Hatchling painted turtles (Chrysemys picta) survive exposure to subzero temperatures during hibernation by avoiding freezing

Hatchling painted turtles (Chrysemys picta) were placed individually into artificial nests constructed in jars of damp soil and then were cooled slowly to temperatures between-7.7 and-12.7 °C. Distinct exotherms were recorded in all jars when water in the soil began to freeze at temperatures between-0.9 and-2.4 °C. A second (animal) exotherm was subsequently detected in some of the jars when water in hatchlings also began to freeze. An animal exotherm occurred in the temperature records for all 23 hatchlings that died in tests terminating at temperatures between-7.7 and-10.8 °C, but no such exotherm was apparent in the temperature records for the 23 turtles that survived these treatments. Moreover, the 4 hatchlings that produced exotherms in tests terminating between-11.5 and-12.7 °C failed to survive, but 5 of 7 hatchlings that produced no exotherm in these tests also died. Thus, turtles that die at subzero temperatures above-11 °C apparently succumb to freezing when ice propagates across their integument from the frozen soil, but animals that die at temperatures below-11 °C generally perish from some other cause. These findings indicate that hatchling painted turtles overwintering inside their shallow, subterranean nests survive exposure to subzero temperatures by avoiding freezing instead of by tolerating freezing.     

Supercooling,ice inoculation and freeze tolerance in the European common lizard,Lacerta vivipara

The European common lizard (Lacerta vivipara) is widely distributed throughout Eurasia and is one of the few Palaearctic reptiles occurring above the Arctic Circle. We investigated the cold-hardiness of L. vivipara from France which routinely encounter subzero temperatures within their shallow hibernation burrows. In the laboratory, cold-acclimated lizards exposed to subfreezing temperatures as low as -3.5°C could remain unfrozen (supercooled) for at least 3 weeks so long as their microenvironment was dry. In contrast, specimens cooled in contact with ambient ice crystals began to freeze within several hours. However, such susceptibility to inoculative freezing was not necessarily deleterious since L. vivipara readily tolerated the freezing of its tissues, with body surface temperatures as low as -3.0°C during trials lasting up to 3 days. Freezing survival was promoted by relatively low post-nucleation cooling rates (0.1°C·h^-1) and apparently was associated with an accumulation of the putative cryoprotectant, glucose. The cold-hardiness strategy of L. vivipara may depend on both supercooling and freeze tolerance capacities, since this combination would afford the greatest likelihood of surviving winter in its dynamic thermal and hydric microenvironment.Abbreviations bm body mass - SVL snout-vent length - T_b body surface temperature - T _c crystallization temperature     

Equilibrium freezing of leaf water and extracellular ice formation in Afroalpine ‘giant rosette’ plants

The water potentials of frozen leaves of Afroalpine plants were measured psychrometrically in the field. Comparison of these potentials with the osmotic potentials of an expressed cellular sap and the water potentials of ice indicated almost ideal freezing behaviour and suggested equilibrium freezing. On the basis of the osmotic potentials of expressed cellular sap, the fractions of frozen cellular water which correspond to the measured water potentials of the frozen leaves could be determined (e.g. 74% at -3.0° C). The freezing points of leaves were found to be in the range between 0° C and -0.5° C, rendering evidence for freezing of almost pure water and thus confirming the conclusions drawn from the water-potential measurements. The leaves proved to be frost resistant down to temperatures between -5° C and -15° C, as depending on the species. They tolerated short supercooling periods which were necessary in order to start ice nucleation. Extracellular ice caps and ice crystals in the intercellular space were observed when cross sections of frozen leaves were investigated microscopically at subfreezing temperatures.Symbols T temperature - water potential Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Geographic variation of freeze-tolerance in the earthworm Dendrobaena octaedra

Freeze-tolerance and some of the underlying biochemical defence mechanisms in the earthworm Dendrobaena octaedra was investigated. Survival after slow cooling to -2 degrees C, -4 degrees C, or -6 degrees C was analysed in D. octaedra from three geographic regions representing large differences in winter temperature (Denmark, Finland and Greenland). A large variation in freeze-tolerance between the three populations of D. octaedra was found. Earthworms from the northern populations (Finland and Greenland) tolerated lower temperatures (-6 degrees C) than earthworms from the Danish population (poor survival at -4 degrees C and -2 degrees C). In the Finnish population, freezing led to the production of high concentrations of glucose, which reached values much higher than controls (94 mg g(-1) vs. 2 mg g(-1) dry weight). Other potential cryoprotectants were not elevated after freezing. The Danish and Greenlandic populations had substantially lower mean glucose levels after freezing than the Finnish population (about 15 mg g(-1)). Danish earthworms rapidly frozen did not accumulate glucose, and did not survive freezing at -2 degrees C. Danish earthworms exposed to osmotic stress in Ringer's solutions, containing different concentrations of glycerol, showed significantly elevated glucose levels, but did not survive rapid freezing. It was determined if freezing had an influence on the reproduction of the earthworms. After warming to summer temperatures (15 degrees C), survivors of freezing produced viable cocoons. In a field experiment it was tested if natural acclimatization during autumn and winter months had an effect on freeze-tolerance in the Danish population. There was a significant increase of post-freeze survival during this period. The results of the freezing experiments are discussed in relation to the general ecology of D. octaedra.     

