Ethanol Preservation Research Articles

Mother-egg stable isotope conversions and effects of lipid extraction and ethanol preservation on loggerhead eggs.

Carbon and nitrogen stable isotope (δ(13)C and δ(15)N) analysis has been used to elucidate foraging and migration behaviours of endangered sea turtle populations. Isotopic analysis of tissue samples from nesting females can provide information about their foraging locations before reproduction. To determine whether loggerhead (Caretta caretta) eggs provide a good proxy for maternal isotope values, we addressed the following three objectives: (i) we evaluated isotopic effects of ethanol preservation and lipid extraction on yolk; (ii) we examined the isotopic offset between maternal epidermis and corresponding egg yolk and albumen tissue δ(13)C and δ(15)N values; and (iii) we assessed the accuracy of foraging ground assignment using egg yolk and albumen stable isotope values as a proxy for maternal epidermis. Epidermis (n = 61), albumen (n = 61) and yolk samples (n = 24) were collected in 2011 from nesting females at Wassaw Island, GA, USA. Subsamples from frozen and ethanol-preserved yolk samples were lipid extracted. Both lipid extraction and ethanol preservation significantly affected yolk δ(13)C, while δ(15)N values were not altered at a biologically relevant level. The mathematical corrections provided here allow for normalization of yolk δ(13)C values with these treatments. Significant tissue conversion equations were found between δ(13)C and δ(15)N values of maternal epidermis and corresponding yolk and albumen. Finally, the consistency in assignment to a foraging area was high (up to 84%), indicating that these conversion equations can be used in future studies where stable isotopes are measured to determine female foraging behaviour and trophic relationships by assessing egg components. Loggerhead eggs can thus provide reliable isotopic information when samples from nesting females cannot be obtained.

Volume 85A, Number 10, October 2014 Cover Image

On the cover: Arthropods are the most biologically successful animals due to their unsurpassed diversity and adaptability to different Earth environments. For the same reasons, it's hard to obtain and transport fresh or flash frozen samples of arthropod tissues for accurate genome size estimation, leaving a large chunk of this super‐phylum out of the Animal Genome Size Database. A quickly dried leaf is a good source of the intact plant genome for the size estimation, but this approach is not suitable for animal samples. Feulgen densitometry and a good old ethanol preservation appear to be a perfect combination to overcome this issue as shown in two test studies, allowing the addition of 29 species of crustaceans to the database. Read the accompanying article by Jeffery and Gregory in this issue.Cover design by Bärbel Beran [www.beran‐design.de].

The Effects of Ethanol Preservation on Fish Fin Stable Isotopes: Does Variation in C:N Ratio and Body Size Matter?

AbstractAlthough chemical preservation of stable isotope samples has been studied in a variety of species and tissue types, the effects of ethanol preservation on fish fin tissue have not been examined. Using caudal fin samples from juvenile Chinook Salmon Oncorhynchus tshawytscha and Rainbow Trout O. mykiss or steelhead (the anadromous form of Rainbow Trout), we investigated how storage time (2, 4, and 6 months), fin composition (C:N ratio), and fish body size (50–130 mm FL) influence preservation‐induced changes in δ13C and δ15N. In both species, we found that treatment fins (frozen and later preserved in ethanol) exhibited higher δ13C than did paired reference fins (frozen). The changes in δ15N, however, were smaller in magnitude and less consistent. Preservation‐induced increases in fin δ13C, but not δ15N, were significantly correlated with the change in C:N ratio (treatment–reference) in both species. In addition, these increases in δ13C were more highly correlated with body size in O. mykiss than in Chinook Salmon. Storage time had a significant effect on the shift in treatment fin δ13C and a small, but insignificant, effect on δ15N in O. mykiss. However, storage time was not a significant factor for explaining the isotopic shifts observed in Chinook Salmon fin tissue. This is the first study to document variation in preservation‐induced changes in δ13C within a species and to link this variation to C:N ratio. Future studies using species‐specific and tissue‐specific models to correct for preservation‐induced shifts in stable isotope ratios should be aware that these models do not account for intraspecific variation in tissue composition.

