CALUX AND HIGH RESOLUTION GC/MS ANALYSIS OF DIOXINLIKE COMPOUNDS IN CHLOROPHENOXY PESTICIDEFORMULATIONS
Janice K. Huwe,1 George C. Clark,2 Andrew C. Chu,2 and Vincent Garry3
1USDA, ARS, Biosciences Research Laboratory, PO Box 5674-University Station, Fargo, ND
58105-5647 USA
2Xenobiotic Detection Systems, 1601 East Geer Street, Durham, NC 27704 USA
3University of Minnesota, Environmental Pathology Laboratory, Minneapolis, MN 55409 USAIntroduction
An epidemiology study to evaluate the health effects of pesticide exposure is being conducted bythe University of Minnesota on a cohort of pesticide applicators from the Red River Valley ofMinnesota. An initial report from a sub-cohort of this study has suggested a correlation between
pesticide exposure, testosterone levels, and the sex ratio of the applicators’ offspring.1 Because
polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) have been known to effect thesex ratio of offspring2,3 and are found in various chlorinated pesticides,4,5,6 a number of pesticides
commonly used in the Red River Valley of Minnesota were collected and assayed for dioxin-like
activity by a reporter gene bioassay (CALUX). Of the twelve pesticides assayed, ten had
measurable dioxin toxic equivalency (TEQ) and four had TEQs of over 1 ppb.1 In order toconfirm the presence of PCDD/Fs and provide a congener profile of dioxins in these pesticides,
we have now performed high-resolution GC/high-resolution MS (HRGC-MS)analysis on severalof these same pesticides. This paper will compare the results from the CALUX bioassay to that ofconventional HRGC-MS analyses.
Materials and Methods
Pesticides. Samples of chlorophenoxy pesticide mixtures that were sprayed in the Red River
Valley were collected in the years 1993 and 1998 under the guidance of the University ofMinnesota. Samples were stored at 4°C until sent for analysis. Sub-samples of eight of thesepesticides were shipped to Xenobiotic Detection Sytems and to the USDA, ARS, Biosciences
Research Laboratory for analysis by the CALUX bioassay for dioxin-like TEQ and for HRGC-MS
congener-specific dioxin analysis.
CALUX bioassay. The CALUX bioassay is a reporter gene assay in which a mouse hepatoma cellhas been stably transfected with a vector that contains the luciferase gene under transactivational
control of the Ah receptor.7,8,9 Combined with a patent-pending sample processing system toreduce contamination with non-dioxin-like agonists for the Ah receptor, the CALUX bioassay canbe used for estimation of TEQ contamination with dioxin-like chemicals of environmental,biological, and chemical samples.10,11,12 In this study, a 0.5 g sub-sample of each herbicide
mixture was dissolved in hexane and processed through our patent-pending cleanup procedure.10
The isolated extract was exchanged into dimethyl sulfoxide and applied to 96 well platemonolayers of our genetically engineered cells and incubated for maximal induction of the firefly
luciferase within the cells. Light produced by the firefly luciferase enzyme was measured with anassay kit from Promega and quantified on a Berthold Orion Microplate Luminometer. Dilution
analysis was performed for range finding of the approximate concentration of the processed

