Honey Bees: Estimating the Environmental Impact of Chemicals - Chapter 2 doc

ronments contain compounds released by the bees themselves e.g.pheromones, other chemicals released to repel pests and predators,metabolites, etc., compounds from hive stores e.g.. honey

2 Volatile and semi-volatile

In the process of monitoring organic contaminants, it has also beennecessary to characterize the complex background of organic compoundsfound naturally inside beehives Beehives located in uncontaminated envi-

ronments contain compounds released by the bees themselves (e.g.pheromones, other chemicals released to repel pests and predators,metabolites, etc.), compounds from hive stores (e.g honey, beeswax,pollen, and propolis), and volatile compounds from the materials out ofwhich hives are constructed (wood, paint, plastic, etc.) We show here thatbeehive atmospheres also contain compounds from vehicles, farms, indus-tries, and households in the hive vicinity.

This paper summarizes the types of compounds found by our techniquewhile biomonitoring for a variety of volatile and semi-volatile organic con-taminant residues Briefly, hive atmospheres were drawn through multibedsorption traps and subsequently analyzed by thermal desorption/gaschromatography/mass spectrometry (TD/GC/MS)

Methods and materials

Fingerprinting studies

Fingerprinting studies for hive components were conducted at the sity of Montana’s research apiaries on seven dates during July 1996 (Table2.1) Ambient air was concurrently sampled so that contaminants present

Univer-in the urban airshed of our apiary could be identified and accounted for Univer-inall other samples

To fingerprint the active physiology of honey bees by themselves, astainless steel cage was fabricated to contain about 4000 individuals The

Volatile organics in beehives 13

Table 2.1 Fingerprint studies

Hive stores

Unoccupied 1995 hive box (no bees or frames) 7/5, 7/6, 7/7

Unoccupied hive 56 (no bees, with frames) 7/11

top of the cage was outfitted with a syrup bottle to feed the bees duringthe 8- to 10-hour pumping periods Pumping was done in the open air, free

of any hive enclosure that could contribute extraneous substances

Hive stores were evaluated by pumping on a previously occupiedupper-story box with and without honey frames These samples had con-tributions from both hive stores and hive materials Two samples of propo-lis from Missoula colonies were placed in loosely capped glass vials forpumping

Hive components profiled included unpainted wood, painted wood,machined plastic parts, vinyl-coated screen wire, and completely instru-mented “condo” units [14] The effect of aging on the loss of volatile andsemi-volatile components from hive boxes was assessed by comparingunpainted wood from 1995 and 1996 lumber inventories We also com-pared a condo used during the 1995 field season to a newly completed

1996 model

Air sampling

Carbotrap 300 thermal desorption tubes (Supelco) or four-phase Carbotrap

400 tubes These sorbent tubes house a sequence of graphitized carbon andmolecular sieves of increasing activity that sorb volatile and semi-volatileorganic compounds over a molecular size range from C1to C30

Desorption tubes were connected to constant flow pumps set at ratesbetween 0.080 and 0.150 dm3/min The distal end of the sorption tube wasattached to copper tubing (2 mm ID3mm OD) with a brass compressionfitting and a vespel/graphite ferrule The copper tube was inserted directlyinto the hive interior between the wooden frames that support the waxcombs (Figure 2.1) The outlet end of the sorbent tube was connected to aconstant flow pump (SKC, Inc.) with a 1-m section of 5 mm ID8mm ODTygon tubing Pumping periods ranged from 8 to 12 hours

14 G.C Smith et al.

Figure 2.1 Schematic diagram of a hive with air-sampling train.

Sample tubes were sealed in individual vials and stored in a dedicated4°C sample refrigerator until analyzed.