Distribution of Arterial Supply to the Large Intestine in the Anteater (Tamandua tetradactyla)

The blood supply in the large intestine of seven specimens of the lesser anteater, Tamandua tetradactyla, studied. The method included preparation of the macroscopic collection report, perfusion of the arterial network with water, injection of colored latex, fixation in formaldehyde, and preservation in ethanol. For our description and analyses, we performed dissections under mesoscopic light and made photo documentation of our observations. The large intestine of T. tetradactyla is irrigated by the caudal mesenteric artery (rectum, left colic fold, descending colon and transverse colon) and cranial mesenteric artery (right colic fold, cecal pouch). We observed that the large intestine in these animals is implied in the abdominal wall without becoming affixed to the wall, or developing adhesions on individual segments. The caudal mesenteric artery feeds the straight collateral branches (primary, secondary, and tertiary) and a few juxtacolic arched branches (first and second order). The straight branches emerge from the arched branches, bifurcate, and embrace the intestinal loop to irrigate it. The presence of anastomoses between the CaMA and the CrMA apparently ensures a relatively stable flow in the event of failure of either. This is very important, as the peritoneum in this species is completely dependent on blood from these two arteries. The model of vascularization and fixation of the large intestine into the abdominal wall of T. tetradactyla is different from that in other vertebrates.

Preservation effects on the isotopic and elemental composition of skeletal structures in the deep-sea bamboo coral Lepidisis spp. (Isididae)

Trace elements and stable isotopes (δ13C and δ15N) in deep-sea coral have been used as proxies to reconstruct past climate, and to investigate food web structure. However, there is a paucity of information regarding the effect of preservation on the chemical integrity of archived coral. In this study a live-caught colony of Bamboo coral (southern Australia), genus (Lepidisis), was sectioned into three pieces and stored for approximately one year to investigate the influence of preservation in ethanol and preservation in seawater, that mimics the early stages of fossilization, against a dry preserved control. Storage and preservation have no significant effect on the isotopic signature of Δ14C, bulk δ15N, δ15N and δ13C of individual amino acids, or C:N in the fibrillar protein matrix, with the only offset (~0.2‰) being observed in part of the δ13C record. In the high-magnesium calcite lattice Ba/Ca appears to be significantly different after storage in ethanol, whereas Δ14C, B/Ca, Mg/Ca, Sr/Ca, and U/Ca remain largely unaltered. Possible mechanisms responsible for these observed differences center around the decomposition, or contamination, by organics and we recommend further investigation, and caution when comparing samples with differing preservation histories.

Short‐Term Effects of Formalin and Ethanol Fixation and Preservation Techniques on Weight and Size of Fish Eggs

AbstractPreservative‐induced changes in the weight and size of fish eggs distort information in studies of fecundity and egg production, but the nature of changes that occur during short‐term preservation is not well understood. To determine the nature of these effects and to provide a basis for correcting for such changes after sample processing, we examined the short‐term effects of 10% formalin and 70% ethanol on eggs from gravid female white suckers Catostomus commersonii and fantail darters Etheostoma flabellare collected in two tributaries of the New River, Virginia. A sequential treatment with formalin and then ethanol was also tested to simulate effects expected in a typical fixation and preservation schedule when eggs are fixed and preserved within the fish. Formalin resulted in a significant gain (36%) in egg dry weight during the first 12 h of treatment. Ethanol had no detectable effect on egg dry weight after 9 d of fixation and preservation. Eggs that were fixed in formalin and then preserved in ethanol showed dry weights that were equivalent to those of eggs that were fixed and preserved only in ethanol, implying a reversal of the effect of formalin. We hypothesize that the action of ethanol on egg proteins unbinds the formaldehyde, with the effect of reversing formalin‐induced weight gain in eggs that have been fixed with formalin. Reductions in egg diameter were similar (∼17% after drying) between ethanol and formalin treatments, but there was an additional 5.4% shrinkage when eggs were fixed in formalin and then preserved in ethanol. In comparison with changes in egg diameter, changes in egg weight had a wider range among treatments and less variability within treatments. The experimental results indicate that egg weight is a more reliable indicator of preservation‐induced changes and a more consistent characteristic than egg diameter for comparing egg production among fishes.

