NATURAL HISTORY COLLECTIONS las texturas delapiel losespecímenes. mejora significativa de los perfiles originales y una y espécimen del cuerpo del alcohol del eliminación la flexibilidad, la de mejora una especímenes, los de color del recuperación rol. Entre los resultados positivos se incluía la glice de acuosas soluciones de secuenciales cambios de densidad de la solución en baños los y peso de aumento del seguimiento un hizo etanol.Se de acuosas soluciones en dos conserva bien o formaldehído con fijados tiburones en glicerol de impregnación la zar anali para estudio un presenta artículo Este Resumen des textures depeaudesspécimens. et originauxprofilssignificative lioration des dans le corps du spécimen, ainsi qu’une amé imbibé l’alcool de l’élimination flexibilité, re tour de la couleur des spécimens, une meilleu re un incluent positifs résultats Les aqueux. surveillées dans une série de bains de glycérol été ont solution la de densité la de variations de l’éthanol aqueux. La hausse du poids et les fixés avec du formaldéhyde et conservés dans requins des dans glycérol du l’imprégnation Cet article présente une étude préliminaire sur Résumé textures ofthespecimens.skin cant improvement of the original profiles and signifi and specimen the of body the from alcohol of removal and flexibility, proved im specimens, the of colour the of return a of aqueous glycerol. Positive results included changes were monitored insequential baths density solution and increase weight The fixed and aqueous-ethanol-preserved sharks. impregnation of glycerol into formaldehyde- the on study scoping a presents paper This Abstr nism glycerol,ous Keywords: *Author for correspondence Leiden, The Netherlands Leiden Centre University Medical Leiden MuseumofAnatomy Andries J. van Dam [email protected] Welshpool, Western Australia, Australia &ResearchCollections Centre Western Australian Museum Ian D. MacLeod* a

ct sharks, ethanol removal,ethanol impregnation, mecha aque ------preserved preserved human tissue in 65 vol% of glycerol following the principles of storage the effective of years 100 glycerol. than more on aqueous based was with decision This Megamouth impregnate to be would problem to alcohol the solution a that possible it decided was authors, the between discussions extensive and Leiden to visit a Following 2008). (MacLeod time washing of logarithm the with kinetics linear showed sharks large reported that the mechanism of removal of formaldehyde from small and have studies Previous 2006). al. et Burroughs 1990, Hutchins and (Berra remove the excess formaldehyde associated with the initial fixing process exhibition tank in the 1996, the into megamouth shark had insertion been properly its washed to to Prior found. be to had environment storage new a so spaces, open public to proximity close in even and buildings public inside ethanol of volumes large of use continued the exclude regulations liable for a personal fine of $250,000 and six months imprisonment. Present employee museum the and non-compliant container storage the rendered with of associated the revised Goods regulations Dangerous Codes, which second major factor dictating the relocation of the specimen was the impact plastic exhibition container and its 12 mm-thick laminated glass cover. A and falling debris could have irreparably damaged the fibreglass vibrations during demolition of the seven-storied reinforced concreteshark building from its present storage location in 10,000Museum in Perthlitres necessitated the removal ofof the 705.2 m, vol%700 kg megamouth ethanol, since of of the the main building demolition Western The impending Australian Introduction the Maritime Museum in Fremantle while on public exhibition. in treatment undergoing now is Megamouth which in positions, viewing adult and child at portholes with lined tank steel stainless massive a of the of CEO The Western the building commissioned Museum has subsequently Australian experiment. the of outcome successful the on reports specimen, experiments with two small precious sharks were conducted as a the trial and this paper destroy to about not were conservators the that staff curatorial assure to order In tank. exhibition and treatment designed the kinetics that will be observed in treating the massive shark in a custom- trial the impregnation of small sharks with aqueous glycerol to determine in formaldehyde-fixed tissue samples. With bio-deterioration this knowledge, it visible was decided no to with 1959) Edwards and Edwards 1896, Kaiserling method, enabling the conservation in natural colours (Kaiserling solutions to a shark for tr A migr queous glycerol s fromethanol a ansfer of tion mechanism ‑ reinforced 1 NATURAL HISTORY COLLECTIONS concentration of65vol% glycerol 296days after Sharks oftreatment inafinal Figure 1b glycerol solutions before ofthesharks Image treatment in Figure 1a Australia, of Western region Kimberley the from shark reef a and larger 27232.002, WestNorth near Cape, from shark Ahammerhead small Experiment a linear fashion with regards to their colligative properties. The plot of plot The properties. colligative their to regards with fashion linear a in combined liquids two the that and solution glycerol aqueous the into was that assumed the fall in was density due to of the ethanol mobilisation In order to the assess of of preservative. amount it fluids, exchange bodily ethanol the displacing solutions glycerol incoming the with consistent is was an increasingly strong smell of alcohol coming from the tub and this had plateaued after approximately two months. As the density fell, there weight the until rate same the at density in fall to continued solution the to remove accumulated solution. This caused a drop in the density, after until which “massaged” was area abdomen fashion cut the and regular manipulated were a specimens in fell density the treatment, of weeks two is apparent from the increased weight of the sharks that there is a largera is there that sharks the of weightincreased the fromapparent is it correct, is mixtures) glycerol-water the with interact chemically not do mixtures ethanol-water the that the (i.e., additiveand are solution liquor outgoing impregnation the of densities the that assumption the If their muscles. from and cavities body the within from released being solution storage solution of 1.0937 g.cm glycerol solution volume of 27 litres with an initial density of the glycerol g.cm 0.9162 of density initial an using calculated was treatment the during released solution storage of ethanol volume former their estimated The 2). (Figure time immersion impregnation of logarithm the the with of fit best phases the had initial the in density of change of rate the which typically had a reproducibility of ±0.0002 g.cm soaking the of solution was monitored density using an The Anton Paar portable digitalsolution. densitometer impregnating the which retain to skin, the tended of surface the on solution glycerol excess from sharks the dry” “towel to necessary was it data, reproducible obtain to order In solution and were monitored for weight at approximately weekly intervals. a 33% glycerol tub containing treatment polyethylene 20 rectangular litre a in placed and measured weighed, photographed, were specimens The that they had been in storage for more than 12 years (Figure 1). of photographs the sharks at the time of their recovery, but records showed no were there Unfortunately storage. during to shrinkage owing wrinkled storage drums. The specimens were very stiff and the surfaces were highly the sharks were fixed with bends in them that replicated the radius of the drum, black polyethylene wide-necked the from recovered When drum. rinsed under running water for being several hours and then before placed in the days storage three for formaldehyde wt% 4 the in soak to left and collection specimens, they had been gutted and injected with formaldehyde ethanol storage environment. As part of their normal preparation as museum 2349

