WEF Discussion Forums
Laboratory Management and Technical Issues
DO meter Calibration help
We currently use our DO meter (HACH LDO probe) for reading the DO of samples (temps range between 18-27oC) and also for BOD measurements. We have only started doing BOD testing and I am having problems with the blank reading. Our lab is not temp controlled and I think the high blanks are due to the probe calibration. We normally do a water saturated air calibration daily (1 inch of water in 300ml BOD bottle shaken for 1 minute and read straight away) and then do a zero calibration weekly (300mg of Sodium sulfite in 300ml water). These calibrations are carried out at whatever temp the lab is at in the morning as there is no way of regulating the lab temp. Should we be doing a calibration at lab temp for the DO reading of the samples (usually at around this temp) and then perform a second calibration using water stored at 20oC for the BOD testing?
Also, we have a specified DO that our samples should reach (3.5mg/L). The meter used in the lab has a built in Temp/pressure probe but I notice that the DO reading of the same sample will vary at different temps. Shouldn't we then be asking for a specified DO reading (ie 3.5mg/L or above) when the sample is at a specified temp? Hope this makes sense?
Hi, Tia. I think that the Hach method for use of the LDO probe will answer most of your questions except maybe the "3.5 mg/L" thing in your second paragraph. Whatever it is you are talking about there is new to me, but I will try to make some sense out of it. You can find the current Hach LDO method at...
...but I don't think it will be much help to you. It talks about an "accuracy check" and speaks of 8.0 and 15.0 mg/L standards, but doesn't tell you where to get such standards (to the best of my knowledge, you can't). I think you would be better off to review the old (original) method. The PDF is too big to attach here, and I'm too dumb to figure out how to cut it down to a size that will fit, but it's available on my web site. If you want to check the original method, go to
You will find a link toward the bottom of the page.
Take a look at para 8.3.1. which is the calibration check that must be done with each batch. It is NOT done in water saturated air...the method refers you back to para 6.2 which tells you to completely fill a bottle with water and test it. Again, you have to know the actual temperature and pressure, and refer to a table listing DO saturation levels for various temps and pressures (the only variable in method 5210B is temperature and it assumes one atmosphere of pressure (760 mm). If your lab temperature varies widely, I suggest that you keep your reagent grade (distilled, DO, or whatever) in the incubator (at 20°C) overnight, add your nutrients/buffers the morning of the BOD test by gentle swirling so as not to create air bubbles, stick it back in the incubator for an hour with the top not on the bottle, allowing it to come to equilibrium and keeping it at 20°. On Day-5, the blank bottle (and other bottles) will be at 20 degrees when you take them out of the incubator. By doing that, you should eliminate the temperature problem as far as the blank goes. But don't overlook the temperature requirements for all other bottles as stated in the method...all must be at 20 degrees ±1 (or ±3 in the 21st edition).
By the way, the old Hach method refers in several places to "room temperature ± 2 degrees. They don't define what "room temperature" means. Would it mean that if your room temperature is 28° you could do a calibration check at anywhere between 26 and 30°?? I don't think so!! I think they meant to say 20° ±2 degrees. They avoid that dilemma in the new method by not saying much about temperature (or pressure).
Now, to your second paragraph. I have never heard of any lab worrying about 3.5 mg/L with regard to anything to do with the BOD test. The method requirement is that a sample deplete at least 2.0 mg/L, and leave at least 1.0 mg/L at Day-5. Where 3.5 mg/L came from, I don't know. But regardless, you are absolutely correct in stating that you see differences in DO levels according to the temperature...that's why the tables I mentioned above exist. (If you don't have a DO table that includes both temperature and pressure, reply to this post saying you need one and I will send it.)
Good luck with whomever told you the DO levels have to get down to 3.5 mg/L...I'm sure they told you "we've always done it that way" but that doesn't mean anything if it's faulty advice!
I, too, have used the Hach LDO for a long time...LOVE it. What I also do to make sure the temps are all consistent is to store the LDO meter and probe in the incubator, along with the dilution water, and then calibrate the meter right in the incubator. That way, everything is done at 20 degrees from the get-go.
Thanks for your replies. Im still a tad confused on some points though (not the brightest spark) as it appears there are 3 HACH methods for calibrating the LDO probe and they are all different:
1. HACH Method 10360 - 1/4 inch water in 300ml Bottle, shaken vigorously for ~30S, left to stand 30 minutes (stopped) then probe inserted (I imagine it must be dried) and calibrated.
2. HACH Method 10360 (same method but different DOC completely) - DOC316.53.01243 - 1cm of water in a 300ml bottle, shaken vigorously for several minutes, remove stopper and insert probe (dried), Alow several minutes for probe to reach equilibrium, press calibrate.
3. HACH Method 10230- DOC316.53.01242 (for BOD) using LDO probe - add 225ml of water to 300ml bottle, shake vigorously for one minute, insert probe (dried) and press calibrate.
I like the idea of calibrating in the incubator (I assume your water is also at 20oC) but which method is the best? I asked the HACH rep and he was pretty vague and basically said you could do any of them? He also said that I should do an air calibration of the probe at least daily and a zero calibration (using at least 300mg sodium sulfite in 300ml bottle (plus DI H2O) so I use 400mg. I do the zero calibration after the air calibration so the sodium sulfite residue wont interfer with the air calibration and then I just wash the probe for several minutes afterwards. Is it necessary to zero the probe so often as I dont see it mentioned much?