Is formalin fixation and ethanol preservation able to influence in geometric morphometric analysis? Fishes as a case study

Geometric morphometric analysis has increased in the recent years, turning into a powerful tool to explore shape and size variation. Several biological studies use specimens that have been through some kind of preservation, mainly formalin preservation, commonly used in biological collections. This study analyzed the effect of preservation in shape on two fish species: Eucinostomus argenteus and Pomadasys corvinaerformis. Twenty-nine individuals of E. argenteus and twenty-five of P. corvinaeformis were collected, photographed twice, preserved in 10 % formalin for 1 week, and then transferred to 70 % ethanol for 83 days. We evaluated three levels of error: (1) error of landmark digitalization, (2) error of taking the picture and storage in JPEG format, and (3) the formalin and ethanol fixation error using Procrustes ANOVA, Discriminant Analysis, and Principal Component Analysis. Significant difference between treatments was observed on both species with Procrustes ANOVA and Discriminant Analysis. In addition, Principal Component Analysis showed a separation between groups of treatment on both species. These results represent the first evidence of preservation effects in studies of geometric morphometrics and show that according to the statistical test utilized, the fixation could affect the shape variations in different ways and could lead the researcher to false results or wrong conclusions. Other methods to explore the shape variation of organisms previously fixed should be tested in order to assess their influence in geometric mophrometric studies.

Geographical characteristics of Chrsyomya bezziana based on external morphology study

Correct identification of Chrysomya bezziana is a fundamental step to evaluate the successfulness of the eradication program based on Sterile Insect Techinque (SIT). However, geographical variation of the fly is being controversial among scientists. The aim of the study was to investigate the effect of preservation method on visualisation of characters of external morphology and to analyse geographical variation of C. bezziana populations throughout their distribution regions. A total of 88 flies collected from 7 populations in Indonesia, 2 populations in Africa and each 1 population from Oman, India, Malaysia and Papua New Guinea (PNG) were tested in the study. All larvae were removed from natural myiasis cases. The larvae were reared at laboratory until they became adult flies. The samples were preserved into two methods, wet (80% ethanol) and dried (pin) methods. Ten external characters of head and body were observed. Data were subjected to principal components and hierarchical cluster analyses in UNISTATO software. The Euclid distance measure was used for the cluster analysis, and the linking method used was the unweighted pair-group method using arithmetic average (UPGMA), to compute the distance between clusters. Results indicated that dried preservation (pinned samples) provided better external character than ethanol preservation. Based on external morphology of adult stages revealed that C. bezziana occurs as two geographical races, African and Asian races. There was no geographical variation of C. bezziana throughout Indonesian archipelago, except for the population from PNG forming its own cluster. Key Words: Myiasis, Chrysomya bezziana , Morphology, Geographical Variaton

Preservation effects on C/N ratios and stable isotope signatures of freshwater fishes and benthic macroinvertebrates