Thermal desorption analysis

Sample tubes were desorbed in a direction opposite to sampling flow.After a 4-min helium purge to remove incidental moisture, tubes weresubjected to a 10-min desorption cycle at 250°C Each tube was then given

desorption tube Make-up helium flow from other paths on the

going into the focusing trap (10 cm Carbopack B graphitized carbon, 6 cmCarboxen 1000 molecular sieve and 1 cm 1001 molecular sieve) The focus-ing trap was desorbed and flushed into the gas chromatograph for 1 min

1:20 thereafter

Chromatographic separations were accomplished on a Hewlett Packard

initial temperature 40°C, ramp 5°C/min to 220°C, 9 min hold time at220°C) Mass spectra were collected over a range of 35 to 450 amu

Computer matches with the National Institute of Science and logy (NIST) database initially identified compounds Many, though not all,were subsequently confirmed using commercial mixtures of analyticalstandards The concentrations of all compounds were computed on a rela-

Compounds of interest to regulatory agencies have been rigorously tified [11–14]

quan-Results

To place our hive atmosphere findings in perspective, we have compiledlists of specific compounds whose presence in bees and beehives have beendocumented in the honey bee literature by previous researchers The datafor these tables come from several review articles and selected papers Wehave not attempted to conduct a comprehensive review of this large body

of work Honey bees exhibit pheromonal parsimony The same compoundmay have different functions in different contexts Also, manypheromones have not been characterized As this knowledge baseexpands, we are changing our interpretation of the function of identifiedcompounds Queen pheromone may not so much inhibit worker’s ovaries

as signal the presence of a queen, and brood pheromones may provide thestimulus to prevent workers from laying eggs [15] Because propolis ishighly variable in its composition, we have included compounds reported

© 2002 Taylor & Francis

to be characteristic of different geographic regions [16] Propolis is aresinous material obtained by bees from woody plants It is made up of anindeterminate number of substances and has no specific chemical formula[17].

A typical hive atmosphere chromatogram from our TD/GC/MS

compounds reported as honey bee semiochemicals Semiochemicals areproduced in glands that secrete to the exterior of the insect, and includepheromones, which are chemicals used to communication between indi-

compounds arising from non-bee sources Within each category,

levels for hazardous air pollutants that have been collected from hives inour studies in the vicinity of Chesapeake Bay, USA

Compounds detected with our TD/GC/MS technique are designatedwith an “X” in the next-to-last column of each table Compounds that wehad analyzed by EPA Methods 8081A (pesticides) and 8082 (PCBs) aredesignated with a “Y” in the tables Whenever possible, we also provideCAS numbers for reported compounds CAS numbers proved difficult toobtain or have not yet been assigned to some of the biologically derivedchemicals (i.e recently discovered semiochemicals and botanicals inpropolis)

Discussion

Chromatographic considerations

Because of the general nature of our sampling technique and the sequent TD/GC/MS analysis, only certain categories of volatile and semi-volatile compounds were detectable – nonpolar organics (alkanes, alkenes,alkynes, cycloalkanes, aromatics, terpenes, PAHs, biphenyls), partiallyoxygenated organics (alcohols, ethers, ketones, aldehydes, acids, esters),organonitrogen and organosulfur compounds (amines, amides, hetero-cycles), and organochlorine compounds (solvents, pesticides) Highly polarmolecules were generally missed with our technique This is a con-sequence of choosing sorbents that target nonpolar species and achromatographic column coated with a substance of intermediate polarity.Use of other sorbents and different column coatings could enhance theability to find other classes of compounds

sub-Masses of compounds ranging from 35 amu up to those associated with

The molecular weight cut-off was constrained by the maximum

ture to which the carbon-based sorbents could be subjected – about 350°C.Higher molecular weights are accessible with silica-based sorbent mater-ials, which can tolerate temperatures up to 600°C This was demonstrated

in side-by-side tests done recently in conjunction with Oak Ridge NationalLaboratory [111] Compounds of higher molecular weights, for examplemany polycyclic aromatic hydrocarbons associated with petroleum andcreosote, were seen more readily

Approximately 25 ng of analyte were needed for detection above ground noise in the mass spectrometer This quantity was usually accumu-

Our current sampling train has added two tubes in front of the Carbotrap

Figure 2.2 Total ion chromatogram of a typical hive atmosphere sample Selected

peaks have been labeled with the identity of the compound and tion time in minutes Seen here are compounds from bees (nonanal at 34.57 min), from plant resins in propolis or hive boards ( -pinene at 28.24 min), and from non-bee contaminants (toluene at 21.89 min and tetrachloroethene, PCE, at 23.69 min).

