Replacing Formalin with Alcohol and its Effect on Preserved Fish Specimens

This study aims to formulate an alternative solution for Formalin for preserving fish as study specimens for long periods. The main reason for finding a solution instead of formalin is to get rid of the negative effects of this solution on those who work with it, as well as to better preserve the bodies of fish. Hence, three new solutions were proposed to replace formalin. Thus, Formalin, in turn, may enter the composition of a small part of these solutions to give better results and for long periods of keeping specimens. All solutions prepared in this study participated in being acidic as in formalin. Two solutions succeeded in compensating for the use of formalin in preserving fish bodies for a period of six months, with a lesser effect on some of the studied biometrics of fish kept in these two solutions.


Introduction
Students of Ichthyology depend on preserving fish either by using preservation solutions such as Formalin or alcohol.Formalin is the most common form of fish for preserving or freezing.This method is also common for preserving fish for study and diagnosis [1][2][3].Formalin is a multi-purpose solution which is used in many scientific and industrial fields as explained by [4,5] and it is used in the manufacture of wood, plastics, dyes and metal purification.Formalin is also used in preserving vital samples and tissues as well as in embalming.In addition to its use as an antiseptic and an exterminator for many pathological microorganisms such as bacteria, fungi and protozoa in addition to insects alike, as indicated by [6], it also has a wide range of medical uses, especially in tissue preparation and preservation, dentistry, and kidney dialysis.It is a preservative for many different drug formulations, as shown by [7][8][9].When using formalin in preserving fish models after collecting them, it may cause some biological changes in some of the vital measurements of the bodies of these fish, including shrinkage and contraction of bodies, expansion and contraction, as well as a pale colour of the fish, and this is indicated by [10,11].Preservation in alcohol and freezing are good methods for preserving some vital measurements of fish bodies preserved as confirmed by [12] .As the length increases in general in the bodies of preserved fish, it expands by the methods of freezing and preservation with alcohol than those preserved in Formalin.Also, the preservation of most Biometrics in their good form is better with these two methods than preservation in Formalin traditionally, and this is confirmed by each of [13][14][15].The 1259 (2023) 012085 IOP Publishing doi:10.1088/1755-1315/1259/1/012085 2 specimens preserved by the methods of preserving with alcohol and freezing are more vibrant and fresher.Formalin is one of the most dangerous solutions to public health.It is a cause of many diseases, especially cancers of the upper respiratory tract, such as skin and nose cancer.Scholars like [16][17][18] mentioned it in their studies and talked about the danger of formalin as a solution or the gases emitted from it alone or when interacting with other substances.

Materials and Methods
Three solutions containing alcohol, glycerine, and formalin were prepared in the composition of three treatments: T2, T3, and T4, respectively, and compared with the control treatment T1, which was formalin at a concentration of 10%.Twenty-five Planiliza abu fish were kept for each treatment (preservation solution) in this study to verify some of the biometrics of these fish represented by the total length TL, head length HL. and BTH body thickness.Also, comparisons were made on the vital measurements among these fish in the four solutions proposed in this experiment, and selection of the solution was made that is more safer for public health, and that gives the best results in preserving some vital measurements of the fish kept in these four solutions, provided that it does not cause high physical changes in the bodies of these preserved fish.The experiment took six months, during the period April 2022 up to October 2022.Readings were taken monthly for some biometrics of fish kept in the four solutions.The statistical analysis of the different data received from this experiment was conducted using the SAS statistical program and conducting some statistical tests associated with this program to obtain high accuracy in the data for the studied measurements, and compare them with each other to reach the best results agreed upon by the statistical analysis between the four solutions of this experiment to reach the best solution preservation possible to come out of this study.

