i n t a i l s ,  tents were recorded; up to 53 and 63 ppm  higher l i v e r conwith only average  dosage of three or four p e l l e t s . After elimination of the lead p e l l e t s the l i v e r lead drops sharply to normal or just above normal.  The l e v e l of  l i v e r lead then depends on the rate of elimination from the body of bone lead which i n most cases can be expected to procede gradually.  The rate of elimination of the bone lead  - may be expected to decrease or perhaps temporarily, cease  K5 e n t i r e l y as the hone content becomes smaller.  Mallard N o . 6 l  (Table I I ) , with a bone lead content of 29 ppm  and a l i v e r  ;  lead content of £l  ppm  may  I l l u s t r a t e such a condition.  I n some specimens, such as mallard No.9 adult with a bone lead content, of 4- ppm of K ppm,  (Table I I ) , an  and. a l i v e r content  i t i s d i f f i c u l t to say whether the case i s one  of  almost completed elimination of lead from the bones after exposure or rather, the occurrence of the death of the duck immediately a f t e r the elimination, from the gizzard, of a p e l l e t which had not been retained long enough to make the disease more apparent to analysis.  That the l a t t e r s i t u a t i o n  can take place i s i l l u s t r a t e d by the case of mallard No.97 (Table I I ) , i n which the l i v e r lead content i s 19 ppm bone lead content i s 11 ppm, of the disease,  and  indicating an i n i t i a l progress  elimination of the p e l l e t or p e l l e t s and  subsequent death of the duck shortly a f t e r elimination. bone lead had nated.  the  the The  just begun i t s r i s e when the lead xiras e l i m i -  The duck was  then shot before the l i v e r lead could  f a l l to more normal l e v e l s .  P i n t a i l No,59 ( T a b l e . I l l ) , also  shows such a state before elimination and death.  It was  still  carrying one p e l l e t and had a bone lead content of 11 ppm a l i v e r lead content of 12  ppm.  Mentioned e a r l i e r was  the apparent tendency of p i n t a i l  l i v e r s to suffer higher lead contents than mallard even with fexver p e l l e t s .  and  livers,  I t seems l i k e l y that the loxver sur-  v i v a l of p i n t a i l s from the effects of plumbism may .least part of Its explanation  i n this f a c t .  f i n d at  It x<ms noted  that i n 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 l i v e r lead contents were very high, reaching l e v e l s four and f i v e times greater than the l i v e r lead contents of the leaded ducks of the hunter k i l l  sample.  It i s true that the dosages i n t h i s sample xvere heavier than the average dosages i n the hunter k i l l but at the same time, the p e l l e t s were not eroded to any great extent. No.123, (Table I I ) , of the hunter k i l l ,  In mallard  carrying 27 p e l l e t s  nearly a l l of which were ground to small f l a t discs, the bone and l i v e r contents of lead were only lok and $ ppm as compared to 73 and 113 ppm of mallard No. 132, (Table 10), which contained 23 p e l l e t s .  The poor physical condition of the  l a t t e r specimen due to malnutrition and perhaps other factors, apparently contributed to the exaggerated effects of the p o i soning.  The entire pattern of high l i v e r lead contents  indi-  cates some accentuated condition which resulted i n the very abnormal amounts of laad i n that organ.  This could be a mal-  function of the l i v e r i t s e l f which prevented the prompt transfer of lead to other tissues.  More l i k e l y however, i t i s 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 i n a b i l i t y to deposit more.  In addition, low calcium levels i n the Blood would also  result i n a greater absorption of calcium, and with i t lead, from the alimentary t r a c t .  Both would cause greater concen-  trations of lead i n the blood, and result i n a higher content  47 of lead in the l i v e r . With respect to p i n t a i l s , i t might be added, that i f l i v e r or blood damage i s more easily accomplished by lead i n that species, the higher incidence of lead poisoning apparentl y existing may have been due to the adverse effect of the disease on their f l i g h t and escape a b i l i t i e s .  As mentioned  e a r l i e r , i t s t i l l remains to be shown whether f l i g h t , while s t i l l possible for a poisoned duck, i s nevertheless affected to the extent that It i s more prone to shooting than Is a healthy Individual.  I t would remain necessary then, i n 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 i n the- p i n t a i l s alone during that year. S h i l l i n g e r and Cottam (29) ing  mentioned that lead poison-  seems to have more severe results i n \ •••••winters.  It can  be expected that any adverse period r e s u l t i n g in malnutrition w i l l exaggerate  the severity of the condition and result i n  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 a c t i v e l y leaded and tiro lesser scaup apparently having been previously leaded, (Table IV). latter.two showed bone lead contents of 73 and 77 ppm.  The Liver  contents were low, £l in one case and 3 PP** in the other. 