Cold hardiness of larvae of the southwestern corn borer, Diatraea grandiosella

The cold-hardening capacity of field-collected larvae from southeast Missouri and laboratory-reared larvae of the southwestern corn borer, Diatraea grandiosella Dyar, was examined. Supercooling points of non-diapause and diapause larvae collected from maize plants grown in Missouri (36°30 N lat.) were ca.-7.0°C. The hemolymph melting points of diapause field larvae (-0.8°C) were significantly lower than those of non-diapause larvae collected in July (-0.5°C). The supercooling points of hemolymph from non-diapause and diapause field larvae ranged randomly from-10° to-18°C. Supercooling points of non-diapause laboratory larvae increased from-13° to-10°C prior to pupation, whereas those of diapause larvae increased similarly before the onset of diapause, but then decreased during diapause to ca.-17°C. No change in supercooling points or capacity to survive in the presence of ice was observed in diapause laboratory larvae acclimated at 4°C for 63 days. Laboratory and field larvae began to freeze at ca.-1.5°C in the presence of ice, but survived to several degrees below their melting points. The high supercooling points of field larvae appeared to be due to the presence of an environmental ice-nucleator. Although data for laboratory larvae indicate sufficiently low supercooling points to permit winter survival in southeastern Missouri, considerable larval mortality occurs in the field due to inoculative freezing and the presence of an ice-nucleator.     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Cold hardiness of the eggs of the plaice,Pleuronectes platessa

Summary The pelagic eggs of the plaice, Pleuronectes platessa, at the stage of first heart beat, can tolerate freezing into solid sea ice at temperatures down to-6°C. A long term freezing period of 5 days, at sublethal temperatures of-3 to-4°C, had only a minor effect on the eggs. When the ice was absent from the medium, the eggs supercooled readily to temperatures around-15° to-20°C. However, no thermal hysteresis agents were present in the eggs at low temperature and the cold hardiness therefore seem to rely on a high tolerance to brine solutions at low temperatures.     

Intracellular Freezing in the Infective Juveniles of Steinernema feltiae: An Entomopathogenic Nematode

Taking advantage of their optical transparency, we clearly observed the third stage infective juveniles (IJs) of Steinernema feltiae freezing under a cryo-stage microscope. The IJs froze when the water surrounding them froze at −2°C and below. However, they avoid inoculative freezing at −1°C, suggesting cryoprotective dehydration. Freezing was evident as a sudden darkening and cessation of IJs'' movement. Freeze substitution and transmission electron microscopy confirmed that the IJs of S. feltiae freeze intracellularly. Ice crystals were found in every compartment of the body. IJs frozen at high sub-zero temperatures (−1 and −3°C) survived and had small ice crystals. Those frozen at −10°C had large ice crystals and did not survive. However, the pattern of ice formation was not well-controlled and individual nematodes frozen at −3°C had both small and large ice crystals. IJs frozen by plunging directly into liquid nitrogen had small ice crystals, but did not survive. This study thus presents the evidence that S. feltiae is only the second freeze tolerant animal, after the Antarctic nematode Panagrolaimus davidi, shown to withstand extensive intracellular freezing.     

Physiological adaptations to low temperature and brine exposure in the circumpolar amphipod Gammarus wilkitzkii

Summary The amphipod Gammarus wilkitzkii does not survive being frozen totally into solid sea ice. When the animals are cooled in air or freezing seawater, they will freeze and die at a temperature of about-4° C. However, during sea ice growth, the amphipods may tolerate to stay in the vicinity of the ice by conforming to the ambient brine in a salinity range of 34 ppt to about 60 ppt. A passive relationship between the concentrations of the haemolymph and seawater Na⁺ and Cl^-, lowers the melting point of the body fluids of the animals, thus preventing internal ice formation at low temperatures.     

