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Um diagrammes suivants illustrant la mtthoda. 1 2 3 1 2 3 4 5 6 MicKocory resoiution mt chaut (ANSI and ISO TEST CHART No. 2) 1^ IZ-B |50 ^* y^ i£ If 1^0 iy£ 1111.8 ^ /APPLIED IIVHGE I ^K 1653 East Uoin Street g"iJS Rochester, New York 14609 USA ^S ('16) 482 - 0300 - Phone ^S ("6) 288-5989 - Fa« Il ' fl UNIVERSnY 1 V*^-" '>JL.o •Mxi a J ^"^ WS^^^LI \LI RESERVE OF MARINE FISH AND o \ \ JBRARY INVERTEBRATES ^^ THfe^XCRETION OF CARBON DIOXIDE C\f/ BY J. B. COLLIP (From the Marine Biological Station, Departure Bat, Canada) <;f' RiPBiinno FBOH THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. XLIV, No. 2, November, 1920 ffK^^MX^imittttlBB^II^ Raprinted from Ta* Journai. or Biolouical Cbemivtiit, Vol. XLIV, No. 2, November, 1920 THE ALKALI RESERVE OF MARINE FISH AND INVERTEBRATES. THE EXCRETION OF CARBON DIOXIDE. Bt J. B. COLLI P. {From the Marine Biological Station, Departure Bay, Canada.) (Received for publication, September 3, 1020.) INTRODUCTION. The results of an investigation to determine the carbon dioxide content of the blood and body fluids of such marine forms as were available for study at or in the vicinity of the Marine Biolog- ical Station, Departure Bay, Vancouver Island, British Columbia, are herein reported. As the Van Slyke-Cullen (1) apparatus furnishes a convenient and ready means of determining the car- bon dioxide content and capacity of blood and thereby the alkali reserve of the same, it was used exclusively in this investigation. Methods. The representd,tive forms studied were, for the most part, col- lected personally. Great care was taken to insure that the indi- vidual specimens, the blood or celomic fluid of which was to be examined, should be bled while in a fresh condition. As will appear more fully in a subsequent communication this is a very essential point especially as regards various molluscan types. An endeavor was made to secure specimens representative of as many of the invertebrate phyla and orders of the Pisces as was possible. The methods employed in obtaining blood or celomic fluid from the forms studied will now be detailed. 32B 330 Alkali Reserve Cmlenterata. Two typcH of MedusjB and the sand anomonc were examined. The results recorded for these specimens can only be taken as approximate since the methods here adopted were of a special character. Medus'. Thoy wore first drained free of sea water hy siispendinR ovor a wido funnel l>y means of a double fold of checsr-cloth. Tliey wcrr llioii Konlly miicerated and p;isw»(l llintUKli the chcpsi'-clotli. The jelly-like mass was col- lected ill ii clean, dry test-tulM-. TIum was shaken vigorously for '.i minutes, air being admitted on several occasions. I cc. of the fluid was then transferred to the Van Slyke-Cullon apparatus and the analysis made in the usual manner. The evolved carbon dioxide was absorbed by the use of 10 per cent sodium hydroxide. The volume of gas was reduced to cubic centimeters of carbon dioxide at 0°C. and 760 mm. pressure held by 100 cc. of fluid. Owing to the difficulties in the way of collecting blood or celomic fluid from marine forms without loss of carbon dioxide it was thought best to examine all specimens under uniform con- ditions. The various samples were therefore shaken for 3 min- utes in the test-tulx" or 25 cc. Luer syringe with atmospheric air wliich was renewed on several occasions. The samples thus equilibrated with atmospheric air were submitted at once to analysis. In many instances concurrent samples were also ecjuilibiuted with alveolar air of the normal human subjfect after ihv manner suggested by \'an Slyke and Cullen (2). Such sp(icimens were tlicMi transferred by means of an Ostwjdd pipette to the apparatus for analysis without appreciable loss of carbon dioxide. .SV(i Aneniont.s. The s|}ecinien was dug from a sand flat at low water and carried to the laboratory in a container filled with fresh sea water. The animal was then allowed to contract in a dry open vessel. A slit was next made in the side by means of a sharp scalpel and the cavity drained free of the fluid. The soft parts were macerated in a mortar and finally .suspended in an equal volume of fresh distilled water. 