1  One lesser scaup had lead contents of 9 ppm 4 ppm  i n the bone and  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.  I t 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. I n s u f f i c i e n t numbers of specimens makes this section of l i t t l e value for d e f i n i t e conclusions,, that animal-eating  However, i t can be expected  diving ducks, feeding i n possibly lead-  bearing estuary waters, may carry more lead i n their bones than pond ducks.  Chapman (9)  states that molluscs and crus-  taceae found i n the mouths of i n d u s t r i a l r i v e r s may accumulate lead.  This could f i n d i t s way into the systems of ducks  feeding on such animal matter and at the same time, the high calcium l e v e l to be expected i n the blood of such ducks would facilitate  the deposition of lead i n i t s harmless state i n  the bone. The lower figure f o r previous leading i n Juveniles, (Table VII), probably indicates lower rate of occurrence of the condition rather than lower survival a f t e r 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 i d e n t i f i c a t i o n s were i n adequate f o r d e f i n i t e conclusions, out of the a c t i v e l y leaded ducks i n the samples obtained f o r this study, the majority were juveniles.  Out of the seven mallards  carrying lead and  i d e n t i f i e d as to age, tv/o were d e f i n i t e l y i d e n t i f i e d as juveniles and another three were identified, as mot probably  ^9 Juveniles.  Out of the eight p i n t a i l s leaded, f i v e were  Juveniles.  Elder (13),  analysing the s i t u a t i o n among blue-  winged t e a l , mallards and p i n t a i l s found that the figure f o r active leading was  always higher among the Juveniles.  How-  ever, i t i s s t i l l l o g i c a l 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 i n their bones for a period of years and thus swell the numbers of those individuals entered as survivors.  A d e f i n i t e comparison of the survival expectancy of  juveniles and adults would constitute a valuable and i n t e r e s t ing study. The r e s u l t s reported i n Table VIII suggest that there i s no difference i n the incidence of lead poisoning between sexes and that the survival rates of male and female juven i l e s 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  Report of the I l l i n o i s Natural History Survey (19)  1950  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 i s 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. l i z e d for use i n egg production  As body calcium i s mobi-  the general calcium  level  50  in the body should decrease.  The mass of evidence  to be found  in the l i t e r a t u r e suggests that this fact would be calculated to  increase blood lead, both through accelerated absorption  of any lead s t i l l carried i n the gizzard and the mobilization of inert lead carried i n the bones.  Increase in the blood  lead at a time when the female i s undergoing the stress of reproduction may play a part i n the greater t o l l of female ducks apparently taken by plumbism.  I t i s s i g n i f i c a n t that  t h i s difference i n death rate between sexes does not take place t i l l after the female i s of an age to have experienced i t s f i r s t nesting. There would appear to be a d e f i n i t e increase i n 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$, i n November 1 6 . 6 6 $ and i n December 1 6 . 0 0 $ .  The percentage of  survivors of previous leading, which had remained i n the t h i r t i e s throughout October and November, rose to 56$ i n December, (Table IX).  In order that such a figure should r e -  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$ f o r 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 r i s e of survivors percentage to 5b.36$ with only  51  a small Increase i n active leading figures suggests that the duration of any individual case of leading could not be very long, that i n most cases  the p e l l e t s are not held for more  than perhaps a week or two.  This would bear out many of the  observations made i n Table II where bone and l i v e r contents indicate the beginning and then the sudden halting of lead poisoning. There i s some correlation between the number of shot present i n the gizzard and bone and l i v e r lead contents, with v i s i b l e gizzard damage.  I f lead i s s t i l l present i n the g i z -  zard of ducks with a high bone and l i v e r content of lead, indicating that the p e l l e t or p e l l e t s have been present for some time, the gizzard almost invariably shows great injury i n the form of corroded and loose l i n i n g s which slough o f f with ease.  On the other hand, i n 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 i s that damage i s due to the actual presence of a heavy dosage of lead or lead which has been held f o r a considerable length of time, and that very quick recovery takes place after the elimination of the lead.  Macroscopic  examination  of gizzard l i n i n g s could not be used as an index of previous leadings.  52  IX.  1.  CONCLUSIONS  There has been an increase i n the incidence of. lead p o i soning i n mallards and p i n t a i l s i n southern  British  Columbia i n the l a s t twenty years. 2.  