Hello everyone.
I'm doing an evaluation of microplastics in several coastal species tissue samples, and what I want to know is what is the volume ratio of solvent:tissue to be used in the digestion process. The articles I read (not all literature, my bad.. my bad) are somewhat cryptic about that. They did mention volumes of solvent mixtures to then put them on tissues, but I'm interested in knowing, for instance, how much volume of KOH or H2O2 is necessary for achieve the digestion of shrimp tissue (such as the abdomen). Some papers mention 10 ml of 10%KOH but seems like is too little, and I found another that mentioned 150 ml. So, is there a precise volume? or you just simply add the solvent until covering the tissue. Or, because the incubation period, even a small volume of solvent is enough for the digestion purpose. Thanks for your time and (hopefully) answers.
Best wishes.
Hi Hiram Herrera - in my experience there is no predetermined volume ratio of solvent:tissue. I have recently digested lobster muscle tissue and used 30mL 10% KOH per ~10g of tissue, followed by filtering and then 10mL of 30% H2O2. I found these ratios by running numerous tests beforehand using different configurations and lobster tissues. You certainly don’t want to use too much or too little solvent, but the amount that will work best depends on many factors such as length of the digestion, how much heat you are applying, whether the tissue has been preserved or is fresh (for example fresh vs frozen vs ethanol preservation has different effects on the proteins within the tissue which can impact how it digests). A good rule of thumb in my experience is to make sure the tissue is completely covered. Sorry I don’t have a more straightforward answer, but ultimately if you’re able to, play around with different ratios and you’re bound to find what works best for digesting shrimp tissues!
Hi Amber LeBlanc , thank you so much for your answer. Indeed, it appears that there is not a predefined ratio, but I will take note of your recommendations and experience. Since you kindly answer my question, and have worked with crustaceans as well, I'd like to take the chance to ask another one: which filter pore size do you recommend to filter the digested solutions?. It appears that is the same case as the volume ratio, some researchers used 0.7 micron micro glassfiber filters, some other used 0.45, 0.20... and so on. Thank you, again, for your attention.
Hi Hiram Herrera, of course, I'm happy to help if I can! The filter pore size you use depends on your research objectives and what analytical equipment you have access to. Also be mindful of greater potential for clogging when filtering through a finer pore size. In my research objectives I set my particle size range to anything less than 8um and greater than 3um, based on the tissue type I am analyzing (muscle tissue) and detection limits of the analytical equipment I am using. I used 2.2um fibreglass filters and saved remaining filtrate (containing particles >2.2um) for potential future filtration using 0.2um silver filters if down the road nanoplastic analysis becomes more accessible. I am analyzing using nile red analysis (detection limit of 3um) and micro-Raman spectrometry (detection limit 1um). Therefore, I don’t have the capacity to analyze anything >1um at the moment and based on limited funds, did not have the capacity to spend more on smaller filters. Hope this helps!
Hello again Amber LeBlanc , well thanks for the answer, it was very helpful the limit of detection issue that I frankly didn't take into account. I was planning to use a filter with a pore size of 0.7 um, under the basis of nothing more that some papers report that size during the digestion of shrimp abdominal muscle. My objective is basically know wether or not there are MP on shrimp abdominal tissue, since is a very valuable food resource locally and regionally, measuring them, classifying by shape, color and then identify them with spectroscopy methods. I have no Raman option but I do have FTIR + ATR, so there's hope. Regarding nile red analysis, I'm not familiarized with that methodology but, to my knowledge, a fluorescence microscope is needed, though I ignore if all of them are suitable. The clogging issue is a really good thing to be aware of, so maybe I'll be working with a wider pore size as a starting point. I really really thank you for the guidance, and I hope your research got much further and interesting as you originally expected. Best wishes!
Would someone please help? I have carried out Fenton pretreatment and that has left out a lot of ferric precipitates on my filter paper. So, i have added 50 to 60 % of sulphuric acid onto my GF filters to dissolve the ferric precipitates. Then, I could see that the precipitates were dissolved immediately and then I have washed the filter paper with plenty of millipore water. But, I would like to know whether this process (less than one min of contact between filter paper and 55 % sulphuric acid solution) would affect the chemical nature of my microplastics? Observation in microscope suggests that discolouration has occurred. But I would like to know does any of you has encountered similar situation? What is your recommendation? Thank you so much.
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