Freeze or dehydrate: only two options for the survival of subzero temperatures in the arctic enchytraeid<Emphasis Type="Italic"> Fridericia ratzeli</Emphasis>

Hygrophilic soil animals, like enchytraeids, overwintering in frozen soil are unlikely to base their cold tolerance on supercooling of body fluids. It seems more likely that they will either freeze due to inoculative freezing, or dehydrate and adjust their body fluid melting point to ambient temperature as has been shown for earthworm cocoons and Collembola. In the present study we tested this hypothesis by exposing field-collected adult Fridericia ratzeli from Disko, West Greenland, to freezing temperatures under various moisture regimes. When cooled at –1 °C min^–1 under dry conditions F. ratzeli had a mean temperature of crystallisation (T_c) of –5.8 °C. However, when exposed to temperatures above standard T_c for 22 h, at –4 °C, most individuals (90%, n= 30) remained unfrozen. Slow cooling from –1 °C to –6 °C in vials where the air was in equilibrium with the vapour pressure of ice resulted in freezing in about 65% of the individuals. These individuals maintained a normal body water content of 2.7–3.0 mg mg^–1 dry weight and had body fluid melting points of about –0.5 °C with little or no change due to freezing. About 35% of the individuals dehydrated drastically to below 1.1 mg mg^–1 dry weight at –6 °C, and consequently had lowered their body fluid melting point to ca. –6 °C at this time. Survival was high in both frozen and dehydrated animals at –6 °C, about 60%. Approximately 25% of the animals (both frozen and dehydrated individuals) had elevated glucose concentrations, but the mean glucose concentration was not increased to any great extent in any group due to cold exposure. The desiccating potential of ice was simulated using aqueous NaCl solutions at 0 °C. Water loss and survival in this experiment were in good agreement with results from freezing experiments. The influence of soil moisture on survival and tendency to dehydrate was also evaluated. However, soil moisture ranging between 0.74 g g^–1 and 1.15 g g^–1 dry soil did not result in any significant differences in survival or frequency of dehydrated animals even though the apparent wetness and structure of the soil was clearly different in these moisture contents.Abbreviations DW dry weight - FW fresh weight - MP melting point - RH relative humidity - Tc crystallisation temperatures - WC water contentCommunicated by I.D. Hume     

The distribution,structure, and composition of freshwater ice deposits in Bolivian salt lakes

Freshwater ice deposits are described from seven, high elevation (4117–4730 m), shallow (mean depth <30 cm), saline (10–103 g l^-1) lakes in the southwestern corner of Bolivia. The ice deposits range to several hundred meters in length and to 7 m in height above the lake or playa surface. They are located near the lake or salar margins; some are completely surrounded by water, others by playa deposits or salt crusts. Upper surfaces and sides of the ice deposits usually are covered by 20–40 cm of white to light brown, dry sedimentary materials. Calcite is the dominant crystalline mineral in these, and amorphous materials such as diatom frustules and volcanic glass are also often abundant.Beneath the dry overburden the ice occurs primarily as horizontal lenses 1–1000 mm thick, irregularly alternating with strata of frozen sedimentary materials. Ice represents from 10 to 87% of the volume of the deposits and yields freshwater (TFR <3 g l^-1) when melted. Oxygen isotope ratios for ice are similar to those for regional precipitation and shoreline seeps but much lower than those for the lakewaters. Geothermal flux is high in the region as evidenced by numerous hot springs and deep (3.0–3.5 m) sediment temperatures of 5–10°C. This flux is one cause of the present gradual wasting away of these deposits. Mean annual air temperatures for the different lakes probably are all in the range of -2 to 4°C, and mean midwinter temperatures about 5°C lower. These deposits apparently formed during colder climatic conditions by the freezing of low salinity porewaters and the building up of segregation ice lenses.     

Low temperature survival in different life stages of the Iberian slug, Arion lusitanicus

The slug Arion lusitanicus Mabille (Gastropoda: Pulmonata: Arionidae) is an invasive species which has spread to most parts of Europe. The area of origin is unknown, but A. lusitanicus seems to cope well with the local conditions in the countries to which it has migrated. It spreads rapidly, occurs often in high densities and has become a serious pest in most European countries. Therefore there is an urgent need for better knowledge of the ecophysiology of A. lusitanicus, such as the influence of climatic conditions, in order to develop prognostic models and strategies for novel pest management practises.The aim of our study was to investigate the influence of subzero temperatures in relation to winter survival. A. lusitanicus is shown to be freeze-tolerant in some life stages. Most juveniles and some adult slugs survived being frozen at −1.3 °C for 3 days, but none of the slugs survived freezing at −3 °C. The eggs survived subzero temperatures (down to −2 °C) probably by supercooling. Juveniles and adults may also survive in a supercooled state (down to −3 °C) but are generally poor supercoolers. Therefore, the winter survival of A. lusitanicus depends to a high degree on migration to habitats protected from low winter temperatures, e.g. under plant litter, buried in the soil or in compost heaps.     