The t o t a l incidence of lead poisoning i s considerably higher than the macroscopic examinations of the gizzard.s for lead p e l l e t s have previously indicated.  3.  The fact that so many ducks have apparently passed off the p e l l e t s 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,, p a r t i c u l a r l y mallards, appear to be able to withstand prolonged  the effects of sometimes very heavy or  dosages, even greater than that described by  Wetmore as "always f a t a l " , namely s i x No.,6 shot. 5.  Plumbism w i l l exert i t s greatest influence and w i l l do i t s greatest damage during period.s of adverse conditions such as heavy snowfalls and r e s u l t i n g famine.  Presumably  decalcifying agents and conditions making themselves  f e l t at such times result i n an increase of lead i n the blood where i t can exert i t s f u l l toxic e f f e c t s . 6.  Further evidence, though very sparse, was  obtained to  indicate that diet has an effect on absorption  of lead  from the alimentary t r a c t , deposition of lead in the bone, amount of lead concentrating  in the l i v e r , in.short,  plays  an important role In the entire course and effects of lead poisoning. 7.  P i n t a i l s appear to suffer much more than mallards from lead poisoning,  both from the point of view of the actual  incidence but p a r t i c u l a r l y from the effects of the ted lead.  inges-  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 effects of lead poisoning  9.  the  than adult males.  There appears to be no difference i n the p r o b a b i l i t y of ingesting lead between the sexes nor of the rate of surv i v a l i n the  10.  Juveniles.  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 able on the summer range.  Fresh lead deposition  the hunting season does, however, result i n an i n the incidence of 12.  be a v a i l during increase  poisoning.  The period of actual retention of lead p e l l e t s i n the gizzard appears, i n most cases, to be considerably  less  54  than one month in duration.  55  X.  In the f a l l of 1949  SUMMARY  the author was  assigned to carry out  further researches into the lead poisoning of waterfowl i n the Lower Fraser River Valley of B r i t i s h Columbia. and most s i g n i f i c a n t portion of the work was  The major  to consist of  the application of chemical analysis to the study of the condition. Previous workers had  shown that the key organs i n plumb-  ism cases are the l i v e r s , which, without delay, r e f l e c t the amount of lead appearing i n the blood stream of the dual, and the bones into which blood lea.d may  indivi-  be transferred  under suitable circumstances and where i t i s , temporarily  at  l e a s t , rendered harmless to the organism. It was  therefore decided to carry out chemical analysis  on the bones and l i v e r s of ducks i n a hunter k i l l  sample, to  determine lead contents and t h e i r possible significance„ The hunter k i l l \  sample consisted of 154  and included 79 mallatds, the remaining 11  ducks of 13  species  35 p i n t a i l s and 40 individuals of  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 i n too poor physical cond i t i o n to escape capture. E x i s t i n g methods of quantitative microanalysis  for lead  56 were found to be unsuitable and a new method was s p e c i f i c a l l y for this study.  developed  It was based on the use of  dithizone (diphenylthiocarbazone) with chloroform used as the solvent. A macroscopic  examination of the gizzards of the ducks  i n the hunter k i l l sample disclosed that an increase i n the incidence of lead poisoning was taking place i n the study area.  A s t a r t l i n g increase i n leading was noted among pin-  t a i l s which were found, to be 22.36$ a c t i v e l y leaded as compared to their 5$ leading In 194-7  as reported by Tener.  Among mallards, Munro had reported 130$ Tener reported 16.1$  i n 194-7  leading in 1935>  and the present study showed  16.4-5$ leading i n 194-1. The results of the chemical analysis showed that many of the mallards and p i n t a i l s shot out of the a i r by hunters were survivors of previous cases of lead poisoning.  The t o t a l  incidence of the sickness was thus seen to be much higher than gizzard examinations have shown i n previous studies. This very fact, however, indicates that a certain rate of survival, dependent perhaps on weather, a v a i l a b i l i t y of food and other factors, can be anticipated and the death t o l l attributable to plumbism should not normally equal the percentage 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 f a t a l . The p o s s i b i l i t y of survival after leading appeared to be  57 considerably higher i n 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 i n this species indicates that plumbism constitutes a f a r greater hazard for p i n t a i l s than for mallards and that l i t t l e of the optimism that may "be f e l t over the survival expectancies of  the l a t t e r 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 deleterious effects on diet which i s seen to be an important factor i n 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 popul a t i o n s 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 p e l l e t s  53 i n any one contamination are carried for periods ably less than one month i n duration.  