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Informed Discussion of Beekeeping Issues and Bee Biology <[log in to unmask]>
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Thu, 3 May 2012 17:33:12 -0400
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[log in to unmask] (mailto:[log in to unmask])   writes:

Wiping  memory
 
Allen, I haven't thought of this for years.  When I first started  doing 
bee research, I was heavily involved with developing test protocols for  EPA.  
At the time, Larry Atkins was the EXPERT in contact toxicity  testing.  He 
developed the protocol published in the US Federal  Register.  Basically, 
you put the bees to sleep, administer a drop of  carrier containing a known 
amount of a test chemical, place the dosed bees in a  cage in an incubator, 
monitor longevity.
 
When this type of testing began to go to private labs in the  US, Larry's 
protocols got changed - mainly for cost  savings.  One of the major private 
bioassay labs initiated  the change from using CO2 to nitrous oxide, and they 
used it over and over  for each trial.  Every time they handled the bees, 
they hit them with  it again. (I'm guessing their techs didn't want to get 
stung or  chase bees).  Problem is, their control bees had higher losses than 
my  testing.  So I called Larry.  In fact, we had several  conversations.  
He pointed out several problems with the protocol  modifications.  There were 
five real issues:
 
1) All tests were based on groups of bees from a SINGLE hive.  Larry's  
protocol called for a minimum of 3 sets of bees from each of 3 colonies for a  
total n of 9.  However, subsamples of bees from same hive are arguably  not 
true replicates.  So, the power of his test may not be as great as he  
supposed.  However, conducting a pesticide label registration on a single  hive 
really does strain the definition of replicates.  As Larry said, we  know bee 
colonies vary in their susceptibility to any given pesticide, it is  
probably genetic.  Using a SINGLE colony for the whole test runs the risk  of 
having a particularly susceptible colony, or a resistant colony, and just  maybe 
its actually representative of most colonies.  Whatever, it's bad  science.
 
2) They used tenereal adults (bees just emerging from pupal cells), rather  
than bees of mixed age.  Tenereal bees aren't fully developed, still need  
pollen, and ARE not representative of field bees.  Larry wanted all ages of  
adult bees in the bioassays,  That's what happen in real colonies, bees of  
varying ages are exposed.  Using only young bees does not represent what  
happens in the field, where the foragers (old bees) are likely to be the 
first  points of exposure, and if these bees don't die in the field, they bring  
the  chemical home to the hive bees, and it often gets distributed  
throughout the colony.
 
3) Larry used CO2 once and only once, and he put the bees under from less  
than 60 seconds.  The private labs used nitrous oxide when they removed  
adults from brood frames, when they administered the test compound, when they  
transferred the bees to holding cages, and they knocked bees out for several 
 minutes at a time.  To this day, I don't know why they switched from CO2,  
nor why they repeatedly put the bees to sleep.
 
4) Larry used micropippetes and a bare minimum of carrier solvent to  
administer the drops of pesticide for the contact bioassay.  The private  labs 
used LOTS of carrier by comparison.
 
Add all of this up, and the private labs had high losses in their  
controls.  Larry and I strived to hold control losses to 5% or less.
 
 
I got the bee sleep time down to below 60 seconds, with a goal to hold  it 
to 30 seconds.  I could knock down the bees, dump them on a grid,  place a 
drop on each of 30 bees, and get them into their holding cage before  they 
woke.  Looked a bit like I was on speed, but it did the trick.  
 
PS, any bee that stopped breathing BEFORE the drop was administered was  
eliminated from the test.  Handling plus C02 can be rough on bees.  We  dusted 
bees with soft brushes off frames, off our sheet into cages.  Never  
vacuumed them, but others just suck them up.  And, not  surprisingly, they get 
high losses of controls.
 
This type of testing is VERY sensitive to handling technique.   When you 
can hold your control losses over the duration of the trial to  below 5%, 
consistently, you've arrived.  That's what Larry and I strove  for.  Sometimes, 
one has to accept higher, but 5% is achievable.
 
Jerry
 

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