The Curious Beekeeper archives

September 2014

No September Article

 

August 2014

Sampling for Pesticide Residues, Part 1:

Deciding What to Sample

by TIMOTHY J. BROWN and SUSAN E. KEGLEY, PhD
Pesticide Research Institute

(excerpt)

Introduction
In the last Curious Beekeeper article, we talked about the different kinds of samples that could be taken—bees, pollen, wax, honey, plants, soil, water—and described the advantages and disadvantages of analyzing each. In this installment of The Curious Beekeeper, we provide logistical guidance on taking samples from your apiary without contaminating the samples through contact with other materials or another sample. We also outline best practices for documenting the process and provide insight on interpreting and using sample results.

Documenting the Process
Properly documenting the sampling process is essential to drawing meaningful conclusions from your results. Although it is not guaranteed that pesticide residue analysis will solve your honey bee mystery, improper or inadequate documentation will very likely diminish the utility of your results.

One of the most important things to do when you are sampling hive materials is to compile detailed information concerning your samples. Document the reasons for sampling, the collection date, sample location (as detailed as possible), and the type of samples taken (pollen, wax, brood, bees, honey, nectar, or plants). Also record any other relevant observations about the bees, the brood, the queen, hive strength, recent pesticide applications, the crops within flight distance, and/or anything that may be relevant for assessing what went wrong with the hive.

A sample name or number should be assigned to each individual sample. This step helps to ensure proper correlation of results from the laboratory to a specific sample. More detailed information for a given sample name should be recorded in your personal records (described above), but not shared with the lab. Make sure that the sample name is consistent between your records and the materials you send to the lab (see below).

The final documentation step is to fill out the Chain of Custody (COC) form provided by the laboratory doing the analysis. Most labs have one that can be downloaded from their web site. The COC provides a list of the samples contained in the shipping box by sample number and sample type (pollen, wax, bees, etc.), as well as the type of analysis requested. The COC form also is a legal document providing documentation of the movement and location of the samples from the time they were taken until the lab receives them, and includes the date the samples were taken from the apiary, shipped to the lab and received by the lab, the signature(s) of the people who have handled the samples, as well as information on how the samples were handled and stored (preferably kept cold at all times to prevent degradation).

If there is a legal challenge related to a bee-kill incident, the COC form provides essential documentation of sample handling. Samples taken by the beekeeper may not be accepted by enforcement agencies or by a court as valid even with a COC, but if there is no COC, the results will certainly not be accepted as valid.


Clean Sampling Technique is Critical

If you wish to obtain an accurate sampling of pesticides inside the hive, it is critical to ensure that the sample is not contaminated through the sampling process. Potential sources of pesticide contamination include the hive body, beekeeping gloves, the hive tool, and sampling tools. Beehives that are placed in orchards and near fields are subject to spray drift or direct sprays that contaminate the outside of the hive. Pesticide residues can be transferred from the hive body to gloves when the beekeeper touches the hive. The hive tool and sampling tools can transfer pesticide residues between samples.

The potential for contamination can be greatly reduced by protecting your work surface with a clean (unscented) plastic trash bag, wearing single-use latex or nitrile gloves over beekeeping gloves when taking the sample, and cleaning the hive tool and any sampling implements between samples with rubbing alcohol.

It is usually easier to keep a clean workspace indoors, so preliminary sample collection should be done in the apiary by taking a frame out of the hive (for pollen, wax, brood, and honey samples), shaking the bees off and placing the frame in a clean plastic trash bag to take home for further processing. Adult bee samples can be taken directly in the apiary by placing them in a Ziploc® bag, but avoid mixing twigs, grass or dirt into the sample. If you cannot process the samples right away, freeze them as soon as possible to preserve them.

Sample Preparation
Proper sample preparation begins with labeling and requires attention to detail. Careful labeling of the sample containers with indelible ink will ensure that different samples are readily identifiable and the results can easily be correlated to a particular apiary and hive. The sample name or number should be indicated on the sample container(s), the COC form, and any personal records for reference later in the sampling process (see above).

It is equally important to provide sufficient material for analysis. Most labs will be able to analyze samples as small as three grams (about a tablespoon of pollen, honey, or compressed wax, see Figure 1), but providing more ...