consider-  59  XI.  SUGGESTIONS FOR FUTURE INVESTIGATIONS  The present study, being the f i r s t of i t s kind and suffering from the lack of essential information which might be more c a r e f u l l y specified and aquired in future work, i s one that could be usefully progressed further.  Various controlled  experiments would be of great value i n confirming and amplifying some of the trends and observations making themselves apparent during the present investigation. For instance, the different survival expectancies between p i n t a i l s and mallards, the fate of lead p e l l e t s , the duration of contamination and extent of erosion taking place i n that time are several aspedts of the work which would lend themselves to controlled investigation. I f a hunter k i l l sample can be obtained from persons f u l l y q u a l i f i e d to make invariably accurate  identifications  of sex and age other detailed work w i l l be possible. to determine,  In order  for example, just where the greatest damage i s  done i n the entire population of any one species, the actual number of p e l l e t s 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. results can then be considered  The  against other evidence obtained  on the ingestion and survival rates of the different sexes  60 and ages. An i n t e r e s t i n g and useful study i s suggested by the knowledge that lead i n the body follows a path similar to the calcium stream I t appears l i k e l y that when calcium i s withdrawn from the bones of the female for use i n egg production, lead may also be l i b e r a t e d from the bone.  S t i l l following the  calcium stream, this lead may be deposited, with calcium, i n the egg s h e l l s . sis  It i s thus conceivable that chemical  analy-  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 i s suggested.  A student, desiring to carry  through the entire project could begin, i n the f a l l of h i s graduate year, by inducing plumbism i n domestic and captive wild mallards.  These ducks could then be mated i n various  combinations and the influence on mating, reproduction and the survival of nesting females determined.  The r e s u l t s 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 be tested for the presence of lead.  part of the experiment could Should the test prove  positive the student can make his study of the l i t e r a t u r e and write the preliminary protion of his MA thesis during his first  post-graduate session. A c o l l e c t i o n of egg shells from the nesting grounds,  which could have been i n i t i a t e d during the previous summer,  61 All  survey parties entering the f i e l d can be instructed to  make c o l l e c t i o n s 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 postgraduate 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 p a r t i c u l a r l y indebted to Dr. R. Delavault and Miss Ruth I r i s h of the Department of Geology, for their a s s i s t ance with the chemical aspects of the project and to Dr. W.S.  Hoar and Dr. W.M.  space and f a c i l i t i e s .  Cameron for the use of laboratory  I also wish to extend my thanks to my  wife and to V. Vishnlakoff for the many long hours spent with me i n preparation and analysis of specimens. For their c r i t i c i s m s , advice and assistance I am gratef 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 B r i t i s h Columbia Game Commission for their f i n a n c i a l support of the project.  63  Table I. Species  Incidence of lead shot i n hunter k i l l No. of Percentage Total Total No. of Ducks with No. of Ducks with Lead Shot Lead Shot Found Shot  Mallard  79  Pintail  35  Widgeon  15  0  Green-winged Teal  2  G-adwall  sample. Average No. of Shot per Duck  16.4-5  62  KU  22.86  26  3.25  —  —  —  0  —  —  .—  2  0  —  —  —  Shoveller  1  0  —  —  —  Lesser Scaup  6  1  —  1  —  Greater Scaup  4-  1  —  2  —  Barrow s Golden eye  2  0  —  —  —  Bufflehead  2  0  —  —  —  Scoters  3  0  —  —  —  Red-breasted Merganser  2"  0  —  —  —  Ring-necked Duck  1  0  —  —  15^  23  13  1  TOTALS  1^.93  91  —  Table I I . Results of Chemical Analysis f o r Lead i n Mallards (Cata-  logue No.  No. of shot i n G-izzard  Lead i n P a r t s / M i l l i o n of drv tissue Liver Bone  Sex  Age  Month when Shot  ?  Ad.  Oct.  Ladner  0  5  Ad.  Oct.  Nicomen Island  0  12  Place  2  1  2  2  3  3  5  cri-  Ad.  Oct.  Nicomen Island  0  iz  3  4  g  er  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  a"  Ju.  Oct.  Canoe Pass  0  Z2  Z i  Ju.  Oct.  Canoe Pass  0  b  1  Ad. . Oct.  Canoe Pass  0  7  2  7  13  7  Z i  g  lb  9  23  %  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  15  45  lb  5b  ?  1  9  Ju.  Oct. "  Nicomen Island.  0  Z2 Z2 Z2  Z i Z i Z i  Table I I . Cont'd. Catalogue . Sex No.  ?  Results of Chemical Analysis f o r Lead In Mallards. No. of shot i n G-lzzard  irts/Mllllon Lead i n Pg of dr\r tissue Liver Bone .  Age  Month when Shot  Place  Ju.  Oct.  Nicomen Island  0  Z2  Zi  Ad.  Dec.  Canoe Pass  1  62  15  17  57  13  60  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  u.  