Dear Abid,

Probit analysis to calculate LC50 of your insecticide (Finney, DJ 1971).

Sofwares like Poloplus (LeOra software) or SPSS will help you to get this work done.

The attached reference may help you, explained in a simple and effective way to perform Probit analysis.

Good luck.

Abid,

You can estimate EC50 (and other parameters) by performing non-linear regression analysis. If you use R, my suggestion would be to perform this analysis using 'drc' package in R.

First you need a pretest to determine the range of values you need to do dose-response modelling (if you do not already have information about that). You should have a minimum of 5 concentrations for modelling. It would be advisable to use a concentration with maximum and minimum effect, each. Additionally you it would be great if 2 concentrations would be in the approximate vicinity of your EC50, since the sigmoid curve is very steep there. Usually a factor of ca. 3 between concentrations is used (e.g. 0.1,0.3,1,3,10,30 mg/L).

for getting series of concentrations of your plant extract, you presume 1/2, 1/4, 1/8 towards lower side and 2EC50, 4, 8 times towards higher side keeping EC50 in middle. once you have these concen, you can plot the graph or use the software to calculate EC50 values....you can use EC50 calculator software also.

Dear Abid

Please do see this paper

http://jaas.blue-ap.org/wp-content/uploads/2014/03/45-48.pdf

You can calculate it through probit analysis using statistical software, life spss.

Check out the related question on research gate on how to calculate LC50s where there are several detailed answers to your question by myself and several others. POLO Plus is the best program for this type of analysis.

Estimado Abid

Conozco 2 software que calculan CL 50, uno de ellos es POLO, sin embargo este programa funciona bajo el entorno D.O.S, el otro software es el SPSS, en mi perfil publique un articulo al respecto.

What exactly you want methodology for the experiment or formula to calculate EC50 under lab. condition.

Probit analysis in any specialized mathematical software.

Dear Archana Srivastava

Actually, i want to know about the methodology. Especially, the parameter to determine the EC50 values. I know very well that Polo PC or Probit analysis is for such kind of analysis. For LD50/LC50 determination, dose/concentration mortality response is recorded. Could you please guide me regarding the parameter to record the data for EC50 determination.

Thanks

Abid,

You need to get ahold of the J. Robertson book on pesticide bioassays that runs you through the steps and parameters. It can be found at:

http://books.google.com/books/about/Bioassays_with_Arthropods_Second_Edition.html?id=t0QWS_Jn0-cC . This is a more detailed version of what in in the manual of the POLOPlus software program which she also wrote. As a quick generalization, to generate an LC50, you need 5 concentrations that give mortality between 20-90%. You can use a concentration that gives you 100% or less than 10% mortality only once in a pinch. You should run 30-50 individuals in each concentration and check mortality should be below 10%. POLOPLUS is great because it will not give 95% confidence levels on LC levels if there is too much variability or check mortality. Bioassays still report the non-overlap of 95% CL as a criteria for determining if the responses are different, but it is much better to use an option in the program that will use the variance-covariance matrix of the error terms of both lines to generate 95% confidence limits between the ratio of two populations (lines) such as resistance ratios and instar toxicity ratios (see my resistance and synergism papers) which is the correct way for most IPM journals. It can also run synergism comparisons where on compound is harmless by itself (see my recent PLOSOne bee toxicity paper. You really need to get a copy of the book and read it, because if you wish to get accurate LC90 values you need to lump the analyzed concentration towards the higher end of the mortality curve to be above 50%. The Rousch & Tabashnik book I mentioned in the other LC calculation thread is useful for interpreting the results on such analysis.

Dear Abid

Mainly there are two parameters : one is dose/concentration mortality reponse ( that you have already mentioned) and another is dose/concentration Growth inhibition response. According to me, Important things are selection of broad range of dose/ concentration, number of dose/concentration and stage of the larvae taken for the exp.

You may refer to Bioassays with Arthropods, 2nd edition, CRC Press by Robertson et al. 2009, chapters 1-5. Chapter 4 is a guide to POLO sotfware for probit analysis. From my point of view it is the best reference book dealing with this issue

Abid,

Could you tell us what exactly is your observation? Are you looking for mortality of larvae?

or studying any other adverse effect of your insecticide/plant extract on larvae (ex. growth regulation). Are Looking for a particular type of defect after treatment at various doses? as explained by fellow researchers involved in this discussion.

The main purpose is to study the adverse effects of plants extracts/insecticides on the larval growth of the insect of my interest.

So, If I understood your objective correctly, you will be using sub-lethal doses of your toxicants to study their effect on growth of your insect.

The following are generally observed parameters on this aspect (larva to adult stages) in comparison to control.

1. larval weight (at each instars or before pupation)

2. larval stage duration

3. pupal weight

4. pupal duration

5. any deformity at eclosion

6. fecundity of females (number of eggs laid per female)

7. sex ratio

8. longevity of adults (both male and females)

In my opinion there is no limit for any parameters (customize according to your test insect) to observe, however be open to suggestions of other researchers. I too would be happy to learn if any other parameters could be added.

Good luck.

The book, probit analysis by Finney, 1971 will provide all the information you required for determining EC50 of test chemical/bio-compound

Further, for testing the efficacy, you need to prepare a series of doses/ concentrations to test for efficacy against insect larvae and by plotting mortality against log dose/concentrations to find out the dose/concentration-mortality combination where 50% mortality response occurs.

Come on guys, Finney is 43 years old now! Not even sure it is in print anymore. He was the pioneer, but a lot of new statistical techniques and even more importantly are the stats programs that have come out since then so you don't have to do everything by hand.

POLO Plus from LeOra Software has gone through several improvements since it came out about 15 years ago and even better for the novice is that the CRC books by Jacqueline Robertson on Arthropod Bioassays that Dejan cited earlier. It was written for students and will walk you through the setup of experiments, data entry, analysis, and interpretation. One of the most useful techniques in it is putting a confidence limit around a ratio of two dose mortality curves rather than using a rather crude non-overlap of 95% confidence limits. The program uses the variance-covariance matrix from each line to generate this. Having done it by hand as a student, I can attest that this is a real time saver and the most accurate way to determine differences between line whether it be a resistance ratio, synergistic ratio or instar toxicity ratio or most of the metrics that Seenivasagan mentioned earlier. Although some use SAS for probit analysis, I believe at least in the earlier versions there were some problems with their assumptions according to Robertson.

1) IRRI book :Research Methods in Toxicology and Insecticide Resistance Monitoring of Rice Plant hoppers by Heong etal, available on google books is also helpful

2) Polo is also probit / logit based program ...

To know EC50, you have to conduct lab assay and data collected to be analysed using probit analysis.

Very simple. Just determine an 'all response' concentration ie Concentration that gives you over 95% mortality. That is after exposing an adequate number of larvae (>10) and replicating these 3 - times. Then serially dilute (either 10-fold ie 1 in 9 of solvent) this concentration 5-6 time until you get close to a region of 'no response'. I will recommend concentration that induces less than 10% mortality. Convert the concentration to Log10 of dose and % mortality to probit units. Plot probit vs log conc. The concentration that corresponds to the probit of 5 is your LC50 or LD50 depending on the type of assay you used.

hi you use probit analysis for doing this

Www.leora-software.com is the new website for the PoloSuite software. The 3rd Edition of Bioassays with Arthropods will be available this fall.

By using Polo Plus Software, Leora Berkeley, CA using range of LC50/LC90 as standard (Leora, 2006).