This study examined the effects of fluid preservatives on carbon and nitrogen stable isotopes (δ13C and δ15N) and C/N ratios of freshwater animals. Brotia hainanensis snails, Caridina cantonensis and Macrobrachium hainanense shrimps, and Pseudogastromyzon myersi, Liniparhomaloptera disparis, and Ctenogobius duospilus fishes were collected from seven Hong Kong streams, so as to incorporate natural variations in isotopic signals among conspecifics. Samples were preserved with 10% formalin, 70% ethanol, or formalin‐ethanol solution (fixation in formalin then storage in ethanol). We compared sample molar C/N, δ13C, and δ15N with frozen conspecifics after 30, 60, 90, 180, and 360 d. Increases in C/N were evident in formalin‐fixed shrimps and fish only, whereas ΔC/N attributable to ethanol and formalin‐ethanol preservation was insignificant in all species. Chemical preservation generally caused δ13C depletion in fishes and Δδ13C significantly declined over time in formalin‐ethanol‐preserved L. disparis. Formalin‐induced δ13C shifts were observed in shrimps (C. cantonensis: −1.54‰; M. hainanense: −0.80‰) and snails (−0.25‰) and were relatively consistent when preservation was ≤ 60 d. The influence of formalin‐ethanol on C/N and δ13C was smaller than that of formalin for all species and more consistent than ethanol preservation. δ15N of all species was unaffected (within ±1‰) by chemical preservation. Effects on isotopic signals were more predictable among fishes than shrimps or snails. Corrections of +1.11‰ and +1.24‰ should be applied to δ13C of fishes preserved with formalin and formalin‐ethanol (respectively) during trophic analysis. We recommend using formalin‐ethanol for macroinvertebrates to limit isotopic shifts, especially those preserved for > 60 d.

Change in body size of juvenile marbled sole Pseudopleuronectes yokohamae after preservation in ethanol

We evaluated the extent of shrinkage in body size of juvenile marbled sole Pseudopleuronectes yokohamae after preservation in 70% ethanol for 6, 12, 24, or 48 h, or 4 weeks. Standard length (SL, range of the analyzed specimens: 22.82–53.01 mm) decreased by 5.6% after 12 h and body weight (BW 0.174–2.964 g) decreased 27.8% after 24 h preservation in 70% ethanol. There was no further decrease in SL or BW after 12 or 24 h of preservation, respectively. The original body size could be estimated based on the size after preservation in 70% ethanol for 4 weeks using the following equations: SLoriginal = 1.05 SLpreserved + 0.37 and BWoriginal = 1.36 BWpreserved + 0.04. The condition factor calculated using the body size of preserved individuals was 16.0 and 15.8% lower than that calculated from the original body size and the body size back-calculated using the linear regression equations described above, respectively. Thus, our data suggest that the shrinkage of body size due to preservation in ethanol can cause errors in estimation of the condition factor. Our results can be used to improve the accuracy of estimating size-related biological parameters based on juvenile marbled sole that are preserved in ethanol.

Effects of lipid removal and preservatives on carbon and nitrogen stable isotope ratios of squid tissues: Implications for ecological studies

Stable isotope analysis of carbon (C) and nitrogen (N) in animal tissues is an important approach to investigate the tropic status and habitat of marine species. Some biases due to lipid extractions and preservation can hinder the interpretation of results, yet their effects have not been investigated in squid. In this study, we evaluated the effects of lipid extraction and preservatives (dimethylsulfoxide (DMSO), 70% ethanol, and 10% buffered formaldehyde) on the δ13C, δ15N and C:N ratios in squid muscle. Beaks were placed under the same treatments with the exception of DMSO. Muscle and beak samples remained under treatment for 375days and 416days, respectively. Our results indicate that lipid extractions increased the mean values of unpreserved samples by 0.8‰ for δ13C and by 0.68‰ for δ15N. Preservatives also affected the isotopic composition in muscle at different magnitudes. DMSO remarkably reduced and increased the variability for δ13C and δ15N values among samples, formalin mainly reduced δ13C values by 1.5‰, whereas ethanol increased both δ13C and δ15N by ≤0.8‰. Lipid extractions eliminated the effect of DMSO and ethanol for δ13C and δ15N, and formalin only for δ15N. In beak, negligible shifts in δ13C, δ15N and C:N ratios were recorded after preservation in ethanol and formalin. Although lipid extractions can be recommended to reduce the effect of preservation, further research is needed to develop correction models for isotopic shifts associated with both lipid extractions in unpreserved and preserved muscle tissues. Lipid extractions per se could introduce a bias that may have important implications for ecological studies.