 

July 2014

Sampling for Pesticide Residues, Part 1:

Deciding What to Sample

by TIMOTHY J. BROWN and SUSAN E. KEGLEY, PhD
Pesticide Research Institute

(excerpt)

Introduction
After the large bee kill in almonds this spring, many beekeepers were interested in finding out more about what role pesticides might have played in the losses. In this installment of The Curious Beekeeper, we provide some guidance on sampling for pesticide residues, with a focus on what type of sample should be taken, either from inside the hive or from the environment in which the bees may have been exposed. We also provide perspective on the advantages and disadvantages of the different sample types. In a subsequent article, we’ll talk more about the actual logistics of sampling, analysis, and interpretation of results.

Why Take Your Own Samples?
If pesticides are suspected as the possible cause of a bee-kill or hive failure, sampling and analysis for a range of pesticide residues will help determine what pesticide or pesticides were responsible for the kill. The results could also indicate that the hive failure was unrelated to pesticide use, which allows the beekeeper to focus on other possible causes.

If you have reported a bee-kill incident to your local Agricultural Commissioner or Department of Agriculture’s enforcement office (the State Lead Agency), they will want to take their own samples. In general, enforcement staff will not use samples taken by beekeepers for enforcement actions. However, if they cannot visit the apiary until a week or two after the incident, it is possible that the pesticides that affected the bees will have already been degraded by microbes, sunlight and oxygen, especially in hot weather, and the results may not provide an accurate accounting of the pesticides that caused the damage. The best approach in this case is to take samples as soon as you notice something wrong and store them in the freezer until they can be analyzed. At a minimum, take a frame containing pollen from one of the affected hives, place it in a plastic trash bag and put it in the freezer. If the State Lead Agency staff also takes samples, you will both have a point of comparison.

Another reason to take your own samples is that you can select a lab that will analyze for a wide range of pesticides, with the ability to detect low concentrations. Your local State Lead Agency might have access to a lab that can analyze for only a limited number of pesticides, and/or has limited ability to detect low levels of pesticides. If the pesticide that caused the hive failure or bee-kill is not included in the analysis or the detection limits are too high, the results may be presented as “no pesticides were detected,” and the State Lead Agency would likely dismiss pesticides as a possible cause. But in fact, it may have been that their analysis wasn’t specific enough or sensitive enough to see the pesticide in question.

In any event, it is critical to take samples as soon as you notice a problem and get them into a freezer as soon as possible.

What to Sample?
You go out to check on your apiary and find many dead bees on the ground in front of the hives, with some of them still alive, but twitching and spinning on the ground in front of the hive. Pesticides are a likely suspect. The question is, what do you sample—Bees? Pollen? Wax? Honey? The plants that were sprayed? In an ideal world, you would be able to sample all of the above, but with each sample costing over $300 to run, some choices usually must be made.

Bees: A bee sample is easy to take, but only provides information on pesticides that the bees were exposed to in the very recent past. Sampling bees is best for very high exposures, such as when bees have foraged on flowers contaminated with dust from the planting of treated seeds that contain high levels of pesticides. For any bee samples, it is essential to sample them as soon as possible, preferably while they are still alive and twitching in front of the hive.

There are several disadvantages that make bee samples not the

 

June 2014

 

Metabolites and Breakdown Products:

The Enduring Legacy of Bee-Toxic Pesticides

by TIMOTHY J. BROWN and SUSAN E. KEGLEY, PhD
Pesticide Research Institute

(excerpt)

Introduction
When it comes to the safety and efficacy of pesticides, the parent pesticide generally receives the lion’s share of attention from researchers, the chemical industry and regulators. This isn’t too surprising given that the mode of action for pesticide products is intimately tied to the chemical and toxicological profile of the active ingredient. However, pesticide active ingredients do break down in the environment to one or more different chemicals. While some degradation products are less toxic than the parent chemical, higher toxicity degradates may also be generated. This means that, when evaluating the impacts of pesticides on honey bees, it is important to know exactly what chemicals are present.
In some cases, foraging bees are exposed to toxic mixtures of the active ingredient and its various degradates. Inside the beehive, stored pollen or nectar that was brought into the hive containing a single pesticide active ingredient may later contain a mixture of the active ingredient and the degradation products that formed over time. This mixture may pose a significant risk of colony impairment for hives using stored food sources during fall and winter months.

In this installment of The Curious Beekeeper, we delve into the intricacies of pesticide degradation products—what they are, their relative toxicity to honey bees, and implications for monitoring pesticide residues in the environment—for several widely used, bee-toxic pesticides.

