VDK+ Test Kit: What does it mean when you see a dark circle?

First off, make sure you take photos so that we can help troubleshoot! There are some troubleshooting steps we can take to diagnose what is happening. It is possible that this is a real result. 2,3-pentanedione and/or acetoin, which are vicinal diketones (VDK's) similar to 2,3-butanedione (diacetyl) sometimes show up as a dark circle, especially with certain types of yeast. You won't necessarily detect any diacetyl via sensory because 2,3-pentanedione and acetoin are not as potent as diacetyl. However, before we can say that, I want to make sure everything else about the test is running smoothly.

• Are you writing with Sharpie on the lid/cap? That can bleed into the filter paper.
  
• Have you run a water blank? Run the test with 10 mL of distilled water (or even tap water). Do you still see the dark circle?
  
• When you see the dark circle, can you detect any diacetyl sensory-wise? That will help lead troubleshooting.
• Is the height of the water in the water bath above the level of the liquid in the vials?
• Is the dark circle occurring in real beer samples or samples with controls? If controls, what controls are you using? We have found that controls from Aroxa do not actually contain diacetyl and manifest as that dark circle because they are using one of the other VDKs mentioned above.
• Have you measured the actual water temp of the water bath? I have seen the dark circle when the water bath is hotter than 70C.