reten-© 2002 Taylor & Francis

Table 2.2 TD/GC/MS detection of volatile and semi-volatile organic compounds previously reported as honey bee semiochemicals and

brood recognition?

inhibits queen rearing attracts drones, recognizes queen

S-9-Hydroxy-(E)-2-decenoic acid C10H18O3 186 queen retinue formation

R-9-Hydroxy-(E)-2-decenoic acid C10H18O3 186 queen retinue formation

Nasonov gland

Table 2.2 Continued

Koschevnikov gland

Table 2.2 Continued

Venom sac (Venom oil)

Wax gland

Table 2.2 Continued

Tergite gland

inhibit queen rearing attract drones, orientation

at flowers

Tarsal (Arnhart’s) gland

Worker-repellent pheromone

o-Aminoacetophenone C 8 H 9 ON 135 young queen repel other queens 551-93-9 Brood pheromones

inhibit worker ovaries Drone pheromones

Beeswax (comb) pheromones

Oxygenated organics

Notes

*Extracted from young queens, does not occur in the alarm pheromone of workers, promotes aggressive behavior of workers towards supernumerary queens References: Data by category – general review of bee pheromones [15, 18–25]; mandibular gland [17, 20, 25, 26–50]; Nasanov gland [17, 24, 51–58]; Kuschevnikov gland and venon sac [17, 22, 24, 59–79, 99]; tergite and tarsal glands [25, 79–85]; worker repellent [86]; beeswax pheromones [87–92]; brood and drone pheromones [93–98].

Table 2.3 Volatile and semi-volatile organic compounds found in hive stores

N-Methylpyrrole C5H7N 81 x 96-54-8 Tiglic acid C5H8O2 100 x 80-59-1 5-Methyltetrahydrofuran-3-one C5H8O2 100 x 34003-72-0 Benzoic acid C7H6O2 122 [101] x 65-85-0 Benzaldehyde C7H6O 106 x 100-52-7 Methyl benzoate C8H8O2 136 x 93-58-3 6-Methyl-3,5-heptadien-2-one C8H12O 124 x 1604-28-0 Phenethyl alcohol C8H10O 122 x 60-12-8 Acetophenone C8H8O 120 x 98-86-2 4-Methylenecyclohexylmethanol C8H14O 126 x 1004-24-6 1-Octanol C8H18O 130 x 111-87-5 Vanillin C8H8O3 152 [101] 121-33-5 Cinnamic acid C9H8O2 148 [101] 621-82-9 Hydrocinnamic acid C9H10O2 150 [101] 501-52-0 1-Nonyne C9H16 124 x 3452-09-3 3,7-Dimethyl-1,3,6-octatriene C10H16 136 x 29714-87-2 Eucalyptol C10H18O 154 x 470-82-6

-Myrcene C10H16 136 x 123-35-3 1-Phenyl-2-butanone C10H12O 148 x 1007-32-5 Palmitic acid C16H32O2 256 [101] 57-10-3 Benzyl cinnamate C16H14O2 238 [101] 103-41-3 Kaempferid C16H12O6 300 [101]

3,4-Dimethoxynaringenin C17H16O6 316 [101]

Betuleol C17H14O7 330 [101]

1-Nonacosanol C29H60O 424 [101] 25154-56-7 Tetracosyl hexadecanoate C40H80O2 592 [101]

Table 2.4 Volatile and semi-volatile organic compounds from hive construction

components

Sources Formula MW Ref TD CAS no.

Wood boards

2,3-Dimethyloxirane C4H8O 72 x 1758-33-4 2,2,3,3-Tetramethylhexane C10H22 142 x 13475-81-5 2-Ethylcyclobutanol C6H12O 100 x 35301-43-0 Hexanal C6H12O 100 x 66-25-1 1-(1-Methylethoxy)-2-propanone C6H12O2 116 x 42781-12-4 1,2-Diethylcyclobutane C8H16 112 x 61141-83-1

cis-1-Cyclopropyl-2-ethenyl- C9H14 122 x 61141-61-5 cyclobutane

Volatile organics in beehives 23

© 2002 Taylor & Francis

Table 2.5 Volatile and semi-volatile organic compounds from non-bee sources

Sources Formula MW Refs TD CAS no.