Results and Discussion
All solutions prepared for preservation in the current experiment participated was shown to be acidic.The pH of these solutions was less than 7 degrees.In addition to fixing the total dissolved solids in these four solutions for this study, the temperature of these solutions over the duration of the experiment was (28 C) as shown in Some of the biometric measurements were identified for preserved fish which are the total length TL., Head length HL. and BTH body thickness.We see from Table (2) the rates of biometrics studied for fish specimens preserved in the four solutions, including the three new solutions proposed in this study T2, T3 and T4, respectively.Table 2.The rates of Biometrics studied for fish samples preserved in the four solutions.When studying the measurement of total body length TL. of the studied fish specimens and the comparison between the four solutions to save these specimens, the T1 control of formalin had the superiority on the rest of the studied total lengths in at a highly significant level.The value of the total length of formalin was the highest compared to the rest of the solutions.This was followed by the fourth treatment, T4, in affecting the fish specimens preserved in this solution.While the effect of the two solutions represented by the second treatment T2 and the third one T3 decreased on the characteristic of the total length TL of the models kept in them as shown in figure (1).

Figure 1.
Compare the total length between the four solutions.
And this characteristic of formalin preservation is one of the undesirable characteristics due to the contraction and expansion of the bodies of fish preserved in it.It can cause obvious contraction and contraction in most of the newly collected fish specimens.It may also be a reason for the change and disappearance of the color of the fish when kept in it.Thus, [3] in their study on carp fish specimens have indicated a shrinking of the bodies of those fish and a fluctuation in the rates of total lengths of common carp fish when preserved in Formalin at a concentration of 10%.The same change occurred in the lengths of the fish studied in the current study , especially for the specimens preserved in 10% formalin, despite the difference in the preserved species between the two studies, as the common carp fish were the models for the study of the above researchers, while the Planiliza abu fish were the specimens for this study, and the effect of shrinkage and shrinkage of the bodies was clear due to the preservation in Formalin, despite the different fish samples of the two studies.The fourth treatment, T4, ranked second after the control (formalin) in its effect on the measurement of the total length of preserved specimens, knowing that this solution in treatment T4 partially included formalin in its composition, mixed with two other solutions, alcohol and glycerin.The effect of formalin is clear even when mixed with other ingredients.Despite this, we should not forget that formalin possesses the highest quality in stabilizing many physiological matters of the tissues preserved in it, especially for fish Larvae.This is shown by [19] in their study about the fixation of genes for fish Larvae that are kept in formalin at a concentration of less than 10%, which may reach 5 or 4%, due to the fact that the bodies of fish larvae do not tolerate high formalin concentrations, unlike large-sized fish.Formalin here achieved a positive result in favor of the genetic and physiological analyses.The effect of the two solutions in treatments T2 and T3 on fish bodies decreased in terms of contraction and expansion in the total length of the fish kept in them.It is due to the presence of alcohol in their composition.Ethanol alcohol is less stressful on the bodies of fish of all ages, from the larval stage to the adult.This is confirmed by [20] in their study when they preserved the fish with a low concentration of ethyl alcohol and low temperatures.It did not even change the larval physiological composition.This result is considered close to the two solutions of the two treatments T2 and T3, which did not change the total length of the fish kept in them.[21] indicate that keeping fish and even small crustaceans in preservative solutions of different compositions may cause shrinkage and contraction in these bodies, especially when the Ethanol concentration is higher than 90%.This decrease did not occur in the total length of the fish bodies preserved in the two treatments T2 and T3 sharply than it is in the total length of the fish bodies kept in Formalin even when reducing its concentration.Figure (2) shows the effect of preservative solutions on measuring the head length of fish kept in these four solutions.The second preservation solution T2, was superior to the rest of the other preservation solutions followed by the control treatment T1 formalin solution 10%.While the effect of the two solutions in the two treatments T3 and T4 decreased the length of the head of the fish kept in these four solutions.One of the most important components of the second preservation solution T2 is glycerin.It is known as a viscous liquid with a distinctive, non-disturbing smell, with a sweet, non-toxic taste and it is used in many different industries.Among its characteristics is that it is amorphous at high temperatures, hygrophilous, and causes expansion in some tissues when used, as shown by [22,23].Also, it may be the reason for the expansion of the bones of the skull and thus giving measurements of the length of the total head which is higher than the rest of the other preservative solutions, and this what distinguishes the bodies preserved with glycerin and its high ability to absorb and thus the expansion of those bodies, especially the head of fish when preserved with it for long periods, and the saturation of those bodies with glycerin after the long period of the preservation for those objects such as fish.And this is shown by [24] when they restored the size of the fish head when preserved with Glycerin for a long time.Figure (3) represents BTH body thickness measurement data.As the fourth treatment or the fourth solution T4 which overcame the rest of the other three solutions of the experiment.The reason for the increase in the thickness of the body may be the entry of glycerin into the composition of the three solutions T2, T3 and T4 and the saturation of the bodies of the specimens with glycerin.Since part of the composition of these solutions was varying between these solutions to find the best solution that preserves the bodies of the specimens and with the least obvious shrinkage on them.In their study, [24] indicate an increase in the saturation of the fish body with glycerin when it reaches a concentration of less than 60%.It seems clear in this study that the results of the two studies are identical when the T4 treatment even when the glycerin concentration reached less than 30% of the total solution concentration.It is also noted that the lower concentration of glycerin in the composition of the solution, the lower the thickness of the body, as shown in figure [3] in the treatments T2 and T3.As the concentration of glycerin in the T3 treatment reached more than 70%.This is what makes glycerin a catalyst for body swelling, according to the fish species preserved and saturated with it, as confirmed by [24].While the effect of the body thickness did not appear clear, rather the body thickness was affected slightly in the control treatment or Formalin solution in treatment T1.In addition , the difference in measuring the body thickness of the fish kept in the treatments T4, T3 and T2 was downward and directly proportional with the concentration of glycerin in these treatments.The overlapping of the effect of alcohol in a single composition as a solution with glycerin may be a cause of increasing the thickness of the body of the preserved models of what is known about alcohol as a solvent for some types of fats.And since glycerin is a fatty solution, it may make its penetration into the preserved bodies and being saturated with it more faster and better when alcohol is present in this composition.And this is what happened with [25,26] when the bodies of the specimens preserved in alcohol and glycerin solutions returned to their real shape, especially when the glycerin concentration in these solutions was less than 60%.The specimens preserved in the two solutions in which glycerin and alcohol were included, namely T3 and T4 that gained a good shape and high flexibility for the fins and the general joints of the body , as compared to the solutions in which formalin was included in the composition, namely T1 and T2.These results were good and were in consistent with that got by [27][28][29].