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  ?  J  Table I I .  Cont'd.  Catalogue No.  Sex  32  101  ?  33  102  3^  104  35  -  Results of Chemical Analysis f o r Lead In Mallards Age  Month when shot  Place  No. of shot i n Gizzard  Lead i n P a r t s / M i l l i o n of drv t i s s u e  Bone  Liver  —  Dec.  Ladner  0  —  Nov.  Ladner  0  Z2 Z2  —  Dec.  Ladner  1  3  6  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 .  -Zi  4i  111  ?  Nov.  Ladner  0  12  Zi  42  114  a*  —  Oct.  Ladner  0  102  Zi  43  116  cT  —  Oct.  P i t t River  0  Zi  44  119  ?  Ad.  Dec.  P i t t River  0  Z2 Z2  45  120  Ad.  Dec.  P i t t River  -  2  Zi  46  121  Ad.  Dec.  P i t t River  0  /2  Zi  ?  ?'  —  Zi Zi  Zi  cr  Table I I . Catalogue No.  Cont'd. Sex  ?  Results of Chemical Analysis f o r Lead i n Mallards. Age  Month when shot  Place  No. of shot i n G-i z zard  Lead i n P a r t s / M i l l i o n of drv tissue Bone Liver  ——  Oct.  Westham Island  0  Z2  Zi  Ad.  Oct.  Westham Island  21.  104  g  Ju.  Nov.  North Arm Fraser  0  13  Zi  Ad.  Dec.  P i t t River  0  Ju.  Dec.  P i t t River  0  6o  140  Ad.  Dec.  P i t t River  0  11  1  53  142  —  Dec.  Ladner  0  22  2  64  143  Ad.  Dec.  P i t t River  0  6  55  144  ?  Ad.  Dec.  P i t t River  0  41  4  56  145  ?  Ju.  Dec.  P i t t River  0  7  2  57  146  Ad.  Nov.  P i t t River  0  Z2  Z2  47  122  48  123  49  124  50  137  51  138  52  or*  ?  Zi io  Zi  Table I I I . Results of Chemical Analysis f o r Lead i n P i n t a i l s Catalogue No.  1 2  Sex  Age  Place  No. of shot i n G-lzzard  Ju.  u  ?  Ad.  Oct,  North Aym Fraser  0 2  ?  Ju.  Oct.  North Arm Fraser  Ju.  Oct.  Canoe Pass  14 15  Month when shot ct.  Canoe Pass  3  • 17  4  19  5  20  <y  Ad.  °ct.  North Arm Fraser  6  26  ?  Ju.  Oct.  North Arm Fraser  7  27  ?  Ju.  Oct.  8  38  ?  Ju.  9  40  .?  10  41  n  Lead i n Parts/Million of dry tissue Bone Liver 4  Zi  151  5  5'"  49  22  1.  120  53  45  -  Z2  North Arm Fraser  0 0 0  1  Zi Zi  Oct.  Canoe Pass  4  53  63  Ju.  Oct.  Canoe Pass  Z2  ?  Ju.  Oct.  Canoe Pass  44  ?  Ju.  Oct.  ^adner  Z2 2 12  12  46  ?  Ju.  Oct.  North Arm Fraser  13  49  ?  Ju.  Oct.  Canoe Pass  0 0 0 0 0  14  54  ?  Ju.  Oct.  Ladner  » 15  55  Ju.  Oct.  Canoe Pass  n  0  3  Zo  Z2  Zi Zi  84  34  Z2  Z2  4  Table I I I . Cont'd. Sex  Catalogue No.  Results of Chemical Analysis for Lead i n P i n t a i l s . No. of shot i n Gizzard  Month when shot  Place  Ju.  Oct.  Canoe Pass  1  11  12  9  Ad.  Dec.  North Arm Fraser  1  1  9  2  9  Ju.  Nov.  North Arm Fraser  0  Z2  Zi  9  Ad.  —  Lulu Island  0  5  Zi  44  Zi  ?  16  59  17  63  12  75  19  79  20  95  cf  —  Nov.  Ladner  0  21  93  cf  —  Dec.  Ladner  0  22  112  —  —  Ladner  —  —  P i t t River  23  141  Lead i n P a r t s / M i l l i o n of drv tissue Bone Liver  Age  '  ¥  0  Z2 Z2  Zi  0  4  Zi  Zi  Table IV.  Results of Chemical Analysis for.Lead i n Miscellaneous  Species.  Catalogue No.  Species  Sex  Age  Month when shot  Place  13  Widgeon  ?  Ju.  Oct.  N. Arm.Fraser  21  Widgeon  Ju.  Oct.  N. Arm Fraser  3  30  Widgeon  Ju.  Nov.  N. Arm Fraser  4  33  Widgeon  Ju.  Oct.  Canoe Pass  5  42  Widgeon  Ju.  Oct.  N. Arm Fraser  0 0 0 0 0  6  4g  G-reenwinged teal  9-  Ju.  Oct.  N. Arm Fraser  0  Zi  Zi  Shoveller  ?  Ju.  Oct.  N. Arm Fraser  0  Z2  4  Ad.  Nov.  North Vanoauver  0  22  Zi  . °ct. North Vancouver  0  Zi  Zi  1 2  7  35  g  11  9  25  Lesser Scaup  10  66  Lesser Scaup  n  12  67 77  cf  £  Lesser Scaup  Lesser Scaup Lesser Scaup  ?  %  Ju.  No.of shot Lead i n PPM of dry tissue i n G-izzard Bone Liver  4  2  Z2 Z2  3  Zi Zi  4  Zi  —  Dec.  N. A m  Fraser  0  9  4  —  Dec.  N. Arm Fraser  0  ZZ  1  Ju.  Nov.  N. Arm Fraser  1-  Z2  r  Table IV. Catalogue No.  19  Place  No. of shot in Gizzard  Lead"in PPM' of dry <3Ufi  Bone  Liver  12  Zi  Ad.  Dec.  N. Arm  Fraser  2  4  Zi  Barrow's Golden Eye  Ju.  Nov.  N, Arm  Fraser  0  9  Zi  50  Bufflehead  Ad.  Oct.  North Vancouver  0  3  2  33  w.w. Scoter  Ad.  °ct.  Canoe Pass  0  9  Zi  Ju.  Dec.  Lulu Island  0  3  5  Ju.  Oct.  N. Arm  0  3  Zi  64  13  Month When Shot  0  14  17  Sex Age  Species.  North Vancouver  22  16  Species  Results of Chemical Analysis for Lead in Miscellaneous  Nov.  13  15  Cont'd.  74  34  35  G-reater Scaup Greater Scaup  RedBreasted merganser Redbreasted merganser  9  9  ?  Fraser  72 Table V.  Total incidence of lead poisoning (active and previous cases).  Species  1  2  3  4  Mallard  79  13  bb  49  ;  8  35 Doubtful  7  24. 24  37*24  47.14  20.93 28.19  41.19  52.14  3 .  5.0b  13.06  37.31  3*18  5-36  13.3b  33.17  —  —  —  -  -  18  <T  Doubtful cases-S 18x8 ~$^"~2.93 added. Pintail  b  5  lb  27  cases-1  ^g- = .18;added. Lesser Scaup  6  1  Greater Scaup  4  1  124  23  Totals  . 8  5  4  3  3  1  0  101  70  -  -  -  -  1.  Total number of ducks i n sample.  2.  Number of ducks a c t i v e l y leaded.  3.  Number of ducks not a c t i v e l y 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 l i v e r ,  b.  Number of previously leaded ducks raised to the t o t a l of lead-free ducks. (5x3.. )  ( 4 * 7.  b  )  Total number of ducks a c t i v e l y or previously leaded (2 + b).  . 8.  Percentage  of ducks affected.  Table VI.  Distribution of AffectedMallards and P i n t a i l s of the Hunter K i l l .  Area  Mallards 4 5  54.26  12  3. 9  5  0  0  25.00  2.7  11.6  54.00  5  1. 4  4  l  1  40.00  —  —  37-5  3  0  3  1  .12  —  --  —  -  -  -  -  -  5  1.4-  1*7  39.45  11  2  5  4  4  2  2  50.00  -  -  -  -  1  -  1  Canoe Pass  12  .3  9  Ladne r  27  3  2k  12  Lulu Island.  2  \  5  0  Matsqui  1  0  1  1  l  5  0  .7 (,2.7-  Nlcomen Is.  4-  N.Vancouver  1  0  1  1  12  0  12  11  Surrey  3  1  2  0  -  -  Westham Is.  2  1  1  0  -  -  -  Unknown  3  1  2  1  -  -  -  P i t t River  Totals 1. 2.  I: 5.  No. of No. of No. of No. of No. of leaded.  7  3-5  3  f  6  7  2  6  5  6  1  N.Arm Fraser  Pintails 2 3 4  4.7  79 13 66 49 ducks taken i n area. ducks a c t i v e l y leaded. ducks not a c t i v e l y leaded. ducks chemically analysed. ducks apparently previously  5.1  1  43.64  2 . 1 1  .54 ^ 12.00 3.2  -  -  56.36  -  l  -  6. 1.  1  35 2 27 16 No. of ducks apparently previously leaded raised to t o t a l no. of ducks not a c t i v e l y leaded. Percentage of ducks affected i n area <6 *  (  100>  2  1  x  x  1 0 U  )  h.  7  Table VII. Previous leading of mallards i n r e l a t i o n to the sex and age (using onlylthose individuals which have been s u f f i c i e n t l y i d e n t i f i e d and chemicalLy analyzed). Male  Female  Total Identified  26  23  Previous Leading  13.101  Percent survivors of previous leading  Table VIII.  50.3&1  Juvenile  17  7.335  34.06  Adult  l4  . 5.101  30.00  6.5  48.73  Previous leading i n mallards i n r e l a t i o n to sex and age taken together (using only those individuals which have been s u f f i c i e n t l y i d e n t i f i e d 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 survivors of previous leading  59-17  35.01  29.53  30.37  75  Table IX. Number of mallards affected i n r e l a t i o n t t o 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.  Catalogue No.  Results of Chemical Analysis on 11 Mallards Found Sick and unable to Fly or Dead. Sex  Age .  Condition When Found  Place  No.of shot i n gizzard  Lead i n P a r t s / M i l l i o n of dry tissue Bone Liver  1  126  cf  Ju.  Dead  Lulu Island  0  Z2  Zi  2  127  cf  Ad.  Dead  Lulu Island  0  3  Zi.  3  12g  cf  ~~•  —  Lulu Island  0  4  129  cf  Ju.  Sick  Lulu Island  0  Z2 Z2  5  130  cf  Ju.  Dead  Lulu Island  0  96.0  6  131  cf  Ju.  —  Lulu Island  14  44  g2  7  •132  cf  Ad.  Sick  Westham I s .  23  7g  113  g  133  cf  Ad.  Dead  Westham I s .  21  42  164  9  134  Westham I s .  35  22g  176  10  135  cf  Ju.?  Westham I s .  12  ii  136  cf  Ju.?  Westham I s .  0  Ju.  — — --  14.3  Z2 1  133 lit  Ii  Table XI. Observations of gizzard conditions of p i n t a i l s i n r e l a t i o n to extent of active leading and bone and l i v e r contents. Catalogue No.  No. of Shot i n Gizzard  15  2  17  5  19  .3  33  4  Bone *  One p e l l e t worn. and discolored. •  Lead i n ppm of dry  Observations  Gizzard l i n i n g loose  Llver  151  5  49  22  120  53  Some p e l l e t s worn. Gizzard l i n i n g loose and discolored. Gizzard distended with much animal and some plant food.  53  63  P e l l e t s various sizes and shapes. Gizzard l i n i n g loose and flakey. Liver a l i g h t color. Gizzard f u l l of animal food. P e l l e t s very worn. Gizzard and f u l l of plant food.  sloughing  54  5  Gizzard l i n i n g loose and gizzard stuffed with green plant food.  34  34  59  1  Gizzard showing no effect.  11",  12  Table XI. CoA/r'a.  Catalogue No.  117  Observations of gizzard conditions of p i n t a i l s i n r e l a t i o n to extent of active leading and bone and l i v e r contents.  No. of Shot i n G-izzard  Observations  Lead i n p pm of dry t.1 S S us Bone Liver  1  G-izzard discolored  19  2  5  Three p e l l e t s worn. Proventriculus and gizzard stuffed with oats.  —  —  Table XII. Observations of gizzard conditions of mallards in r e l a t i o n to extent of active leading and bone and l i v e r contents. Catalogue No.  No. of Shot in G-izzard  23  0  24  0  Observations  Lead i n ppm of dry tissue Bone  Liver  G-izzard showing slight e f f e c t s associated with lead poisoning Gizzard showing slight e f f e c t s associated with lead poisoning  7  2  195  15  187  44  28  8  Gizzard l i n i n g loose and brown i n color  34  6  Two'-pellets worn to d i s c s . Gizzard l i n i n g loose and discolored. Gizzard f u l l of seeds and oats.  44  31  45  1  Gizzard not v i s i b l y affected.  Z2  Zi  60  1  Gizzard not greatly affected.  62  15  76  3  One p e l l e t very worn. Gizzard showing some sloughing of the l i n i n g .  127  41  78  0  Gizzard l i n i n g showing d e f i n i t e e f f e c t s associated with lead poisoning.  34  3  104  1  3  6  129  5  io4  8  105 123  P e l l e t worn. Gizzard discolored and contained, seeds i n proventricular end  4  P e l l e t s worn and small i n size. l i n i n g loose.  Gizzard  27  Nearly a l l worn small and f l a t . l i n i n g loose; putrid odor.  Gizzard  Table XII. Cont'd. Observations of gizzard conditions of mallards i n r e l a t i o n to extent of active leading and bone and l i v e r contents. Catalogue No.  