Wood/vegetation combustion sources

Oxygenates

2-Butenal C4H6O 70 [104, 105] x 4170-30-3 2,3-Butanedione C4H6O2 86 [104, 105] x 431-03-8 Furfural C5H4O2 96 [104, 105] x 98-01-1 2,3-Pentanedione C5H8O2 100 [104, 105] x 600-14-6 2,5-Dimethylfuran C6H8O 96 [104] x 625-86-5 2-Hexanone C6H12O 100 [104, 105] x 591-78-6 Isoamyl acetate C7H14O2 130 x 123-92-2 2-Methylbenzaldehyde C8H8O 120 [104] x 529-20-4 Anethole C10H12O 148 x 104-46-1 l- -Terpineol C10H18O 154 x 10482-56-1

cis-Linalool oxide C10H18O2 170 x 5989-33-3

Other

Chloroform CHCl3 119 [103, 106] x 67-66-3 1-Butanol C4H10O 74 [107] x 71-36-3 Isoprene C5H8 68 [103, 106] x 78-79-5 Benzene C6H6 78 [103, 106] x 71-43-2 Toluene C7H8 92 [103, 106] x 108-88-3

o-Xylene C8H10 106 [103, 106] x 95-47-6

m-Xylene C8H10 106 [103, 106] x 108-38-3

p-Xylene C8H10 106 [103, 106] x 106-42-3 1,3,5-Trimethylbenzene C9H12 120 [106] x 108-67-8 Naphthalene C10H8 128 [103] x 91-20-3 1,1 -Biphenyl C12H10 154 [108] x 92-52-4 Vehicle emissions/creosote

n-Hexane C6H14 86 x 110-54-3

n-Heptane C7H16 100 x 142-82-5 3-Methylhexane C7H16 100 x 589-34-4 2,2,4-Trimethylpentane C8H18 114 x 540-84-1

n-Octane C8H18 114 [102] x 111-65-9 2-Methylheptane C8H18 114 x 592-27-8 3-Ethylhexane C8H18 114 x 619-99-8 2,3-Dimethylhexane C8H18 114 x 584-94-1 4-Methyloctane C9H20 128 x 2216-34-4 2,4-Dimethylheptane C9H20 128 x 2213-23-2

n-Nonane C9H20 128 x 111-84-2 2-Methylnonane C10H22 142 x 871-83-0

n-Decane C10H22 142 x 124-18-5

n-Dodecane C12H26 170 x 112-40-3 Tridecane C13H28 184 x 629-50-5 4,6-Dimethylundecane C13H28 184 x 17301-23-4

Cycloalkanes

1-Methyl-2-methylenecyclo- C5H8 68 x 18631-84-0 propane

Table 2.5 Continued

Sources Formula MW Refs TD CAS no.

Cyclopentane C5H10 70 x 287-92-3 Ethylcyclobutane C6H12 84 [102] x 4806-61-5 Methylcyclohexane C7H14 98 x 108-87-2

cis-1,2-Dimethylcyclohexane C8H16 112 x 2207-01-4 1,2,4-Trimethylcyclohexane C9H18 126 x 2234-75-5

Alkenes

Divinyl ether C4H6O 70 x 109-93-3 2-Methylpropene C4H8 56 x 115-11-7 1-Pentene C5H10 70 x 109-67-1 3,3-Dimethylcyclobutene C6H10 82 x 16327-38-1 2-Hexene C6H12 84 [102] x 592-43-8 4-Methyl-1-pentene C6H12 84 x 691-37-2 2-Heptene C7H14 98 x 592-77-8 4,4-Dimethyl-1-pentene C7H14 98 x 762-62-9 1,3,5,7-Cyclooctatetraene C8H8 104 x 629-20-9 3-Methyl-1-heptene C8H16 112 x 4810-09-7