Vascularization of the small intestine in lesser anteaters, Tamandua tetradactyla (Xenarthra: Myrmecophagidae)

The blood supply in the small intestine of seven Tamandua tetradactyla (Linnaeus, 1758), was studied. The method included preparation of the macroscopic collection report, perfusion of the arterial network with water (40ºC), injection of colored latex (Neoprene 650®, 2350-0003 Suvinil® dye), fixation in formaldehyde (10%), and preservation in ethanol (50%). For description and analyzes, dissection under mesoscopic light and photo documentation were performed. The small intestine of T. tetradactyla is irrigated by the cranial mesenteric artery, the ventral visceral branch of the abdominal aorta. The artery emerges from the retroperitoneum and disperses between the layers of the common mesentery, parallel to the caudal mesenteric artery. The primary cranial collateral branches irrigate the pancreas, duodenum, jejunum (13 arteries), ileum (14 vessels), and the cecocolic region. The arteries anastomose with adjacent vessels to form arches. Terminal branches are derived from these peri-intestinal arcs that reach into the intestine through the mesenteric boundary and form capillaries within the lining. The vascular pattern of the lesser anteater differs from those of other previously described vertebrates, but is similar to the pattern found in fetuses of domestic mammals during early intestinal development.

FTA Card Utility for PCR Detection of <i>Mycobacterium leprae</i>

The suitability of the FTA® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA.

Long‐Term Groundwater Monitoring Results at Large, Sudden Denatured Ethanol Releases

Hundreds of groundwater samples were collected at E95 (95% ethanol, 5% gasoline) train derailment spills in Balaton and Cambria, Minnesota and South Hutchinson, Kansas. Most samples were analyzed for benzene, toluene, ethylbenzene, and xylenes (BTEX), ethanol, methane, acetate, terminal electron acceptors, and field parameters. At each site, maximum groundwater ethanol concentrations at percent levels were restricted to the release area and downgradient ethanol transport was not detected. A shallow, anaerobic groundwater zone characterized by the absence of dissolved oxygen, low nitrate (less than 1 mg N/L), high Fe+2, and high dissolved methane (more than 10,000 μg/L) and BTEX formed and spread downgradient from each release area. BTEX appeared to be persistent. Methane appeared to be generated within the capillary fringe and very shallow groundwater and migrate laterally. Methane's high oxygen demand promotes anaerobic conditions within the shallow groundwater. Estimated and measured methane soil gas concentrations exceeded the lower explosive limit. Long‐term monitoring at South Hutchinson and Cambria using 1 to 5‐foot (0.3 to 1.5 m) well screens straddling the capillary fringe and the shallow water table effectively demonstrated the presence of high ethanol (∼1%) and benzene (more than 250 μg/L) concentrations 5 years after the release. The wells appear impacted by long‐term ethanol inputs from the vadose zone where ethanol has persisted for years after the initial release. Toxicity, volatile fatty acids, excess hydrogen production, and/or exudate coatings could be responsible for ethanol's preservation. High acetate and hydrogen concentrations at South Hutchinson indicated that fermentation was actively occurring in the very shallow groundwater and/or in the lower capillary fringe. Short‐screened (1 to 5 feet; 0.3 to 1.5 m) nested wells were pivotal to improving our understanding of ethanol's behavior.