No. of Shot i n Gizzard  154  2  P e l l e t s worn f l a t . green plant food.  159  k  P e l l e t s worn. plant food.  Observations  Gizzard f u l l of  Gizzard f u l l of green  Lead i n ppm of dry tissue Liver Bone —  -  —  Observations of gizzard conditions of 11 mallards taken sick or dead in January 1950 i n r e l a t i o n to extent of active leading and bone and l i v e r contents of lead.  Table XIII.  Catalogue No.. 126  127  No. of Shot i n G-izzard  Observations  Found dead. G-izzard undersized and empty with l i n i n g . f i r m and dark i n color.  0  0  G-izzard empty and undersized with l i n i n g firm and dark In color.  129  0  Emaciated. G-izzard l i n i n g sloughing s l i g h t l y , almost empty and containing g r i t of a small size.  0  Bone  Liver  Z2  Zi  Found dead. Emaciated. G-izzard undersized and. empty with l i n i n g firm and dark i n color.  0  128  130  Lead i n ppm of dry tissue  •  3  Zi  Z2  14.3  Z2  Found, dead. G-izzard very small and l i n i n g black i n color.  131  14  P e l l e t s f u l l size.  Gizzard l i n i n g loose.  132  23  P e l l e t s f u l l size. condition.  G-izzard l i n i n g in poor  Z2 I  44  78  82  113  Table XIII.  Catalogue No.  Cont'd. Observations of gizzard conditions of 11 mallards taken sick or dead i n January 1950 i n r e l a t i o n to extent of active leading and bone and l i v e r contents of lead.  No. of Shot i n Gizzard  Observations  Lead i n ppm of dry tissue Bone Liver 42  164  223  176  133  21  Proventriculus stuffed with oats.  134  35  Gizzard l i n i n g loose. green plant food.  135  12  Proventriculus stuffed with oats. Gizzard containing oats and green plant food.  47  136  0  Emaciated. * Gizzard empty.  11  Gizzard f u l l of  -  -  133  ii  Table XIV. Catalogue No.  Chemical analysis f o r Copper, Iron and Zinc.  Description  No. of Shot per Duck  Copper  Lead  Iron  Zinc  Bone  Liver  Bone  Liver  0  4.1  Zi  41  142  Tfrabe  415  330  142  63.0  44  121  Trace 6922  142  699  Bone Liver Bone Liver  14 .  Pintail, normal  32  Pintail, leaded  4  53-0  77  Lesser Scaup  1  Z2  Z2  -  64  -  2670  -  252  64  Greater Scaup  2  4.5  Zi  -  33  -  430  -  21  50  Bufflehead  0  3  2  -  37  -  99  -  161  23  White winged Scoter  0  9  Zi  -  57  .2674  -  201  Green-winged Teal  0  Zi  Zi  —  94  —  —  142  35  Shoveller  0  Z2  4  -  34  -  2237  -  207  22L.  Greater Scaup  0  Z2  Zi  -  26  -  720  -  76  42  222 .  Table XIV.  Cont'd.  Cata- Description logue No.  Chemical analysis f o r Copper, Iron and Zinc. No. of Shot per Duck  Co]Dper  Lead  Zinc  Iron  Bone  Liver  Bone  Liver  _  5S  Bone  Liver  Bone  Liver  490  _  137  21L.  Widgeon  0  Z2  Z2  74  Barrow's Golden eye  0  9  Zi  —  5b  _  2064  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  927  -  85  9b  .  Mallard  _.  .37  0  Z2  Zi  79  -  Mallard (survivor)  0  11  19-  36  •-  101  Mallard  0  Z2  Zi  81  102  Mallard  0  Z2  Zi  71  97  2g0  1780  -  1429  -  513  -  1824  -  -  1671  -  1393  -  825  -  1800  •  -  Table XIV.  Cont'd.  Cata- Description logue No. 123  Mallard. (Leaded)  127  Mallard, (Dead)  Chemical analysis for Copper, Ironrand Zinc. No. of Shot per Duck  Bone  27  104  0  Lead  Z2  Liver  Copper Bone  Liver  134  Bone  Liver  1400  51  Z2  Iron  -  . 1637  Zinc Bone  Liver  114  -  215  -  86  Figure 1.  S7  XIII.  PREPARATION OF REAGENTS  Preparation of the Dithizone Reagent. The dithizone reagent i s prepared by simply dissolving 30 mgms of dithizone (DIPHENYLTHIOCARBAZONE) i n 1 l i t r e of chloroform, the chloroform being added slowly due to an exothermic reaction taking place during the mixing.  The pre-  paration-- sould be carried out i n 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 2000 ppm (gammas) of lead per cc.  containing 2 mgms or 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 f l a s k . Care was taken during the transfer, the pipette being f i r s t washed and rinsed i n lead-free d i s t i l l e d water and then rinsed with the lead standard. D i 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 ammonium c i t r a t e and hring the volume of the solution to 100 ccs. Lead impruities i n thehbuffer were extracted by shaking with a s u f f i c i e n t quantity of dithizone i n a separating  funnel.  When the dithizone ceased to turn red on addition and shaking, but rather retained i t s green color, i t was allowed to s e t t l e and was then drawn o f f , leaving the lead-free ammonium c i t r a t e intthe funnel. Preparation of the Potassium Cyanide Solution. D i s t i l l e d water was used tocldissolve 10 gms of potassium cyanide and bring the volume of the solution to 100 ccs. Since the solution was found to contain p r a c t i c a l l y no lead, extraction with dithizone was considered  unnecessary.  Preparation of the Color Standard. To 2 ccs of the d i l u t e d lead standard  i n the bottom of  a 50 cc mixing cylinder, 2 ccs of 25$ ammonium c i t r a t e buff e r and 1 cc of 10$ potassium cyanide solution wgre added. This was t i t r a t e d with the dithizone, about . 2 ccs at a time with the mixture being vigorously shaken between additions of the dithizone.  