As for the 3.5mg/L DO - Im refering to a minimum DO that our samples should reach for quality indications (along with other water quality parameters) - it is not a guideline but an indication of water quality of our field effluent (nothing to do with BOD). Since our technicians in the filled are all over the place (different temp and pressure), is it acceptable to have a flat DO value, as wouldnt the temp of the sample effect each reading (I have never used the DO method the technicians use in the field but its a simple method based on titration). Sorry for all the questions but we have just started BOD testing and my Blanks are ridiculous and Im trying to figure out the problem (starting with the probe calibration!).
Anonymous, if you could tell use exactly what you do in setting up your blanks, we might be able to help you. There is a good chance that the bad blanks you are experiencing have nothing to do with meter calibration, but a lot more information is needed to make a reasonable guess on what might be causing them.
Why don't you tell us the following as a start...
What source water do you use (e.g., distilled in lab, DI, RO, purchased distilled, whatever)?
How do you prepare the dilution water (e.g., when and how do you aerate your source water...when and how to you add the buffers and nutrients...do you "store" it...how is dilution water poured into the BOD bottles (if you are using the bottle method)...etc.
What are your typical Day-1 DO levels for the blank? Day-5?
What is the average of your last 20 GGA results (for single bottles, but if you do more than one GGA per batch, give all values...not the average.
What is the standard deviation for those same 20 results?
You may wonder why you should be concerned about the GGA results when your big problem is blanks. The blank results can have a large impact on you GGA average, and they can also affect the standard deviation of the GGA results. If your standard deviation is small (say <15 mg/L), bad blanks are almost always caused by the source water, for example.
And it would be helpful if you let us know who "Anonymous" is by adding your e-mail address at the end of your posting (once you register with WEF...a freebie...we can send you e-mails without knowing your actual e-mail address, so you might want to register instead of adding your e-mail address to your postings).
Ok, here goes....
Dilution water prep- made by adding 3L of distilled water to a 5L carboy and incubating with a perferated foil cap O/N in incubator (I originally was shaking this water prior to incubation but stopped as blanks were out and thought I might be supersaturating it). Then 1 hr prior to BOD prep, I remove the carboy, add a 3L nutrent buffer pillow (HACH), invert the carboy end over end a few times to mix and pop the carboy back in the incubator.
1 hour later, I remove the dilution water, give it a shake and pour it into a BOD bottle to the neck (glass 300ml) - I used to pour it straight from the carboy but now pour it into a beaker first then into the carboy (the beaker is washed in the same manner as the BOD bottles ( soaked for 1hr in phosphorus free detergent and hot water, scrubbed, rinsed free of bubbles, acid washed overnight with tap water and sulfuric acid to ph<,then rinsed well with Distilled water and dried in oven). I try and pour it down the side of the bottle so as not to introduce bubbles.
As for BOD readings - Blanks are always in the mid 8s 8.56 is the average.
on removing the bottles Do is high 7s around 7.8 -7.9 usually. We have also had to change the incubation period to 7days due to staffing issues but we were getting errornous blanks at 5days also.
We have only just started running the test a few weeks ago and are trying to fine tune our SOP (I can email you the draft SOP if easier), therefore we have only run one GGA standard (we were waiting on the chemicals which is why we have more blank data than GGA). Anyway, the GGA was ok, around 210 from memory. We are not seeding our samples but use raw inffluent (settled O/N in incubator) for the seed. Our samples have sufficient seed (not disinfected) and results appear to be inline with what we are expecting (regardless of blanks being out).
Another question I had was when I make up the sample dilution, The BOD bottle actually holds 310ml. I require the sample to reach the bottom of the neck of the bottle so the probe is in sufficent water but then when I stopper the bottle, a small amount of the sample is dispelled into the lip of the flask. I dont really know how to avoid this though and just top it up with Distilled water and cap. I am registered (Tia) see first post, but just havent logged in to reply.Thanks so much for your help.
comments on your blank issues:
some bottled distilled water (if you haven’t already) to compare to your
existing source water. We have been using Poland Spring distilled for the past
couple of years with good blank results.
a calibration log for your meter and record temp, pressure, initial DO,
calibrated DO and % DO saturation. If you see wide variations in your
day-to-day difference between initial and calibrated DO review your calibration
method to make everyting as uniform and standardized as possible to reduce
We shake our dilution water before putting in the incubator, swirl to mix
buffers on day of test but never shake it on test day. It should be at
saturation after a day in the incubator with a loose cap or perforated foil
bottle cleaning method seems to be overkill. A good detergent wash followed by
tap & distilled water rinse should be fine. Acid wash if you see water
droplets inside the bottle after the distilled rinse.
fill BOD bottles to the frosted stopper line, insert stopper and cap the
don’t care about staffing issues. BOD is a 5 day test and I can’t imagine any
agency signing off on a 7 day incubation period. Even if you’re doing the test
just for process control the BOD-7 seems pretty useless. If you don’t have
staff you can send it out to a commercial lab…that will take care of the blank