2 cc. of the suspension t^^j^jk J. B. CoUip 331 were analyzed and the results interpreted as being approximately indicative of the carbon dioxide content of the tiHsues of the animal. JirachioiHMlit. The specimens were collected at low water from the rocks closely adjacent to the lalwratorv and examined immediately. It was found that from 1 to 2 cc. of fluid could be obtained from these small forms by aspirating into a Luor syringe from the celomic cavity through a needle passed through the soft tissue ) have pointed out, with the varying intensity of pholcsynthe.sis by microscopic organisms. Surface and depth tciw.s made daily during the summer months by Mouncc' showed great varia- tion in the relative amounts, and in the distribution of diatoms. The falling off of the degree of alkalinity of .sea water with incireas- ing depth is suggestive in this connection. Sorensen (3) found that deep sea water was less alkaline than that at the surface. The total carbon dioxide content of surface samples of sea water obtained in Departure Bay during the summer of 1920 was found to vary between 2 and 4 volumes per cent, the amount varying directly with the specific gravity. It is therefore of interest to find that the amount of carbon dioxide in the blood or 'Unpublished results quoted by courtesy of Miss Irene Mounce. 334 Alkali Reserve .& 3 a el pi X -b' § i • u '%■ a. BO u S § 1 3 ^1 *? 1 1" *» Is O "o •s -^ iS £ s a ' 1 * 1:1 o 3S3S3SSSS ' •§« ' b ft> <• U .4^ X 03 » is 9i 1 1 , ll » ^ > » ? -5 a ^ ^ Ih •o u 3 09 333333333 ^' - a a — •? 1 '5 •is 1? "3 .s 03 1 a: M O o o3o ^ w 9 11 .= ctaH 1 fiZ. £js u 00 GO '0» cont DOcc. o equilib wit alveola si oi OO u~ •33, c * 5: -K »c O « «0«'5'^"t«b'(5"5 50 d 6 t>i S " 3 £ 582 ;; 00 •« S ^^ .* 1 « .. >. 8' ' c _c 1 1 e I ■1. •5 e 1 >; -a a g . i ! § S 5 s e It i •§ ^ ; 1' ^ ^ ^ ^ § 1 ! 5 1 3 "i flo 3 ■s -r : i| \ . §3 3 3 3 3 S -5 i ii f ' §^ i^ 1 cScC Q S 1 3 CO «s ca cS 0) •-9 S, tn V 03 J. B. Collip 335 S? n i Q o t> b O te J 5 o u a> a '* 1 o .a O >> g h , .S ' ?i .s'g *-• a c5 CQ -s -? 4) • - ■?£ a e .5 d •s| 83 03 «3 C4 <3 tf .O s ^ 50 5 o- o o o O S 2 ?5 2 8 gj S 8 S S 2 2 S 00 CO o> o <-> 00 Oi 03 00 C4 U3 00 a> O O 00 S S 5 . » 2 2 S 3 03 u 5 5 I. « O a ■a &9 ^ *3 u *^ s iS C c3 e e ^ , , 3 - :: s ;: ^ ^ <3 J Si Is to C3 3 « a a o ^1 a > .2 ^ - S o.S >,.5 o e3 o •a S O V 2 I .2 !*< §•> a o 2 GO OS 9 ^ ii5 CQ O O C3 03 o a O go « OS O o o -w 3 S 'C GO'S « a oi.-S g..S: OS" d □oooaocbdcbte'-! -- ce o ■5 1 =...,.,« I §> § §:§ :§ 3 «*" O N '^ "S » § -^ S e « « ~» « •« »^ ^^ J •^ 63 a, !§ S •'S o 5- s O us lO J. B. CoUip 337 2" >, <^ >. -d a C 1i a o o >• 0 *• ai - "1 , = 11 2^ Q t , e! o >5 g n-; ^ C3 a - e 1 '? /<; — 1 +J C " ^ m «_ y: 5S i5 -2 05 -3 a •a Q _a '5 -a a O us 3 O -J t- O 1^ i1 CO CO ^ si CO Q) •» 3 .2 I e •* r; C3 i- .a .2 -i- o S = 2 £ ss £ .» r (£ 3 ~ I f rs s 1^ o Q SQ I 338 Alkali Reserve « >, * n 0 £ ^: O ' w £ li 03 1- - II 1^- . 11 ^ o. fa « . a a is a S. 03 a 00" Q "sl >^«S . s- a £(£ s XQ J d ^ 2=55S .g^.£35SS •^ « m • S ** «M «■*■***■ CJ - B s ■i ■g a _g _e • w* ot - 083533 333333S3 "3 3 +a ** * .O JB .a O 0 0 -3 •B -3 0 V. * ^*---*^ •*'*• _2 - ^ «• JS J .£ ^ i — s ii C m --^..•.* ^^•.* 5^^^ ■3 ^ ^ C3 j s: ft. ^ C- ^ tent of f fluid rated rair. ci c -o { 1 COtcont 100 oc. o equilib: wit alreob ii ?; 2 ^ "82, o ^ «• 9e C 5 1=1^1. u b» 00 ® to Tf< 0 QC :Z. CO CI C-l CI M 0 COicoi 110 cc. equili; wi atmoa a u CO •«)< C< (N i-c t>io©MO— --C S t^c s "m JS 00 ." 3 'Si « bC:: 3 3 ft. "«> t. S xi c .^ ft. ^ GR &■ c ■§ -^tS ' .3 ja •2b3 -«;a,a,!^ Oft. r snake hamnop tt w g CI M (4 OS "3 0« m H 0 1 J. B. Collip 339 TABLE II. Date. .Sourre of sample. Temper- ature. Deiuity at S'C. Alkalin- ity. Reactiv- ity. tuo •c. ee. O.Ol M ee. 0.01 N NaOH H,SO* June 27 Off landing stage at station. 169 1 0178 1.0 17.5 " 28 *< 17.8 1.0146 11 16.0 " 29 « 19.5 1.0140 1.3 13.8 " 30 M 18 0 1.0159 1.0 15.7 July 1 U 18.2 1 0161 0.7 14.8 " 2 « 18.0 1 0167 13 15 1 " 3 « 176 1.0186 12 16 5 " 4 parturp H;iy. 1 ()217 15 180 U 12 At .'