The t i t r a t i o n 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 recorded.  29  Seven ccs of dithizone were required f o r 2 ccs of the lead standard.  Three decimal f i v e ccs of dithizone were  required f o r 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 . 3 5 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 f i v e 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 i n the rack and d i l u t e d with chloroform  to twice, three times, four times  and five times t h e i r o r i g i n a l volumes.  This color series  was l a t e r used to determine the extent of dithizone loss to • the water layer and any loss of color of the dithizone complex due to reduction by f e r r i c iron, p a r t i c u l a r l y in the l i v e r  present  samples.  Throughout the preparation of the reagents and standards i t was most improtant to guard against accidental contamination by lead.  In the l a t e r chemical analysis of the  samples i t was equally important to guard against such contamination  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 i n d i s t i l l e d water.  Pipettes  were kept standing i n d i s t i l l e d water and before use were rinsed i n d i s t i l l e d water and then i n the solution to be ~  measured or transferred. ~ t i l l e d water a f t e r use.  They were rinsed once more i n d i s -  90  Other precautions observed i n 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 c r y s t a l l i z e d around the top of the bottle or under the stopper; avoiding the loss of any standardizing solution contained i n 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 i n the ashing process.  LITERATURE CITED 1.  Adler, F, E. w. Chemical analysis of organs from leadpoisoned Canada geese. Jour, w i l d l . Mgt., 8 ( l ) . 8 3 - 8 5 . 1944.  2.  Aub, Joseph G, The biochemical behaviour of lead i n the body. Jour. Amer. Med. Assoc., 104(lj:37-90, 1935.  3.  c i t e d from Monier-williams,  G.w.  4.  Aub, J . C., L. T. F a i r h a l l , A. S. Minot and P. Reznikoff. c i t e d from Monier-williams, G. w.  5.  Best, Charles Herbert and Norman Buske Taylor. The physiological basis of medical practice. (A text in applied physiology) 5 t h Ed., Baltimore, 1950. Williams and Wilkins Co.  6.  Blaxter, R. L. and A. T. Cowie. 588.  7.  Bowles, J . H.  Lead poisoning i n ducks.  312-313. 8.  Nature 157, M y 4 , 1946, a  The Auk, 2 5 .  1908.  Calvert, J . Hindle.  Cited from Jones, J . C.  9. 10.  Chapman, A. C. Cited from Monier-Willlams, G. W. Cheatum, E. L. and Dirck Benson. Effects of lead poisoning on reproduction of mallard drakes. Jour. W i l d l . Mgt., 9 ( l ) : 2 6 - 3 0 , 1945.  11.  Cottam, Clarence C.  12.  Cited from Jones, J . C.  Cited from Cheatum, E. L. and Dirck Benson.  13.  Elder, William H. Measurement of hunting pressure i n water-fox?! by means of X-ray. Trans, of 1 5 t h Nor. Am. Wildl. Conf., 490-503.  14.  F e i g l , F. Qualitative analysis of spot tests. Third edition. 1946. Elsevier Publishing Company, Inc., New York - Amsterdam.  15.  G-rinnell, G. B.  16.  Cited from Jones, J . C.  Cited from S h i l l i n g e r , J . E. and Clarence C. Cottam.  17.  Holland, George.  18.  Hough, E.  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 i n 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.  23.  Monler-Willlams, C. W. Trace Elements i n Food. Wiley and Sons, 1949, New York.  24-.  Munro, J . A. Lead.poisoning in trumpeter swans. Canad. F i e l d Nat., 39:160-162, 1925.  25.  Munro, J . A. Studies of waterfowl in B. C.. Jour. Res., D 21, 223-260, 1943.  26.  P h i l l i p s , John C, and Frederick C. Lincoln. Cited from Jordan, James S. and Frank C. Belrose.  27.  P i r n i e , D. M. Cited from S h i l l i n g e r , J . E, and Clarence C. Cottam.  22,  Sandell, E. B. Colorimetric determination of traces of metals. Interscience Publishers, 1950. New York, N. Y.  29.  S h i l l i n g e r , 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. B i o l , 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 experimental lead poisoning. Proc. Soc. Exp. B i o l , and Med., 38(3):435-437, 1938.  Cited from Jones, J . C.  Cited from Jones, J . C.  The Auk, 25; 472,  1902. John  C nad. a  Steiman, S. E. The action of lead on the phosphocreatine i n the muscular p a r a l y s i s of lead poisoning. Amer. Jour. Physiol'., 12b ( 2 ) : 2 b l - 2 b 9 , 1 9 3 9 . Tener, John Simpson. An investigation of some of the members of the sub-family Anatinae i n the Lower Fraser Valley of B. C, A thesis submitted i n p a r t i a l fulfilment of the requirements for the Degree of B.A. i n the Department of Zoology, U.B.C., A p r i l , 1 9 4 g . Tompsett, Sidney Lionel. The influence of certain constituents of the diet upon the absorption of lea.d from the alimentary t r a c t . Further studies on the absorption, mobilization and excretion of lead. B r i t . Jour. Exp. Path., 20(b):512-516, 1939.  Weller, C. V. ' The.blastophthoric effect of chronic lead poisoning. Jour. M d, Res. Vol. 23 (New e  Series, V o l . 23), 2 7 1 - 2 9 3 ,  1915-  Wetmore, Alexander. Lead poisoning i n waterfowl. United States Dept. of Agriculture, Bulletin' No.793, u l y 3 1 , 1919. J  

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