g respectively when they were removed from their nominal 70% 70% nominal their from removed were they when respectively g Carcharhinusamblyrhynchos al -3 , and this value came to 1700 ml of the ethanol 28404.001, weighed 455 weighed 28404.001, from ethanolto a for tr A migr glycerol solutions ansfer ofshrk a tion mechanism -3 . During the first first the During . Sphyma lewini lewini Sphyma queous

g and g s -3 , a , 2

NATURAL HISTORY COLLECTIONS the subsequent increasing glycerol concentration baths were also plotted (R bestsuited theexperimental results. Thehighest correlation coefficients a as plotted were sharks the function of linear time, of square root of time and log time to weights see what format the months, few initial the In in physical dimension). change the behind reason underlying be the would sharks by the glycerol (i.e., the fish were becoming plumper and the apparent selective uptake of the amount of shrinkage in the skin of the fish appeared to be diminishing saturated the surface of the alcohol-preserved sharks. It was also noted that effectivelycolour-had solutions glycerol the as hues yellow and brown in rich being to surface grey dull out washed a being from improved sharks had the of colour the that noticed was it period, impregnation initial this During sharks. the of body the in place taking is adsorption polyalcohol the physical of or bonding chemical of form some that likely is it which indicates that the glycerol has a real affinity for the tissue and that 0.16, ± 1.36 is gain weight calculated the over increase weight real the volume of the glycerol solution replacing the alcohol. The mean ratio of same of the incorporation simple on the anticipated that than gain weight versusPlot ofsolutiondensity square root ofimmersiontimefor thefirsttwo months Figure 2 to its shape and form. flesh of amount greater much a has shark reef the whereas area, surface a large with mass body a thin by characterized is shark hammerhead The cm 540 of of the two sharks, for the hammerhead has a surface area to volume ratio ratio volume to area surface the in differences fundamental the to due is this for reason The weight. steady a reach to time the times 3.5 or 2.91) days ( graph the nine in after plateau weight data its reaches shark the hammerhead the of that shows Inspection 3. Figure in seen is glycerol vol% 33 the in bath treatment weight first the of in time rate log of The function a time. as increase elapsed total the of log the with relationship linear a supported best data the all but functions, time of range a against density, g.cm-3 2 1,081 1,082 1,083 1,084 1,085 1,086 1,087 1,088 1,089 1,091 ) were found for plots of weight against the log of time. Data from Data time. of log the against weight of plots for found were ) 1,09 log t 0 hours -1 , while the reef shark has a value of 330 cm 330 of value a has shark reef the while , 2 ρ , 2.34), while it took the reef shark nearly 33 days ( 33% Densitystage1 0,5 = 1.0910 = 1.0910 - 0.0031 1 log t, t, log Densitystage 2 squished specimens squished R 1,5 log time, hours time, log 2 = 0.8995 1 ρ 33% 2 = 1.0935 = 1.0935 from ethanolto a for tr A migr - 0.0032 0.0032 glycerol solutions 2,5 ansfer ofshrk a tion mechanism log t, t, log -1 R (Figure 1a). 1a). (Figure 2 3 = 0.9964 queous log t s 3,5 hours 3 ,

