Cold Lugol’s solution as an Alternative of Formaldehyde-base Preservative in Preserving Stomach Content of Shellfish  

Tan Kar Soon , Julian Ransangan
Borneo Marine Research Institute, University Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah, Malaysia
Author    Correspondence author
International Journal of Marine Science, 2015, Vol. 5, No. 9   doi: 10.5376/ijms.2015.05.0009
Received: 05 Jan., 2015    Accepted: 25 Jan., 2015    Published: 10 Feb., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Soon and Ransangan, 2015, Cold Lugol’s solution as an alternative of formaldehyde-base preservative in preserving stomach content of shellfish, International Journal of Marine Science, Vol.5, No.9 1-4 (doi: 10.5376/ijms.2015.05.0009)

Abstract

Formaldehyde has long been the standard preservative for stomach content of bivalves in the feeding preference studies of the animal. However, formaldehyde-base preservatives are highly toxic and carcinogenic to human. Many other preservatives particularly Lugol’s solution and alcohol are known to be effective in preserving phytoplankton sample in water. However, the performance of these preservatives in preserving phytoplankton in the gut of mussels is not fully understood. Current study compared the preservative efficiency of formaldehyde, Lugol’s solution, alcohol and filtered seawater on the phytoplankton in the stomach of green mussel. Mussel samples were collected from green mussel farm at Marudu Bay and preserved in mixture of 5% formaldehyde and glutardialdehyde (3:1), 1% Lugol’s solution, 70% alcohol and filtered seawater, then stored in 4°C. The phytoplankton composition in the gut of mussels was identified and counted according to different preservatives. The result demonstrates that the preservative performance of cold alcohol and filtered seawater is not encouraging. However, the cell abundance and composition preserved in Lugol’s solution is similar to that in the mixture of formaldehyde and glutardialdehyde, suggesting Lugol’s solution can be used as a safe alternative to formaldehyde-base preservative.

Keywords
Alternative preservative; Gut content of mussel; Phytoplankton; Formaldehyde; Lugol’s solution

Cilliary-mucus suspension feeder is known to preferentially ingest certain group of phytoplankton (Ciocco and Gayoso, 2002; Ren et al., 2000). The selective behavior of bivalves is usually examined on the basis of comparison between the phytoplankton composition in gut and water column (Tan and Ransangan, 2014). Indeed, it is always the best to examine samples as early as possible after collection. However, in some circumstances, particularly sampling sites are too far from the laboratories and it needs few hours for the transportation, it is necessary to preserve the sample with suitable preservative that can ensure the samples in good condition before analyzing.

Bivalve specimens are commonly preserved in formaldehyde-base preservative (Navarro, 2005). However, the hazardous fumes of formaldehyde are extremely harmful to human (Titford and Horenstein, 2005). Formaldehyde is highly soluble in water. It reacts with the active hydrogen of many compounds in biological systems such as ammonia, amines and phenols to form bis-chloromethyl ether, a known carcinogen to human (Infante et al., 1981). Therefore, a safe alternative to formaldehyde in preserving the gut content of bivalve is urgently needed.
Apart from formaldehyde, Lugol’s solution and alcohol are commonly used preservatives for plankton samples from water column (Mohammad-Noor et al., 2014; Gaytan-Herrera et al., 2011; Adam et al., 2011). However, the effectiveness of Lugol’s solution and alcohol as a preservative in the present of enzyme in the gut of bivalve is yet to be studied. Therefore, current study was carried out to investigate whether cold filtered seawater, Lugol’s solution and 70% alcohol could be used to replace formaldehyde in preserving stomach content of bivalve.
1 Materials and Methods
1.1 Sample collection and preservation
Twenty mussels with the shell length of 5.0± 0.5 cm were collected randomly from a green mussel farm at Kota Marudu, Sabah, Malaysia. The mussels were divided equally and immediately preserved in four bottles containing different fixatives; 70% Alcohol, 5% formaldehyde and glutardialdehyde in the proportion 3:2, 1% Lugol’s solution and filtered seawater (control), respectively. After 10 minutes of exposure of mussels to the fixatives, all the four bottles were stored in ice.
1.2 Phytoplankton identification and quantification
In laboratory (about 4 hours later), the valves were separated apart and the stomach content of specimens was withdrawn by syringe. After recording the volume collected, the stomach content was diluted into 50 ml with filtered seawater and re-preserved in respective fixatives. The condition of phytoplankton cells was observed, phytoplankton were identified until genus level, and the cell abundance was counted using a Sedgwick Rafter chamber at 400x magnification according to Hakansson (2002), Hartley (1996), Tomas (1995), Kramer and Lange-Bertalot (1986), and Hendey (1964).
1.3 Statistical Analysis
Statistical analyses were performed using the SPSS Windows Statistical Package (version 21). Tests were judged to be significant at p< 0.05 level. All variables were tested for normality and homogeneity of variances. Data which satisfy the assumptions of normality and homogeneity were subjected to parametric tests, one-way ANOVA.
2 Results
The highest phytoplankton abundance was recorded in the gut content of mussels preserved in mixture of formaldehyde and glutardialdehyde (7.3 x102 cells ml-1), followed by Lugol’s solution (6.9 x102 cells ml-1), alcohol (1.8 x102 cells ml-1) and lowest in filtered seawater (0.35 x102 cells ml-1) (Figure 1). There was no significant difference (P>0.05) between the phytoplankton abundance recorded in gut of mussels preserved with Lugol’s solution and the mixture of formaldehyde and glutardialdehyde. However, these values were significantly higher than the average cell abundance recorded in gut content preserved with 70% alcohol and filtered seawater (P<0.05).


