Q1: 25

Q2: No, you need the propotions as probit values

Q3: It does not make sense to look for gene expressions after dosing with sublethal concentrations - I would use concentrations that do not cause lethality

Fiarly straightforward answer to this issue.  Toxicity is about time and dose.  For LC50 determinations, the number of starting organisms is the number to use.  One can calculate LC50s at each time point to gain perspective on the temporal pattern.  As time goes on, complete kills simply enter in to all calculations as "0 survivors" at those time points. 

As to the question about whether all animals should die in such an experiment, absolutely not.  To establish a good dose(exposure)-response curve, you ideally desire two partial kills, a complete kill and a no kill.  If controls die, this is in indication that your test system may be flawed or your organisms are unfit for testing and there are procedures to adjust the percentages in the dosed treatments (e.g., Abbot's Correction).  Certain LC50 statistics require 2 partial kills (Probit), or can handle 1 partial kill (binomial), or 0 partial kills (all-or-none responding, Spearman-Karber).  Consult a statistician if needed,

As for your last question, it all depends on what the question is.  If you lack perspective on the importance of exposure response to yuour non-lethal endpoint being derived from the gene expression experiments, then multiple treatments is wise.  However, if you drop to one treatment with a control, you are basically asking a yes/no question once again.   To rely on one single LC50 test to help set this up can be ambiguous. LC50 tests themselves will vary and there is an error term involved.  You ought to really understand the implications of this before proceeding.

Dear Sirs,

Thanks for answering my questions. I shall sure follow the guidelines which Dr Ali Adil recommended. Dr Thomas Shall i use SPSS to calculate Probit analysis? Dr Scott Thanks for the clarification, much obliged  

Hi.

Question 1: like one of the previous respondents, the total number of animals is the number you started with which is 25. Sine you’re interested in the LC50 (dose/conc. that will give 50%), you record mortality daily. At the end of 72hours, mortality which you will use for calculation will be the total. no of dead animals (recorded cumulatively over 72hours) in each dose and in duplicates or triplicates divided by the total no of animals exposed at the beginning of the experiment. Your LC50 can be calculated using probit analysis available on spss software. I believe the link was sent by a previous respondent.

Question 2: yes, it is necessary that the experimental animals die but not all of them. Reason is because you are interested in the concentration that will be letal and not an EC50 (which would be a concentration that will cause/elicit any other response other than death). However, ideally the no. of deaths of experimental animals should increase with increasing concentration of your toxicant. Though, this is not always the case but that is the kind of result you would require to obtain a fairly good LC50. Hence, before the actual acute test, it is expected that you would have conducted a preliminary test with a wide range of doses in order to obtain the best concentrations to work with. Your lowest concentration should cause at most 5% death while your highest concentration should cause 95-100% death.

Question 3: For gene expression analysis, it depends on your research question or hypothesis. If you’re interested in working with environmentally relevant doses or near environmentally relevant doses of your toxicant (if it is an environmental pollutant), you might need to go lower than the LC50 in folds of say 1/10th, 1/100th or 1/1000th depending on your knowledge of the level of such a toxicant in the environment. This is called the sublethal concentration. It means, you are working at concentrations that will not necessarily cause death but will induce or elicit some other response in your test animal which is what your gene expression analysis and other tests can assess. On the other hand, your research question could be that, you want to investigate the kind of gene expression in your tests animals at the LC50 or near LC50 values obtained. This could provide information on the kind of genes expressed in response to toxic or lethal doses of your toxicant and their levels, etc. this is like investigating gene responses in the pathology of your toxicant on your test animals (the ones that survive by the way and not the dead ones).

P/S: Always have controls (test animals without toxicant)

I hope these answers help in addition to the brilliant answers proferred by the previous respondents.

All the best.

Dear Dr Temitope

Thanks for your valuable reply. I am working with nanoparticles. Is there any database that provides the details about permissible level of nanoparticles in environment? Regarding gene expression my research question follows your second question that gene expression at at the lethal concentration of my test animal.

Thank you

Hi.

I'm not sure if there's any regulatory data on permissble levels of nanoparticles in the environment (you can look it up on some databases like google, etc) because they are in the class of "emerging environmental pollutants/contaminants".

However, what you can do is to research or look up published articles on level of nanoparticles detected/detectable in the environmental matrix (air, soil, water,...) you are interested in and then you can use that as a basis for your tests. If there's none, it is best to thus work with sublethal and or near lethal doses of the nanoparticle(s). 

Regards.