{ fatlioiiis ('tT Departure Hay. 1 0220 14 18 5 ii 12 .\t !> fathoms niT Departure Hay. 1 ()222 10 IS 6 ti 12 .\t M) fathiinis olV Departure Hay. 1 ()2;« -0 3 21 S n 12 .\t 20 fathoms otV Departure 1 025;- -14 24 0 I5av. fcloiiiic fluid of tho various marine fitriu.s invest ij^atcd is invari- ably groat or than llio niaxitnuni valuo for total oarbon dioxido in soa water. '['\u) amount of oombinod carbon dioxide in the body ti.ssuos of Modusie and soa anomonos. the colomic fluid of brach- iopod.«, .starfish, certain soa urchins, and a number of mollusks and tho blood of the dogfi.sh and the ratfish i.s relatively low, but in all instances is higiior than that in sea water. Certain types of C'ru.stacea h.ave a relatively hijih carbon dioxide content, such forms as (\tNci r iiKtijista-, ('afic r /n'otliiclux, Kchidnoccru^forinalnx, and III iiHiirtipxnK nmlns boing included in this group. The kelp oral.) Kpinltus produrlh-^, the sand shrimp CpD'jchin pimettcnsis, and the (>;iglc' l.arnacio Uiil'unis lupiiUn are on the contrary (!om- parativoly low in the fi.vod carbon dioxich.' in the celomic Huid but they arc in tlii.s respect somewhat analogous with the toleosts examined. .\ sotiiewlial .similar phenomenon oxi.sts in the case of tho Molhisca. While the majority of the forms studied have a carbon flioxide factor falling within the range between 6.3 and 11 volumes i)er cent certain species such as Puphia slamfmu, and I'mitilht pinitu of tho pelocypod type and Polynias lewisii ramn^BSwaaufai si.'!dBaKi&;;saESB3K^scr J. B. CoUip 341 and Thais lamellosa of the gastropod class have a fixed carbon dioxide content which in nearly all instances is considerably higher than that observed in the former group. As pointed out earUer in the paper it is essential that the blood be obtained while the animal is perfectly fresh. For instance it was noted that specimens of Mya arenaria bled immediately after they had been dug gave a fixed carbon dioxide factor of 6.5 volumes per cent while other specimens carried to the laboratory in fresh sea water gave a factor of 8.3 volumes per cent for com- bined carbon dioxide. The fixed carbon dioxide of the mollusk tends to rise rapidly when the animal is not kept in a large vol- ume of fresh water while the opposite effect was noted in the case of the fish examined. Exposure to air causes the carbon dioxide content to fall while in the dead fish the latter may be near to that of .sea water. If a fish is allowed to remain hooked but left in tiic open water for some little time the carbon dioxide content of the blood falls. The fi.sh are in their reaction to injurv much hke the mammal.- as far as the alkali reserve of the blood is concerned. 'J-he relatively low figure for carbon dioxide in the blood of the elasmobranch Sfjiialus sucklii and the holocephalan Hydroktgux colliei stand.s in .sharp contrast with that for the teleostian types studied. As the hydrogen ion concentration of sea water is in most in.stances lower than that obtaining in the blood of marine forms and as the bicarbonate content of the latter is much higher than that of the former it is evident that the amount of the dissolved carbon dioxide in the blood or body fluids of marine forms must be considerably greater than that occurring in sea water. The tension of carbon dioxide in the blood of marine forms must al.so he jjioimrtionately higher than that in .sea water. This brings up an interesting point. Does a process of simple diffusion furnish an adequate explanation of the mode of elimination of carbon dioxide from the blood or body fluids of marine forms to the surrounding sea water? The maintenance of a definite acid-base balance in the blood and tissues of marine forms is no doubt quite as prime an essential as in. the case of higher forms. In the mammal this is largely effected by the concerted action of the respiratory and renal apparatus. The gaseous exchange JSBSS'S^St-iii^ 342 Alkali Reserve botwpon tho blood and the atmosphere takes place almost entirely in the alvpolar sacks where the tensions