Figure 1 Phytoplankton abundance in the gut of mussels preserved in four difference preservatives

In general, the genus Cascinodiscus sp. was dominated the phytoplankton community in the gut content that preserved in all preservatives. Similar relative abundance of phytoplankton genus was observed in the gut content of mussels that preserved in formaldehyde- glutardialdehyde mixture and in Lugol’s solution (Table 1). However, the composition of Bacteriastrum sp., Chaetoceros sp. and Nitzschia sp. was significantly low in the gut preserved with 70% alcohol and was negligible in the gut preserved with cold filtered seawater.


Table 1 Phytoplankton composition in the gut of mussels preserved in four difference preservatives

3 Discussion
As expected, the gut content of specimens preserved in cold filtered seawater was almost completely degraded and very limited phytoplankton cells can be identified. It is not surprising because phytoplankton has been reported to have high lysis rate (Agusti et al., 1998). The isotonic and low temperature (4 °C) of cold filtered seawater does not effectively prevent the microbial degradation of the organic matter in phytoplankton. The small species of phytoplankton with larger surface area per unit volume particularly Bacteriastrum sp., Chaetoceros sp., and Nitzschia sp. were degraded rapidly and resulting in alteration of the overall relative abundance of phytoplankton in the gut sample. The highest relative abundance of Cascinodiscus sp. in all gut samples could be due to selective ingestion of green mussels (Sivalingam, 1977) or the rigid cell walls of the Cascinodiscus sp. that resistant to enzymatic digestion and physical breakdown (Romberger and Epifanio, 1981).


The result of current study demonstrated partially degradation of phytoplankton cells in the sample collected from the gut of mussels preserved with 70% alcohol. Alcohol is not a suitable preservative in preserving gut content of bivalve, because moderate concentration of alcohol (70%) could kills the bivalve quickly within 1 minute. This results in limited amount of preservative access to the stomach contents of mussel (Navarro, 2005). Although the low temperature slowed down the degradation process, the long transportation time of 4 hours significantly reduced the quality of the samples. Since most of the laboratories are often located far from the study areas. Therefore, preservative that can offer better preservation quality for longer time is desirable.
Phytoplankton cells collected from the gut of mussels in the sample that preserved with Lugol’s solution and formaldehyde- glutardialdehyde mixture were in good condition. Mixture of formaldehyde and glutardialdehyde was found to be the best preservative for gut content of bivalves. In fact, formalin is generally the preferred fluid for fixation and is widely used. The mixture of formaldehyde and glutardialdehyde is believed to be the optimal preservative of animal tissues (formaldehyde) and feature of phytoplankton cells (glutardialdehyde) (Navarro, 2005). Mixture of formaldehyde and glutardialdehyde also can preserve the cell wall structure of phytoplankton and last for many years in good condition without attention. However, The International Agency for Research on Cancer branch of the World Health Organization classifies formaldehyde and glutardialdehyde as carcinogenic in humans and it has been demonstrated to contribute to respiratory pathologies, allergies and respiratory tract cancers (Kerns et al., 1983). Enactment of the Formaldehyde Standard (29 CFR 1910.1048 Formaldehyde) law in the US in 1987 also raised awareness that formaldehyde is a potential carcinogen worldwide (Titford and Horenstein, 2005). Exposure to high levels of formaldehyde can cause accumulation of fluid in the lungs, severe shortness of breath, bronchitis and rapid heart rate. Exposure to low levels can irritate, and burn the eye, nose, throat, and skin (Nogueira et al., 1997; Infante et al., 1981). Therefore, analyzing samples preserved with formaldehyde might put researchers in healthy risk as they are dealing with highly carcinogenic chemicals.
Interestingly, the result of current study demonstrates that the phytoplankton abundance and composition recorded in gut of mussels preserved with Lugol’s solution were similar to that preserved in the mixture of formaldehyde and glutardialdehyde. This suggests Lugol’s solution has high potential as an alternative to formaldehyde and glutaraldehyde for preserving gut content of bivalves. Ten minutes exposure of bivalve to Lugol’s solution allowed the chemical access to the gut contents. Then, storing the mussels in cold water kill the mussel by temperature shock and therefore stop the secretion of new enzyme and minimizing the enzymatic activities in the gut of mussels (Khessiba et al., 2005). In addition, the result of current study also showed that the sample preserved with Lugol’s solution offers better accuracy in quantifying with lower standard deviation compare to sample preserved in mixture of formaldehyde and glutardialdehyde. This is because Lugol’s stains cells a dark brown colour, making counting easier and accurate (Modigh and Castaldo, 2005). The most important is Lugol’s solution is relatively less toxic compared to aldehyde-based fixatives. However, iodine breaks down easily in sunlight and samples preserved in Lugol’s solution do not have a long shelf life (3-6 months) (Gifford and Caron, 2000). The limitation of Lugol’s solution can be overcome easily by storing the samples and stock solution in dark and ensure the sample always have a “tea” like colour by adding Lugol’s solution periodically.
4 Conclusion
It is always necessary to preserve the sample with suitable preservative that can ensure the samples in good condition before analyzing. Cold Lugol’s solution is highly recommended to be used as a safe alternative to formaldehyde-base preservative in preserving stomach content of bivalve. Cold Lugol’s solution not only has comparable preservative performance to the mixture of formaldehyde and glutardialdehyde, but it also makes the counting easier and precise as it stains the phytoplankton dark brown colour. Stomach content preservation studies using different concentrations of alcohol should be undertaken in future.
Acknowledgement
This work was supported by the project NRGS0003, a grant from the Ministry of Higher Education Malaysia to University Malaysia Sabah.
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