Extracellular ice and cell shape in frost-stressed cereal leaves: A low-temperature scanning-electron-microscopy study

Low-temperature scanning electron microscopy was used to examine transverse fracture faces through cereal leaf pieces subjected to frost. Specimens were studied before and after sublimation of the ice. The position of extracellular ice in the leaf was inferred from the difference between the specimen before and after sublimation and from ridges and points which occurred in the extracellular ice during sublimation. Steps in the fracture surface indicated that the fracture plane passed through the extracellular ice crystals as well as through cells and also helped identify extracellular ice. The cells in controls were turgid and extracellular ice was absent. Leaf pieces from hardened rye were excised and frost-stressed to-3.3°,-21° and-72°C, cooling at 2–12°·h^-1. Cell collapse and extracellular ice were evident at-3.3°C and increased considerably by-21° C. At-21° and-72°C the leaf pieces were mainly filled with extracellular ice and there were few remaining gas spaces. The epidermal and mesophyll cells were laterally flattened, perpendicular to their attachment to adjacent cells, and phloem and vascular sheath cells were more irregularly deformed. Leaf pieces from tender barley were cooled at 2°C·min^-1 to-20° C; they were then mainly filled with extracellular ice, and the cells were highly collapsed as in the rye. In rye leaves frozen to-3.6° C before excision, ice crystals occurred in peri-vascular, sub-epidermal and intervening mesophyll spaces. In rye leaf pieces frozen to-3.3° C after excision or to-3.6° C before excision, mesophyll cells were partly collapsed even when not covered by ice, indicating that collapse of the cell wall, as well as the enclosed protoplast, was driven by dehydration. No gas or ice-filled spaces were found between wall and the enclosed protoplast. It is suggested that this can be explained without invoking chemical bonding between cell wall and plasma membrane: when the wall pores are filled by water, the pore size would reduce vapour pressure so making penetration of the wall by ice or gas less likely.Abbreviations SEM scanning electron microscopy     

Cold resistance and metabolic responses to salinity variations in the amphipod Eusirus antarcticus and the krill Euphausia superba

Summary The krill Euphausia superba, unlike the amphipod, Eusirus antarcticus, tolerates being frozen into solid sea-ice at temperatures down to about-4°C. Cooled in air, the amphipod and the krill freeze and will die at temperatures of-11° and-9°C respectively, representing the supercooling points of the animals. The krill is an osmoconformer in the salinity range of 25 to 45 ppt, while the amphipod conforms in the salinity range of 26 to 40 ppt. The animals thereby lower the melting point of their body fluids in the vicinity of the freezing sea ice, preventing internal ice formation at low temperatures. The mean oxygen consumption rates, at raised and lowered salinities, were not significantly different from rates obtained in normal (35 ppt.) seawater, indicating that salinity has little effect on the metabolism of either species.     

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae)

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.     

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between − 3 and − 10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below − 4 °C and cell survival exhibits an optimum at a nucleation temperature of − 6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at − 3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at − 10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.     

Seasonal changes in lipid composition and glycogen storage associated with freeze-tolerance of the earthworm, Dendrobaena octaedra

The earthworm, Dendrobaena octaedra, is a common species in the uppermost soil and humus layers of coniferous forests and tundra in temperate and subarctic regions. The species is freeze-tolerant and may survive several months in a frozen state. Upon freezing, glycogen reserves are rapidly converted to glucose serving as a cryoprotectant and fuel for metabolism. In the present study we investigated the induction of freeze-tolerance under field conditions, and sought to find relationships between temperature, glycogen and fat reserves, membrane phospholipid composition and the degree of freeze-tolerance. Freeze-tolerance was induced when worms had experienced temperatures below 5°C for 2 weeks or more. Freeze-tolerance was linked to the magnitude of glycogen reserves, which also fluctuated with field temperatures being highest in autumn and winter. On the other hand fat reserves seemed not to be linked with freeze-tolerance at all. However, high glycogen alone did not confer freeze-tolerance; alterations in the membrane phospholipid fatty acid composition (PLFA) were also necessary in order to secure freeze-tolerance. The changes in PLFA composition were generally similar to changes occurring in other ectothermic animals during winter acclimation with an increased degree of unsaturation of the PLFAs.     

Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.