Pesticide Metabolism and Degradation
All pesticide active ingredients, whether sprayed on the leaves of plants or applied as a soil drench or seed dressing, will degrade in the environment through one or more mechanisms.1 Degradates that are produced by metabolism of the active ingredient (AI) by animals, plants, and microorganisms such as soil bacteria and fungi are called “metabolites.” In addition, a variety of chemical degradation processes occur in the environment by reaction with water (hydrolysis), reaction with oxygen in the air (oxidation), and by interaction with sunlight (photodegradation).

The chemical reactions that transform the parent pesticide into its degradation products can be classified into two general types (see Figure 1). In the Type 1 process, the parent AI gains a new appendage (known in chemistry-speak as a “functional group”). In the example below, this is represented by the yellow star. Also considered in this category is a change in the number of bonds between carbon atoms. Alternatively (Figure 1, Type 2), chemical bonds between parts of the parent molecule may break to form two or more separate molecules resembling the parent. Some degradation products are a result of both Type 1 and Type 2 processes.

The molecular structure and composition of a given AI as well as the environmental conditions determine which degradation products are formed. Likewise, the molecular structure and biological activity of a particular metabolite or breakdown product dictate the level of toxicity compared to the parent active ingredient.

One important concept here is that it is not possible to have more of the molecular subunits than the number that were initially present in the parent compound. In the example in Figure 1, you could never produce more triangles than you started with, even if the degradation occurred by both pathways. What this means in the real world is that if your bees bring in imidacloprid-contaminated pollen from a corn field and it breaks down in the hive, you won’t find a greater number of the degradate molecules than you would parent compound molecules (unless the bees were bringing the degradate in from another source). In short, the total number of molecular pieces must be conserved throughout the transformation process.

When the Degradate is More Toxic Than the Active Ingredient, or Why It’s Not Always Gone When It’s Gone
Degradation of a pesticide generally brings to mind the idea that the toxicity is decreasing as well; however, this is not always the case. Some pesticides form degradates that are as toxic or even more toxic than the parent compound. Imidacloprid, the most widely used neonicotinoid insecticide, is a prime example of this type of transformation.

The parent compound imidacloprid is associated with a number of damaging effects on bees, including acute bee kills, impaired reproduction, immune suppression, and behavioral abnormalities. However, several of the imidacloprid metabolites are equally or more toxic to honey bees. Imidacloprid (Compound A in Figure 2) is transformed through a Type 1 process to 5-hydroxyimidacloprid  (Compound B), followed by transformation to the imidacloprid olefin compound (Compound C)2. While the hydroxy metabolite B is less toxic than the parent imidacloprid, the olefin compound C is 1.6 times as toxic as imidacloprid to honey bees.3

 

April 2014

 

Going the Distance: Scientific Bee Studies

That Make an Impact

by TIMOTHY J. BROWN and SUSAN E. KEGLEY, PhD
Pesticide Research Institute

(excerpt)

Introduction
Carefully designed scientific bee studies are invaluable tools for the practicing beekeeper. Without them it is nearly impossible to analyze the effects of different stressors on long-term colony health and make meaningful, informed decisions. In our previous installment of The Curious Beekeeper we discussed a series of flawed studies analyzing the effects of honey bee exposure to the new neonicotinoid insecticide sulfoxaflor.1 Some of the experiments designed to better understand the effects of sulfoxaflor exposure on brood development failed to follow a complete brood cycle. In addition, one of the key studies used a control hive (no exposure to sulfoxaflor) that was infested with Varroa to make comparisons against mite-free hives exposed to sulfoxaflor. Because the two colonies were not comparable in terms of mite infestation status, it was therefore impossible to draw any conclusions regarding the effect of sulfoxaflor on brood development from these experiments.

For this fourth article in our series, we counter our previous example with a study that goes the distance, with procedurally sound experiments that lead to defensible conclusions. Here we dissect the experimental design and evaluate the results from a notable 2012 study co-authored by Joseph Riddle and his colleagues at Michigan State University (MSU), Jeff Pettis at USDA, and Xianbing Xie from Nanching University analyzing the effects of long distance transport on the physiology of honey bees.2 As the first investigation of its kind, the study was designed to evaluate the two extremes in the level of transport, where one group of bees was transported while a comparison group (the negative control) was not moved at all.

