Ethanol: a better preservation technique for Daphnia

Abstract

Plankton ecologists often kill and fix zooplankton specimens for future study. The standard protocol used by limnologists is a sucrose-formalin solution first recommended by Haney and Hall (1973). This solution is an aqueous solution of 4% formalin (final concentration) and 40 g of sucrose per liter. Commercially available formalin is a 37% by weight, or a 40% by volume, aqueous solution. Thus, a 4% formalin solution is a 1.5% formaldehyde solution.The 4% formalin solution was recommended because it reduced morphological distortion, even of soft animals like Daphnia, while taking advantage of fixation and preservation properties of formalin. Minimum distortion is particularly desirable in studies of size-selective predation or reproductive rates, in which eggs need to stay in the brood chamber of branchiopods. Whereas there are advantages of formalin fixation, there also exists significant human health concerns for formaldehyde exposure.Acute effects of exposure-The effects of greatest interest to limnologists are caused by inhalation and contact. The greatest acute exposure occurs during analysis of samples when the limnologist looks at plankton samples through a microscope, often for hours at a time. Labs are often poorly ventilated, leading to increased exposure through inhalation of formalde-hyde vapors. This inhalation is highly irritating to the upper respiratory tract and eyes.According to the United States Occupational Safety and Health Administration (OSHA 2001), concentrations of 0.5 to 2.0 ppm (in air) may irritate the eyes, nose, and throat of some individuals. Concentrations of 3 to 5 ppm also cause tearing of the eyes and are intolerable to some persons. Concentrations of 10 to 20 ppm cause difficulty in breathing, burning of the nose and throat, cough, and heavy tearing of the eyes, and 25 to 30 ppm causes severe respiratory tract injury leading to pulmonary edema and pneumonitis. A concentration of 100 ppm is immediately dangerous to life and health. Deaths from accidental exposure to high concentrations of formaldehyde have been reported. AbstractA 4% formalin-40% sucrose solution has been used by limnologists for three decades as the preferred freshwater zooplankton preservative because it kills and fixes cladocera (Branchiopoda) with relatively little distortion. Because of the increasing evidence of health hazards related to formalin, we sought an alternative, safer preservative that satisfies the need for low distortion. Our results suggest the ethanol preservative methods (70% and 95% treatments) are as good or better as using 4% sugar formalin to fix and store samples. Our results indicate the best method is to fix samples in 95% EtOH followed by storage in 70% EtOH. This technique gave us the least frequent distortion, the highest average number of eggs per female, and the fewest embryos lost from the brood chamber. None of the techniques appeared to have positive or negative effects on body length. Using hot water to fix animals before storage is not recommended. *

Deadly competition and life-saving predation: the potential for alternative stable states in a stage-structured predator–prey system

“…Nauplii undergo complete metamorphosis, transitioning from the nauplius to the copepodite stage, which closely resembles adults in morphology but is smaller [22]. All Daphnia were isolated from beakers and preserved in 70% ethyl alcohol (following [32]) for later enumeration.…”

Cost/benefit and the effect of sample preservation procedures on quantitative patterns in benthic ecology

“…13,23]. Formalin vapors have irritating effects on mucous membranes, and contact with the skin results in dermatitis [24].…”

Intercalibration of an acoustic technique, two optical ones, and a simple seston dry mass method for freshwater zooplankton sampling

“…These zooplankton samples had been taken from Lake Pyhäselkä in September 2004 with a plankton pump and had first been fixed with 95% ethanol and then stored with 70% ethanol as recommended by Black and Dodson (2003). The technique does not alter the size of the specimens (Black and Dodson 2003).…”