of the gases concerned arc such that a process of physical diffusions seems to furnish a sufficient explanation for the passage of the oxygen inward and of the carbon dioxide outward (G). In the case of the fish con- ditions are somewhat different. While tho respiratory and renal apparatus here as in mammals is apparently closely associated with the regulation of the reaction of the blood yet the inter- change of gases between the blood and sea water taking place as it does largely through the medium of the gill filaments is on a somewhat different basis from the exchange in the mammal at the lung surface. The tension of dissolved gases in sea water to which the gill filaments are exposed cannot differ greatly from the tensions obtaining in the enveloping medium, unle.ss it is possible that the respiratory movements an; so adjuste in the blood on the one .side, and in the sea water on the other. I'urthcr experiment only can furnish a full expla- nation of this phenomenon. In the case of arthro{)od types, such as Cnnctr mofiister, Echid- noarus format iiy nd others, the combined carbon dioxide is at a much higher concentration than it is in any of the Pisces exam- ined. ,\ relatively greater difference must therefore exist be- tween the tension of carbon dioxide in the blood of these forms and sea water than in the case of the Teleostei. If one is to ex|)lain the mode of carbon dioxide excretion in these forms by a process of physical diffusion one must assume that the perme- ability of the gill filaments to carbon dioxide is of a very low order allowing a very steep pressure gradient to be maintained between the two sides of the medium for gaseous exchange. Certain of the Mollusca have a relatively high concentration of bicarbonate in the body fluids but it may possibly be due to the anatomical features occurring here for the carbon dioxide in the sea water tn:«ii» J. B. CoUip 343 which is bathing the organisms to exist at a higher tension than in the open sea. Such a difference does not exist Ijetwceii the hicurhonate con- tent of the body fluids and tissues of Echinodermata, Brachi- poda, and Ccelenterata, and that of sea water as hjis been noted in the forms above mentioned. The existence, however, of a definite pressure gradient for carbon (Uoxide Iwtwcen the tissue and the sea water suggests that a definite mechanism exists for regulating the tension of this gas in the body fluids and tissues. .SI'MMAIIV. 1. The cjirboh dioxide (•(Mitfiil of ||m> blood :iiid llic celornic (liiitls of various m.-irinr forms has bn-ri ilcdTinincd. 2. Tlic cjirlHin dioxidf coiitciil of the blood and (•(•ioiiii(^ lliiid of marine forms cxamiiicil ((luilibralcd with atmosplicrii- air is in all iiistaiKrcs higher than the carbon di((xid(> content of sea water. '.i. The carbon dioxide content of certain of the Artlirop«Kla and Mollusca is relatively very high. 4. The carbon dioxide content of the blood of marine Teleostei is approximately 10 volunics jwr cent. .*>. The carbon dioxide content of the elasmobranch Siinalas liucklii and the liolocef)h;dan Hydrohnjm collici is relatively very low. 0. The alkalinity and the reactivity of several samples of sea water have been determined. 7. Surface .samples of .sea water in the vicinity of Departure Bay are invariably alkaline to phenolphthalein. 8. I( is held tiiat in ord(>r to maintain the constant reaction of th(> bloo considerably higher in the blood .and body fluids than it is in se.i water. n. The (ineslion of carixtn dioxide excretion is tliscu.s.sed. In conclusion I desire to express my thanks to the Curator of the Biological Station at Departure Bay, Dr. C. INFacLean Fraser, for his kind assistance in making the collection of material pos- sible, and for aid in the identification of specimens. ^My thanks are also due to the Biological Board of Canada for defraying the expenses in connection with this investigation. ii^j^ir.^£i4»-." it.i'^i^l&iH 344 Alkali Reserve BIBLIOORAPHY. 1. Van Slyke, D. D., J. Biol. Chem., 1917, xxx, »17. •-». Van Slyke. D. D., and CuUen, G. E., J. Biol. Chem., 1917, xxx, 289. 3. S