Conclusion and Recommendations
The three solutions proposed in this study for treatments T2, T3 and T4 have succeeded in preserving fish specimens for six months.The composition of these solutions differed and varied with the basic components of formalin, Alcohol and glycerin in different proportions of mixing according to the concentrations suggested in this study.Also, the solutions of the two treatments T2 and T3 had the best results because of the lack of effect of the preservation solutions in these two treatments on the bodies of the fish kept in them.Moreover , the preservation solution is considered good whenever its negative effect on the objects preserved in it is reduced, which are represented by cases of expansion, contraction, and pallor of color.Furthermore it is not to deny that Formalin is of great importance in fixing some genetic and genetic characteristics of tissue samples in general and of fish models in particular.
The time factor also has an effect on preservation in these solutions of different compositions.Therefore, the researcher recommends conducting other in-depth and detailed studies that include longer periods for these new proposed solutions in this study as well as using other fish species for preservation in these solutions, regardless of the type of those fish, whether they are scaled or smooth fish alike, to access to good solutions with low toxicity and risk to those working in such studies, in addition to preserving the bodies of fish in their natural shape, size and color as much as possible.

Figure 2 .
Figure 2. Head length comparison between preservative solutions.

Figure 3 .
Figure 3.Comparison of body thickness between preservative solutions.