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MAsample that induced maximal luciferase activity for quantification in the CALUX bioassay.
Dioxin-like TEQ activity was estimated from a standard curve of 2,3,7,8-tetrachlorodibenzo-pdioxin using a four-parameter Hill equation and is reported as the mean ± the standard deviation oftriplicate analyses.
HRGC-MS analysis. Prior to HRGC-MS analysis, 0.1 g of each sample was spiked with fifteen
13
C-labeled PCDD/Fs and three 13C-labeled PCBs (recovery standards) and purified according toEPA Method 1613 using an FMS automated cleanup system (Fluid Management Systems,Waltham, MA). Herbicide samples that were hexane soluble were directly applied to the FMSsystem. Samples that were not readily soluble in hexane were partitioned between hexane and 2.0
M potassium hydroxide with rigorous shaking. The hexane layer was dried with sodium sulfate
before being loaded onto the FMS system. A solvent blank was run with each set of samples. The
PCDD/Fs and three co-planar PCBs were quantitated by EPA Method 1613. All values wereblank-subtracted. WHO-TEFs were used to calculate TEQs.
Results and Discussion
Table 1 shows the TEQs obtained from the CALUX bioassay and from the HRGC-MS analysis ofeight pesticide samples. For six of the samples, the TEQs from both methods correlated quite well,although the CALUX TEQs tended to be higher than the HRGC-MS results by 1.3−3 fold. Twopesticide samples (Bronate and Assure II) differed greatly in the response of the CALUX bioassay
and the HRGC-MS. This divergence is most likely due to the inherent selectivities of the methods.
While the HRGC-MS method is specific for only the tetra- through octa-chlorinated dioxins andfurans, the CALUX bioassay has a broader specificity based on induction of a firefly luciferasegene in response to binding and activation at the aryl hydrocarbon receptor. As a consequence,
the CALUX bioassay can respond to numerous brominated dioxin-like compounds and some nondioxin compounds that activate the aryl hydrocarbon receptor pathway. The active ingredients inBronate and Assure II contain halogenated aromatic moieties that may possess some affinity forthe aryl hydrocarbon receptor and elicit a CALUX response. However, these ingredients appear
unlikely to form PCDD/Fs as by-products during processing.
In general, 2,3,4,7,8-PeCDF and 1,2,3,7,8-PeCDD dominated the TEQs of the pesticides. No
significant amounts of co-planar PCBs were detected in any samples. In addition to the 2,3,7,8substituted dioxins and furans, other isomers were also present in the formulations. The homolog
distribution profile for 2,4-D LV4 is shown in Figure 1. A similar profile was seen for each of theother pesticides. The polychlorinated furans dominated the pattern. Although TCDF was themajor homolog group, no 2,3,7,8-TCDF was found in any of the pesticides. Among the 2,4,dichlorophenoxyacetic acid (2,4-D) type of formulations, TEQs ranged from near background to2,600 ppt. Schecter et al. have reported TEQs ranging from 1-850 ppt for 2,4-D formulations
from various countries.5 The large variability seen from sample to sample may arise fromdiffering degrees of purity in the raw starting materials. A similar explanation has been proposedby Masunage et al. who found elevated TEQs (up to 900 ppb) in chloronitrofen (CNP).6 CNP is
produced from 2,4,6-trichlorophenol, but impurities in the chlorophenol can give rises to toxic
dioxin and furan byproducts.
In conclusion, the CALUX bioassay appeared to be a good screening tool for dioxin-like activity
in a variety of pesticides. Compared to HRGC-MS, the CALUX TEQs were generally higher.
Given the nature of the CALUX assay, however, it may provide a more accurate assessment of theOrganohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MA

potential biological activity of pesticide formulations than the highly selective HRGC-MS method.
The results of this study also confirm the presence of toxic PCDD/Fs in certain 2,4-D pesticides
and support the need for more investigation into possible human health effects.
Acknowledgements
The authors would like to acknowledge Montgomary Botschner, Kristin McDonald, Jean Picard,and Joyce Wold for technical assistance in sample purification for HRGC-MS and Margaret
Lorentzsen for mass spectral analyses. Names are necessary to report factually on available data;
however, the USDA neither guarantees nor warrants the standard of the product, and the use of thename by USDA implies no approval of the product to the exclusion of others that may also besuitable.

Active IngredientsCALUX TEQ
(n=3)

HRGC-MS
TEQ

1993

2,4-D dimethylamine,46.9%

26.2 ± 0.5

8.7

See 2,4-D

1993

2,4-D isooctylester, 61.7%

1637 ± 212

731

2,4-D LV4

1993

2,4-D isooctylester, 66.2%

3392 ± 257

2627

2.4-D LV6

1993

2,4-D isooctylester, 88.8%

1882 ± 311

1379

Weedone LV4

1998

2,4-D isooctylester, 67.2%

45.7 ± 4.1

27.7

34.2 ± 4.1

19.8

800 ± 1800.3

1668 ± 419

4.1

PesticideCollection
Year

2,4-D Amine

Tiller1998

Bronate1998

Assure II1998

MCPA-2EH ester, 32.1%
2,4-D isooctylester, 10.4%
Fenoxaprop-p-ethyl, 4.4%
Bromoxynil octanoic acid
ester, 31.7%
MCPA isooctyl ester, 34%
Quizalofop-p-ethyl, 10.3%

Table 1. Composition and TEQs (ppt) of the chlorophenoxy pesticides analyzed in this study.
2,4-D = 2,4-dichlorophenoxyacetic acid; MCPA = 4-chloro-2-methylphenoxyacetic acid; EH =ethylhexyl.

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MA

120000

100000All isomers
2,3,7,8-substituted
80000
pg/g
60000

40000

20000

0TCDD PeCDD HxCDD HpCDD OCDDTCDF PeCDF HxCDF HpCDF OCDF

Figure 1. Homolog profile of 2,4-D LV4.

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Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MA