NATURAL HISTORY COLLECTIONS the the saturated colour that the original specimens had before being immersed to reflect changed of the the sharks colour had begun, impregnation Once of concentration impregnating solution to minimise stress on the tissues of the specimen. in increases stepwise for procedure standard the with for 3–4 weeks, the concentration of the bath was increased in accordance ahead of the larger shark. When each animal had attained a steady weight to surface it some the higher ratio, weight volume weeks plateau achieved going to be greater than the value. calculated Since the smaller shark had a its area will surface and underestimate so the real ratio of method the two sharks is this shark, hammerhead the than thicker is shark reef the Since relative surface areas assumes that both specimens have the same thickness. factor in the uptake of the glycerol from solution. This method of assessing was 3.2, which indicates that the surface area of the sharks is a controlling standard outline tracing and the ratio of the reef shark to the hammerhead hammerhead usingthe made were sharks than the of area surface faster the of Estimates specimen. 0.54 ± 4.04 at weight its increases shark reef the compared, are plots time log vs. weight the of slopes the When Plot oftheweight ofthereef in33vol% sharks andhammerhead glycerol Figure 3 low vapour pressure which prevents loss of glycerol from the specimen specimen the from glycerol of loss prevents which pressure vapour low attracts humecticant, water and a promotes cell is hydration. Furthermore, it has an Glycerol extremely collections. of years glycerol-preserved 100 during maintaining up built experience the and glycerol of properties the in found be can phenomenon this for explanation Apossible killed. freshly been had as specimen if the looked and turgid fully became shark hammerhead the in eyes collapsed the of One up”. “plumped specimens ethanol decreased with the increasing amount of glycerol in solution as the in the dehydration with associated wrinkles of skin amount The glycerol. of concentration the with increased significantly body and tails the of flexion of degree the and mobile became gills the baths glycerol the in months several after but flexibility, minimal had and hard” “rock were sharks the Initially, years. several for solutions storage ethanol the in

weight, gram 3000 1000 1500 2000 2500 500 0 0,5 Hammerhead Reef shark Reef 2308.4 = 133.03 + Hammerhead + 464.15 = 32.43 1,0 Hammerhead plateau Hammerhead Reef plateau 1,5 log t , R 2 log time, hours time, log = 0.9908 = log t, log t, 2,0 R 2 = = 0.9877 from ethanolto a for tr A migr 2,5 glycerol solutions ansfer ofshrk a tion mechanism 3,0 queous s 3,5 4