The researchers’ hypothesis was that either due to higher mortality of older bees during and after transportation or due to inadequate pollen consumption by young bees, there should be measureable differences between bees that traveled and those that didn’t in terms of premature aging and ability to nurse brood. They chose to evaluate the following physiological parameters in colonies that were transported versus those that stayed in one location:

Levels of juvenile hormone (JH), which are normally low in young nurse bees, but high in foragers nearing the end of their lives. Levels of JH provide a yardstick to estimate premature aging in response to stress.

The size of the acini in the hypopharyngeal gland (HPG), used by the nurse bees to make royal jelly that is fed to larvae early in their development and also to the adult queen, drones, and foragers. The size of the HPG is related to how much food a nurse bee can produce and is genetically determined, but also age-dependent.

Protein content in head and thorax, which provides a measure of whether or not bees are getting enough protein to eat while they are being transported or if their digestion is affected by transportation.

Fat content in abdomen, another indicator of premature aging. Bees that are ready to forage generally have low levels of fat in the abdomen, while nurse bees have high levels.

If transportation were having adverse effects on the bees, the researchers hypothesized that bees that were transported should have higher levels of JH, smaller HPGs, lower protein content in heads and thorax, and lower lipid content in abdomen. So the challenge was to design a study to minimize the effects of other potential factors that might make it difficult to reliably compare the two groups.

Quality Control and Experimental Design
Scientific studies that present the most compelling results are typically designed ...

 March 2014

Going the Distance: Scientific Bee Studies

That Make an Impact

by TIMOTHY J. BROWN and SUSAN E. KEGLEY, PhD
Pesticide Research Institute

(excerpt)

Introduction
Carefully designed scientific bee studies are invaluable tools for the practicing beekeeper. Without them it is nearly impossible to analyze the effects of different stressors on long-term colony health and make meaningful, informed decisions. In our previous installment of The Curious Beekeeper we discussed a series of flawed studies analyzing the effects of honey bee exposure to the new neonicotinoid insecticide sulfoxaflor.1 Some of the experiments designed to better understand the effects of sulfoxaflor exposure on brood development failed to follow a complete brood cycle. In addition, one of the key studies used a control hive (no exposure to sulfoxaflor) that was infested with Varroa to make comparisons against mite-free hives exposed to sulfoxaflor. Because the two colonies were not comparable in terms of mite infestation status, it was therefore impossible to draw any conclusions regarding the effect of sulfoxaflor on brood development from these experiments.

For this fourth article in our series, we counter our previous example with a study that goes the distance, with procedurally sound experiments that lead to defensible conclusions. Here we dissect the experimental design and evaluate the results from a notable 2012 study co-authored by Joseph Riddle and his colleagues at Michigan State University (MSU), Jeff Pettis at USDA, and Xianbing Xie from Nanching University analyzing the effects of long distance transport on the physiology of honey bees.2 As the first investigation of its kind, the study was designed to evaluate the two extremes in the level of transport, where one group of bees was transported while a comparison group (the negative control) was not moved at all.

The researchers’ hypothesis was that either due to higher mortality of older bees during and after transportation or due to inadequate pollen consumption by young bees, there should be measureable differences between bees that traveled and those that didn’t in terms of premature aging and ability to nurse brood. They chose to evaluate the following physiological parameters in colonies that were transported versus those that stayed in one location:

Levels of juvenile hormone (JH), which are normally low in young nurse bees, but high in foragers nearing the end of their lives. Levels of JH provide a yardstick to estimate premature aging in response to stress.

The size of the acini in the hypopharyngeal gland (HPG), used by the nurse bees to make royal jelly that is fed to larvae early in their development and also to the adult queen, drones, and foragers. The size of the HPG is related to how much food a nurse bee can produce and is genetically determined, but also age-dependent.

Protein content in head and thorax, which provides a measure of whether or not bees are getting enough protein to eat while they are being transported or if their digestion is affected by transportation.

Fat content in abdomen, another indicator of premature aging. Bees that are ready to forage generally have low levels of fat in the abdomen, while nurse bees have high levels.
 
Nurse bees use their hypopharyngeal (HPG) glands to produce royal jelly to feed brood. The study evaluated the effect of transportation on the size of the HPG glands. Photo credits: Left, James D. Bull; Right, Maryann Frazier.