Diet–tissue discrimination factors of carbon and nitrogen stable isotopes in blood of Snowy Owl (Bubo scandiacus)

Analysis of carbon (13C/12C) and nitrogen (15N/14N) stable isotope ratios (hereafter δ13C and δ15N, respectively) in animal tissues is a powerful tool in food-web studies. However, isotopic ratios of prey are not transmitted directly to a consumer, as a diet–tissue discrimination factor (denoted Δ) occurs between sources and consumer’s tissues. An accurate assessment of the diet of a consumer with stable isotopes thus requires that the Δ13C and Δ15N of the studied species are known. Our aim was to establish Δ13C and Δ15N values in the Snowy Owl ( Bubo scandiacus (L., 1758)). Moreover, we assessed the potential effect of ethanol preservation of blood samples on δ13C and δ15N values. We kept four captive adult Snowy Owls on a pure diet of mice for ≥6 weeks. We then collected mouse muscle and blood samples from the owls and analyzed their δ13C and δ15N values. Δ13C and Δ15N values (mean ± SE) for owl blood were +0.3‰ ± 0.2‰ and +1.9‰ ± 0.1‰, respectively. These values are the first discrimination factors ever reported in Strigiformes and are lower, for Δ15N, than those obtained in terrestrial carnivores and other bird species, including falcons. Preservation in ethanol did not significantly affect δ13C and δ15N values.

Contamination as the Cause of Erroneous Records of Brochosomes

Brochosomes are ultramicroscopic particles produced in large quantities by the Malpighian tubules of leafhoppers (Insecta, Hemiptera, and Cicadellidae) and applied by leafhoppers as a coat to their integuments. A recent study has described brochosomes on museum specimens of Heteroptera and Psylloidea, suggesting a wider distribution of brochosomes among Hemiptera. Here, I report that the majority of adultSthenarus rotermundi(Scholtz) (Miridae) andKleidocerys resedae(Panzer) (Lygaeidae) reared in captivity and handled with clean tools had no brochosomes on them, suggesting that the earlier records of brochosomes in these and perhaps other species outside of the Cicadellidae were due to contamination. Additionally, simple experiments demonstrated that insects can become contaminated with brochosomes via entomological tools that had been in contact with leafhoppers and via preservation in ethanol together with leafhoppers. Contamination of host plants, predators, and parasites of leafhoppers with brochosomes is also expected but remains to be demonstrated.

Effects of preservation on the δ 13C and δ 15N values of deep sea macrofauna

The aim of the present study was to quantify the effect of formalin–ethanol preservation on the carbon and nitrogen stable isotope values of five deep-sea species ( Abra longicallus, Molpadia musculus, Sipunculus norvegicus, Chirimia biceps and Nephthys hystricis). To examine temporal changes in the effects of preservation and to determine if preservation induces predictable shifts in δ 13C and δ 15N values, analyses were carried out after 1, 6 and 12 months of preservation. The data indicated variable effects on carbon and nitrogen isotope values among species after formalin and ethanol preservation. Formalin fixation did not affect δ 15N values of any of the five species analysed, while it did affect δ 13C values, with a depletion trend observed in all of the species. In general ethanol preservation did not affect nitrogen values for up to 6 months of preservation, and only the δ 15N values of S. norvegicus became depleted after 12 months. Ethanol fixation only affected the carbon isotopic composition of M. musculus, with a progressive depletion from 1 to 12 months after preservation. Due to the different effects of preservation on stable isotope values, the use of a general and systematic correction factor was not possible. Since no effect on δ 15N values of either formalin or ethanol fixation is evident after 6 months, we recommend that these methods be used for short-term (< 6 months) conservation and only if studies are focused on the identification of trophic levels. If changes in carbon and nitrogen isotopic values from long-preserved samples are to be used for reconstructing a food web, it is necessary to establish the effects of preservation in the whole spectrum of species evaluated.

Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies

The purpose of this study is to evaluate whether sample preservation can affect the yield of nucleic acid extracts from environmental samples. Storage of microbial samples was studied using three sediment types of varying carbon contents (10-57% carbon of dry weight). Four different storage solutions were tested at three temperatures. Freezing of samples at -20 °C or -80 °C, either without preservative or in phenol-chloroform solution, retained nucleic acid quantities very efficiently. Storage of samples in phenol-chloroform solution at +4 °C also gave good yields except for sediment with extremely high-carbon content. Ethanol and RNAlater preservation decreased nucleic acid yields drastically at all temperatures. To study how sample preservation may affect the result of microbial community analysis, one type of sediment was selected for length heterogeneity-PCR analysis and PCR cloning of the 16S rRNA genes. Ethanol and RNAlater preservation caused a slight bias towards certain microbial types in the community analyses shown by underrepresentation of Bacteroidetes, Betaproteobacteria and Gammaproteobacteria-affiliated peak sizes and overrepresentation of Actinobacteria, Chloroflexi and Alphaproteobacteria-affiliated peak sizes. Based on the results of this study, preservation in phenol-chloroform solution can be recommended as an alternative storage method when freezing is not possible such as during extended field sampling; however, ethanol and RNAlater may cause serious problems when used as preservatives for environmental samples containing humic acids.

Influence of Storage Duration on Retention of Original Fracture Toughness

Accurate knowledge of bone properties is central in the prediction of failure and the development of injury treatment protocols. Often when bone properties are measured experimentally, bone specimens have to be chemically preserved prior to or during testing. Understanding the effect of the bone preservation method on the material properties is important. Degradation of properties due to preservation methods may lead to incorrect reporting of values of the bone properties. A salient question is, therefore, whether the preservation of bovine cortical bone in ethanol for extended periods of time affects fracture toughness; and if so, whether that affect is reversible. To answer these questions, a three-point bending test set-up was constructed to perform experiments over a period of 9 weeks. A total of 109 specimens of cortical bone of the same orientation were manufactured from the mid-diaphysis of the femur. These specimens were separated into three groups based on location in the femur; namely, medial, lateral, and posterior. The specimens were preserved in ethanol. At the end of each week, a sample of specimens was selected for testing, with the last sample tested after 9 weeks of preservation. It was shown that after 9 weeks of preservation in ethanol, followed by rehydration with physiological saline, the fracture toughness of the specimen was unchanged from that of the control specimen. Omission of rehydration in saline resulted in to an increased fracture toughness of up to 17%.

Correction of the weight and length for juveniles Atherinella brasiliensis (Actinopterygii: Atherinopsidae) after fixation in formalin and preservation in ethanol

The present study compared losses of weight and length in specimens of Atherinella brasiliensis (Quoy & Gaimard, 1825) after fixation in formalin 10% during ten days, and posterior preservation in ethanol 70% for 45 days, in two solvents: freshwater and marine water. Additionally, correction factors were proposed to calculate the correction for weight and length from preserved specimens. The specimens were weighted and measured after collection (fresh), and with 10, 20, 27, 34, 41 and 55 days of fixation plus preservation. The largest losses in weight occurred during the first 20 days (10.35 ± 0.31% - freshwater; 11.29 ± 0.44% - marine water), continuing with lesser intensity up to the 27th day in freshwater dilution, and up to the 34th day in marine water dilution. Weight losses stabilized by the 34th day for both freshwater (11.77 ± 0.33%) and marine water (13.62 ± 0.41%). The largest losses in the total length for the two methods were also observed in the first 20 days (7.42 ± 0.27% - freshwater; 9.76 ± 0.26 - marine water), stabilizing after the 27th day in freshwater (8.60 ± 0.26%) and 20th day in marine water (9.76 ± 0.26%). The losses of weight and total length were significantly dependent on fish size, with smaller individuals suffering the most significant losses. Complete regression equations were proposed for the retro-calculation of fresh weight and total length from the preserved specimens for freshwater (Wfresh = 1.0536W P + 0.0416; TLfresh = 0.9588TL P + 5.8437) and marine water (Wfresh = 1.0868W P + 0.0451; TLfresh = 0.8621TL P + 10.425).