NATURAL HISTORY COLLECTIONS magnification showing pigmentation cells at same skin shark Hammerhead Figure 4b showing denticles andpigmentation cells with0.5mmscalebar skin shark Reef Figure 4a of glycerol intact. Topping up with water is sufficient to restore the the restore to sufficient is water with up Topping intact. properties glycerol (re)-hydrative of and antiseptic the leaving lost, is water only evaporation, it will lead to an increase of the alcohol concentration because by loss of fluid in case In fact, diffusion. and/or to due evaporation tissue or plateaued. In order to get reproducible results, each shark had to be to had shark each results, reproducible get to order In plateaued. or stabilised had weight the until baths for solution the in kept and monitored weight their were sharks the and vol% 65 to 54 45, 33, stepwise from a fashion in increased was baths the in glycerol of concentration The of glycerol Response oftheshark cells located under the skin layer. pigment be to appear similar areas are coloured blue dark The features 1997). al. et (Naresh morphological gross the but different, clearly are sharks two the of surfaces the – 4b) (Figure shark hammerhead the and as seen in Figure 4, which shows the surface of the reef shark (Figure 4a) denticles remain the same size and become a characteristic of the species, grow, cavity. the pulp sharks As central and a dentine covers which layer enamel outer an has denticle Each scales. placoid or denticles called with glycerol. Shark skin is composed of a matrix of tooth-like structures immersion time, the be of it for log either removal the of on formaldehyde or impregnation dependence linear the of cause underlying the be may It is likely that the tortuosity of the diffusion path through the shark skin regenerative properties. so-called has glycerol hydration, cell to regard With re-immersed. being of days few a within form and shape original their restored solution the as specimens, exposed of partially cavities collapsed overcame Anatomy of Museum Leiden the at glycerol with jars up Topping tissue. the specimen of dehydration instant to lead can which loss, concentration the for significantly higher concentration of ethanol has to be added to leads compensate Astrength. antiseptic less solutions and concentration alcohol in decrease ethanol-water a to in evaporation fluid higher water, of significantly is that than ethanol of pressure vapour the Because tissue. specimen the to damage shrinkage of risk any without concentration shark was R R follows: as were shark hammerhead the and shark reef the for equations The solution. the in glycerol of were concentration the to increase proportional of directly rates the that found was it bath, the concentration in the glycerol of the of function a as plotted was shark each for increase baths had typical errors of ± 13% in their slopes. When the rate solutionof weight subsequent but technique, measurement poor to due was this and 18%) (± fit best of line the with associated error greatest the have to found was solution vol% 45 The balance. a digital on weighed and towel drained of excess solution for two minutes before being dried with a paper 0.9953 respectively. In simple terms, this means that the rate of increase log per reef = 34.0 [wt% ]-1240, while the rate of weight gain for the hammerhead t and the R the and hammerhead = 8.3 the where -307, are [wt%] rates in of units gram 2 values for the linear regressions were 0.9895 and and 0.9895 were regressions linear the for values s to increa from ethanolto a sed concentr for tr A migr glycerol solutions ansfer ofshrk a tion mechanism queous a tion s 5

NATURAL HISTORY COLLECTIONS ratio of the surface areas of 3.21. The initial relative rates of increasing increasing of rates relative initial The 3.21. of areas surface the of ratio g.cm 1.26 at glycerol of density the by divided is ratio this If shark. hammerhead the for than shark reef the for faster times 4.04 is sharks’weightthe of of the sharks. Initially, the amount of movement that the curator and curator the that movement of amount the Initially, sharks. the of flexibility of measure the in found was tissue the mobilised had glycerol of how the indicator measurable Another diminished. shrinkage fold skin of amount the and improved specimens the of colour the glycerol, with sharks the of impregnation the during that noted been previously had It Rate ofweight increase for ofglycerol thereef asafunction shark andhammerhead concentration Figure 5 of glycerol into the specimens is related to their surface area. adsorption the for mechanism transport the that supports strongly which into taken are glycerol), wt% (against 4.0±0.5 is slopes the of slopes ratio mean the account, the in variations When solution. storage ethanol aqueous previous the of release rapid the of impact the to due artificially higher been have may solution 33vol% the in sharks the of weight to the length of the shark from the tip of the head to the rise of the tail tail the of rise the to head the of tip the from shark the of length the to fraction is the ratio of the perpendicular distance of the tail under flexion flex The concentration. glycerol of the as a function sharks of the flexion improved flexibility of the specimens is seen in Figure 6, which plots the the of A solution. measure alcohol the and formaldehyde of combination the with solid” “frozen been previously had they whereas sharks, the of back moved and forward. readily By the be end of the could treatment, it was sharks possible to both open the on jaws gills the and flexibility in volumehammerheadareatoratio,the sharkshowedrapidimprovement surface greater its to Owing container. storage previous the of shape the by dictated curve radial fixed a in not and spine the with line in being towards backwards tail the bending other the and head the down holding conservator were able to extract from the sharks was very limited, with one

rate of gram per log time 1000 1200 1400 1600 1800 200 400 600 800 0 reef reef shark 35 -3 , the ratio falls to 3.33, which compares favourably with the the with favourably compares which 3.33, to falls ratio the , 40 hammerhead hammerhead shark 45 50 55 R reef = 34.005 wt% 34.005 = wt % glycerol % wt 60 R hammerhead - 1240, R 1240, from ethanolto a 65 for tr = 8.328 wt% 8.328 = 2 A migr = 0.9895 = glycerol solutions 70 ansfer ofshrk - a 307.4 , R , 307.4 tion mechanism 2 75 = 0.9953 = queous 80 s 85 6