If transportation were having adverse effects on the bees, the researchers hypothesized that bees that were transported should have higher levels of JH, smaller HPGs, lower protein content in heads and thorax, and lower lipid content in abdomen. So the challenge was to design a study to minimize the effects of other potential factors that might make it difficult to reliably compare the two groups.

Quality Control and Experimental Design
Scientific studies that present the most compelling results are typically designed to assess an outcome, while systematically changing a single variable at a time. This study on hive transport used a number of techniques to ensure they were comparing apples to apples, instead of apples to pineapples.

Because the measured parameters change with the age of the bee, it was first necessary to ensure that the comparison between the group that traveled and the group that did not was only between bees of the same age. The scientists did this by collecting newly emerged worker bees and labeling them with Testor’s color paint so they could easily find them again to make the measurements after the bees were transported. Labeling newly emerged worker bees helped the researchers ensure they were comparing only bees of the same age. Photo credit: Christofer Bang.

The strain of bee (Italians vs ....

February 2014

The Dose Makes the Poison . . . or Does It?

Sponsored by the National Honey Bee Advisory Board

by TIMOTHY J. BROWN and SUSAN E. KEGLEY, PhD
Pesticide Research Institute

(excerpt)

Introductions
In our first installment of The Curious Beekeeper, we discussed the differences between the newer systemic pesticides, including neonicotinoid insecticides and certain fungicides, and the previous generation of pesticides, such as organophosphate and carbamate insecticides. Both the newer and older pesticide classes exhibit acute toxicity toward honey bees and other pollinators in the form of bee kills as a result of foliar applications. However, residues of the newer systemic pesticides are incorporated into pollen and nectar because they are taken up through the leaves (from foliar treatments) or through the roots (from soil or seed treatments) and distributed throughout the entire plant. Many of these systemic pesticides are also long-lived, with residues remaining in soils and plant materials for months to years following application. These attributes all contribute to the exposure of bees to low levels of systemic pesticides over an extended period of time, and can lead to significant adverse effects on colony health and productivity.

Overview: Acute toxicity versus chronic toxicity and sub-acute effects
Toxicologists will often quote Paracelsus, a 16th century physician as saying “the dose makes the poison,” suggesting that only high doses of poisons are problematic. However, the time over which the poison is ingested also makes a difference. Exposure to many of the systemic pesticides at sufficiently high doses certainly can kill bees, but low doses can still lead to the impairment of essential pollinator behaviors, reproduction, and immune function. While less immediately obvious, these sub-acute exposures can ultimately be more damaging as they can cause the whole colony death as opposed to simply a loss of foraging bees in an acute event.

Comparison of the acute contact toxicity of the organophosphate insecticide malathion with the most widely used systemic insecticide imidacloprid confirms that both compounds are highly toxic on an acute basis. Indeed, the Lethal Dose for 50% of the bees exposed in acute contact toxicity studies (LD50) are 0.20 and 0.014 mg/bee (micrograms active ingredient per bee) for malathion and imidacloprid, respectively.1,2 With such extreme toxicity at low doses, it’s no surprise that bee kills have been documented immediately following applications of these pesticides.

Unlike malathion, however, imidacloprid is taken up systemically, expressed in pollen and nectar at low levels, and persists for long periods once incorporated into these materials (aerobic half-life = 1.4–2.7 years,3,4 which means complete degradation can take up to 15 years).

An analysis of bee toxicology data shows that systemic pesticides, particularly neonicotinoid insecticides, can produce effects even at very low concentrations, provided there is a sufficiently long exposure period. Long-term, low-level exposure can occur from a short-term foraging event during which bees collect excess pollen and nectar and store it for later use. Even if the pollen and nectar contain only low levels of pesticide, there will be long-term exposure as the colony eats the stored food over time.

Bee researchers have demonstrated that chronic dietary exposure of honey bees to field-realistic levels of imidacloprid as low as 0.1 µg/kg (micrograms per kilogram) in sugar solution led to significant mortality within 10 days.5 This concentration equates to an imidacloprid dose of about 1 picogram (10-9 gram) per bee per day. Figure 1 summarizes the potential for mortality and adverse health effects following exposure to the most toxic neonicotinoids, including imidacloprid (Gaucho®, Admire®), clothianidin (Poncho®, Belay®), thiamethoxam (Cruiser®, Platninum®) and dinotefuran (Safari®, Venom®).