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BOD blanks
Posted: Monday, June 28, 2010 10:45 AM
Joined: 6/28/2010
Posts: 3

For the past month, we have been having varying degrees of depletion of blanks, ranging anywhere from 0.08 to well over 1.0.  We have acid rinsed the bottles, scrubbed bottles, tried different buffers and even bought new bottles and carboy for dilution water.  Any suggestions??

Perry Brake
Posted: Monday, June 28, 2010 10:27 PM
Joined: 12/16/2009
Posts: 69

You need to give us a little more information before anyone can make a reasonable guess at what is happening, st77.



Has there been any change in the procedure/equipment/analyst/reagents/source water since that coincided with the bad blanks?


What was the previous range of your blanks?  Both positive and negative (i.e., did some bottles show excessive increase in DO)?



Did your GGA results change at about the same time the blanks did?



What was the average for 20 GGA results before the blank problem, and for however many you have done after the blank problem?


Same with standard deviation of 20 GGA results before, and after.


Has there been a significant weather change that coincided with the blank problem (atmospheric pressure plays a big role in measurement of DO)?


Have you done any blanks in duplicate?  Results?


Have you incubated any bottles containing only reagent grade (i.e., "source") water?


Answer all those and anything else you can think of that might be a possible cause and maybe somebody can suggest a cause and possible fix.


Posted: Wednesday, June 30, 2010 9:10 AM
Joined: 6/28/2010
Posts: 3

Hi Perry-


We hadn't made any proceudre, analyst, reagent or source water changes before the bad blanks started occurring.  Since, we changed the preparation of the dilution water.  I was reading through your bugs eye view book and saw that it suggested preparing the dilution water 24 hours before use, so we started doing that and that seemed to make no difference.  We also changed the lot of buffer we were using, which also seemed to make no difference.


The previous blank depletions were in the range of 0.02 to 0.2.  We did not have any that had an excessive increase in DO.


The GGA's where fine when the bad blanks started occurring, with the range of 198 +/- 15 mg/L.  Our past few have been consistently 145 mg/L +/- 5 mg/L, and we are running a test today to see if it had to do with our pipetting technique.


There hasn't been any significant weather change in the past month that I could tell of.  We are using the LDO meter from Hach which automatically adjusts for temp and barometric pressure.  I have been checking the calibration against the proble calibration table and it has been spot on.


We did incubate a few bottles containing just source water.  The results were 0.31, 0.09 (duplicate), and then another batch results were:0.39 and 0.27 (duplicate).  We currently use distilled water and are going to try another manufacturer today.  I assumed that with the first results being so far off, that it was a bottle issue, but perhaps I am wrong.


Hopefully, this has cleared up some things.

Perry Brake
Posted: Wednesday, June 30, 2010 6:05 PM
Joined: 12/16/2009
Posts: 69

Thanks for that additional information, st77.  I don't see any glaring indications of what might be causing the blank problems, but there are some things worth mentioning.


First, "A Bug's-Eye-View" doesn't suggest preparing dilution water 24 hours in advance.  It suggests that if you are doing BOD, you may aerate the source water (e.g., distilled, DI, RO, etc.) and place it in the incubator to let it equilibrate its oxygen level for 24 or more hours, then add the nutrients the morning of the test.  It also says that if you are doing CBOD, you may store the aerated dilution water in the incubator overnight.  Storing dilution water over night (or longer) encourages the growth of nitrifiers...not good.  But with CBOD, you inhibit the nitrifiers, so it doesn't matter if they are there.  The method mentions this, by the way.


You say you don't have "excessive" increase in DO" [in the blanks].  Can you be a little more specific.  Do you have any -0.1?  -0.2?  This is important because there are only a couple things that can cause an increase...or an apparent increase...in DO during the incubation.  Photosynthesis can cause an actual increase.  Improper calibration/use of the DO meter can cause an apparent increase in DO (unlikely with your LDO, an excellent system).  Temperature problems can also cause an apparent increase in DO, and this is the most frequent cause of negative blanks.


If you mean by "...198 ± 15 mg/L..." and 145 ± 5 mg/L", that your GGA values were in the range 0f 183 - 213 mg/L, and now they are in the range 140 - 150 mg/L, the standard deviation of your GGA results would be VERY low...<10 mg/L before, and <5 mg/L now, the former indicates EXCELLENT precision, and the latter indicates...quite frankly...UNBELIEVABLY good precision.  Why don't you calculate the actual standard deviations, before, and a second standard after you get 20 current values.