NATURAL HISTORY COLLECTIONS vol%, while the reef shark continued to improve beyond the initial maximum 54 concentration glycerol a at 1.0 of value maximum the reached shark hammerhead the for fraction flex The concentration. glycerol increasing with flexibility in improvement continuing showed shark reef The fin. the interest of natural science curators, who have requested that the work be The initial results from the experiments with the small sharks has awakened C glycerol. of alcohol concentrationlowest the in treatment the of months two ethyl first the during the of that as well as formaldehyde, of smell discernable outbreaks of mould and fungi in the 33 vol% bath, as there was a clearly any for preventing was responsible the glycerol with impregnation initial the during sharks the from coming formaldehyde residual that likely is It Plot and reef ofthechanginghammerhead flexibility withglycerol shark concentration Figure 6 unity. than greater fraction flex a hence and mobility value as the bend in the shark relaxed, which allowed for greater apparent an integrated pulley support system, in order to assess the success of of success the assess to order in system, support pulley integrated through an attached transponders of series a through shark massive the of weighing periodic facilitate to designed been has tank The glycerol. of the same rate as observed with the small sharks in a similar concentration decrease in the density of the 30% glycerol solution is -0.030 g.cm of rate the that show specimen megamouth the of impregnation of weeks six the first from Data percent. of 65 volume up concentration to the final solutions glycerol various in impregnation its during III Megamouth of exhibitionthe permit will that areasviewing porthole glass of number a with of a container and steel storage construction treatment large stainless the for funds provide to management museum senior and curator fish the number of fur-covered marsupials. The trial with the sharks has convinced and a lizards as such snakes, specimens terrestrial-based to trial extended onclusion flex fraction for shark movement 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 20 y= 0,001x 25 flex over natural Reef flex over natrual HH R² = 0,9981 2 - 30 0,075x 0,075x + 1,8268 35 40 vol %glycerol 45 from ethanolto a 50 for tr A migr glycerol solutions 55 ansfer ofshrk a tion mechanism 60 queous -3 65 , which is s 70 7

NATURAL HISTORY COLLECTIONS Acknowledgements absorbed into Megamouth.being is glycerol the which at rate the monitor to and impregnation the Berra, T.M., References on the flexibility of the sharks is greatly appreciated. public exhibition. The assistance of Salvador Gomez with measurements on while treatment for Fremantle to shark Megamouth the relocating in and assistance her support for and purposes experimental continued for sharks her the to for access Morrison Sue thank to like would We of shark skin. Naresh, M.D for the preservation of MacLeod, I.D in German]. preservation of collection specimens to maintain the natural colours. Berlin Clinical Journal, Edw 20(1–2): 49–54. of museum staff to formaldehyde during some wet specimen activities. Burroughs, 14: 651–656 pelagios der natürlichen Farben. natürlichen der K Oxford University Press. aiserling, C. ard (Megachasmidae) from Western Australia. s, J.J., . J. Biosci and ., V. Arumugam, . 2008. Washing formaldehyde from fixed spirit specimens: a mechanism , C. Ha MakosC. K. , G.E., 1896. Über die Conservierung von Sammlungspräparaten mit Erhaltung and J.B. Hutchins. Megamouth III. 22(4): 431–437. M.J. Edw Berliner Klinische WochenschriftKlinische Berliner ard and

AICCM Bulletin 1990. 1990. A specimen of Megamouth Shark, s. R. S 1959. wks, Records of the Western Australian Museum anjeevi. Medical Museum Technology from ethanolto a and 31: 36–43. for tr A migr T.J. R . 33(35): 775–777 [about the the [about 775–777 33(35): . 1997. behaviour Mechanical glycerol solutions ansfer ofshrk a tion mechanism yan. Collection Forum 2006. Exposure Exposure 2006. queous Megachasma Megachasma . . London: s 8