Aromatics

Benzene C6H6 78 [102] x 71-43-2 Toluene C7H8 92 [102] x 108-88-3 Styrene C8H8 104 x 100-42-5 Ethylbenzene C8H10 106 x 100-41-4

tert-Butylbenzene C10H14 134 [102] x 98-06-6

sec-Butylbenzene C10H14 134 x 135-98-8

n-Butylbenzene C10H14 134 x 104-51-8

p-Cymene C10H14 134 x 99-87-6 1,2,3,5-Tetramethylbenzene C10H14 134 x 527-53-7 1,2-Dimethyl-2-butenylbenzene C12H16 160 x 50871-04-0

Acid and acid derivatives

Formic acid CH2O2 46 x 64-18-6 Acetic acid C2H4O2 60 x 64-19-7 Isobutyric acid C4H8O2 88 x 79-31-2 3-Furoic acid C5H4O3 112 x 488-93-7 Methoxyacetic acid anhydride C6H10O5 162 x 19500-95-9 Hexanoic acid C6H12O2 116 x 142-62-1

© 2002 Taylor & Francis

Table 2.5 Continued

Sources Formula MW Refs TD CAS no.

Benzoic acid C7H6O2 122 x 65-85-0 6-Nonynoic acid C9H14O2 154 x 56630-31-0

Amides

Formamide CH3NO 45 x 75-12-7 Acetamide C2H5NO 59 x 60-35-5

Amines

2,2-Dimethylaziridine C4H9N 71 x 2658-24-4

O-Isobutylhydroxylamine C4H11NO 89 x 5618-62-2 Benzothiazole C7H5NS 135 x 95-16-9 2-(2-Aminoethyl)pyridine C7H10N2 122 x 2706-56-1

Aldehydes

Acetaldehyde C2H4O 44 [102] x 75-07-0 Furfural C5H4O2 96 x 98-01-1 4-Pentenal C5H8O 84 x 2100-17-6 Isovaleraldehyde C5H10O 86 x 590-86-3 5-Methyl-2-furfural C6H6O2 110 x 620-02-0 5-(Hydroxymethyl)-2-furfural C6H6O3 126 x 67-47-0 Benzaldehyde C7H6O 106 x 100-52-7 4-Heptenal C7H12O 112 x 62238-34-0 2,4-Dimethylpentanal C7H14O 114 x 27944-79-2 Heptanal C7H14O 114 x 111-71-7 Terephthalaldehyde C8H6O2 134 x 623-27-8 3-Methoxybenzaldehyde C8H8O2 136 x 591-31-1 2-Ethylhexanal C8H16O 128 x 123-05-7 Octanal C8H16O 128 x 124-13-0 Cinnamaldehyde C9H8O 132 x 104-55-2 Dodecanal C12H24O 184 x 112-54-9 Nonanal C9H18O 142 x 124-19-6

Ketones

Acetone C3H6O 58 x 67-64-1 1-Hydroxy-2-propanone C3H6O2 74 x 116-09-6 3-Buten-2-one C4H6O 70 x 78-94-4 2-Butanone C4H8O 72 x 78-93-3 3-Hydroxy-2-butanone C4H8O2 88 x 513-86-0 4,4-Dimethyl-2-oxetanone C5H8O2 100 x 1823-52-5 3-Methyl-2-butanone C5H10O 86 x 563-80-4 2-Pentanone C5H10O 86 x 107-87-9 2,3-dihydro-3,5-dihydroxy- C6H8O4 144 x 28564-83-2

6-methyl-4H-pyran-4-one

2-Hexanone C6H12O 100 x 591-78-6 5-Methyl-2-hexanone C7H14O 114 x 110-12-3 2-Heptanone C7H14O 114 x 110-43-0 Acetophenone C8H8O 120 x 98-86-2 4-Methyl-2-heptanone C8H16O 128 x 6137-11-7 1-Cyclopropyl-2-(2-pyridinyl)- C10H11NO 161 x 57276-32-1 ethanone

Alcohols

Methanol CH4O 32 [102] 67-56-1 Ethanol C2H6O 46 [102] x 64-17-5 2-Propanol C3H8O 60 x 67-63-0 2-Methyl-1-propanol C4H10O 74 x 78-83-1 2-Methyl-2-propanol C4H10O 74 x 75-65-0 1,3-Butanediol C4H10O2 90 x 107-88-0