Your last paragraph indicating significant depletions for just plain old source water indicates a significant problem, and whatever is causing it, is probably the problem for your dilution water as well.  There are (hopefully) no nutrients, organic material, or oxygen consuming bacteria in the source water, so the most likely cause for a significant depletion is that your source water is supersaturated when you put it in the incubator.  With a tiny microphone, you might be able to hear that excess oxygen (actually air) escape as a little "pffftttt..." when you open the BOD bottle after incubation.  Concentrate on making sure your source water is just saturated by aerating (the day before if you want to, but the very first thing on the day of the test, if not), and after you do the last thing to it (which might be adding the nutrients on the morning of the test), let it sit for at least an hour before using.  Every time you let it sit...in the incubator or wherever...the cap should be place lightly on top of the container opening,  but not tightly fixed, so as to allow air in and out.


And one last thing.  You say you are using bottled water.  I hope it is bottle distilled water, and not distilled drinking water (which has chemicals, often ozone, in it).  You might also try "steam distilled" distilled water.  "A Bug's-Eye-View" discusses its advantages.





James Royer
Posted: Thursday, July 1, 2010 8:39 AM
Joined: 9/21/2009
Posts: 98

We always age our source water for about 2 months if we have enough room so that it is always stable.

Posted: Thursday, July 1, 2010 9:16 AM
Joined: 6/28/2010
Posts: 3



I probably should have been more clear on the dilution water preparation.  We aerate just the distilled water for about 15 mins and then let that sit in the incubator overnight to stabilize.  About a hour before our BOD setup, we add the nutrients and just swirl the container to mix and let set again until we are ready to use.


The source water is bottled distilled water.  I haven't seen any increase in DO in the blanks.  We also took out our old meter as well and are running everything in dup, just to make sure we aren't having a meter issue.  Today we are also using another labs source water, just to see if that makes a difference at all.


I will keep track of the next tests of GGA results and calculate the standard deviation for that.  The past few, although low, have been very precise.  We ran a 4mL sample GGA and 5 mL sample GGA in yesterdays batch and we will see how those turn out on Monday.



Thanks again for taking the time to help me out with this.


Eric Rajaniemi
Posted: Wednesday, July 7, 2010 10:34 AM
Joined: 10/7/2009
Posts: 10

I would ask how you deliver your air when aerating the water?  Are you possibly introducing bacteria via that vector?  Another vector you are already checking out:  source water.  I would point out that anyone using Hach nutrient pillows probably will experience the occasional D.O. increase in their blanks.  It is to be expected, occasionally. 


Check to be sure that your probe does not introduce any contamination into the blanks.



Brent Dickey
Posted: Friday, July 9, 2010 1:58 PM
Joined: 9/28/2009
Posts: 18

Good call on the possibility of probe contamination. We use our probe to run MLSS SOUR analyses and quickly learned not to overfill the bottle when running SOURs because it forces the nasty stuff up into nooks and crannies of the probe that don't get rinsed off. It ended up in our BOD bottles causing bad blanks.


I've never been a fan of aerating any sample by air pump to either reduce or increase DO saturation. Way too slow and marginally effective when I tried it. A few good shakes of the carboy prior to incubating our dilution water overnight (or several days) always gives us good initial DO results. Shaking a 2-liter jug half full of supersaturated effluent for 30 seconds will always reduce it to saturation within about 15 minutes.

Posted: Wednesday, July 28, 2010 1:28 AM
Joined: 7/28/2010
Posts: 1

dedicate bottles specifically for method blanks to prevent x-contamination from unknowns. the purist will say NO!!!!!, but if you want to make life easier, have bottles dedicated for blanks, seed, gga, and a set for unknowns.

Pamela Schweitzer
Posted: Wednesday, July 28, 2010 8:59 AM
Joined: 10/5/2009
Posts: 6

st77 you mentioned that you use bottled distilled water.  Here is another item that you might want to look at.  I have noted similar problems with several labs that I oversee and the problem seemed to coincide with the onset of summer heat.  We tried many things, but what seemed to do the trick was in the storage of the bottled water.  Several places stored the water in a garage area or similar that was not air conditioned.  I think when the plastic bottles heat up they may release chemical into the water that has oxygen demand.  When we moved the cases of bottled water into a storage area that was air conditioned the problem went away.  I had another facility that did store the bottle water in AC area but still experienced problem.  There we found the store they purchased from did not have AC in their storage area.  So before onset of summer, they now purchase enough cases of bottled water to see them through to fall as they have limited sources of where to purchase.

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Posted: Sunday, June 26, 2011 5:23 PM
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