FOOD AND FEED I (LEVELS AND TRENDS)

PCDD/DFs AND DIOXIN-LIKE PCBs IN FISH OIL USED FOR FEEDINGREDIENTS IN JAPAN: EVALUATION BY 2005 WHO-TEF AND CALUX ASSAY
Hasegawa J1, Guruge KS1, Seike N2, Shirai Y3, Yamata T3, Nakamura M4, Handa H4, Yamanaka N1, Miyazaki S1
1

National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki, 305-0856, Japan
National Institute for Agro-Environmental Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki 305-0856, Japan3
Food and Agricultural Materials Inspection Center, Shintoshin 2-1, Chuo-ku, Saitama, Saitama 330-9733, Japan
4
Hiyoshi Ecological Services Co., Kitanoshyo 908, Omihachiman, Shiga 523-0806, Japan
2Abstract
Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/DFs) and dioxin-like polychlorinated biphenyls(DL-PCBs) in 41 fish oil samples used for feed ingredients in Japan were determined by using HRGC/HRMS
and CALUX bioassay. The mean TEQ values derived from conventional 1998 WHO-TEF of PCDD/DFs andDL-PCBs were 2.6 and 9.9 pg/g ww, respectively. The levels of TEQ derived from the recently re-evaluated2005 WHO-TEF were slightly lower than those of the former in both groups. Notably, the contribution ofmono-ortho DL-PCBs to total 2005 WHO-TEQ was considerably decreased compared to the case of 1998
WHO-TEQ, resulting from the reduction in its TEF values, while the non-ortho DL-PCBs contribution wasincreased. The mean TEQ determined by CALUX assay for PCDD/DFs was approximately three times higher,
whereas DL-PCBs was approximately two times lower than WHO-TEQ determined by HRGC/HRMS; the sumof PCDD/DFs and DL-PCBs was very similar by both methods. The correlation coefficients between theCALUX-TEQ and 2005 WHO-TEQ were 0.82, 0.89, and 0.90 for PCDD/DFs, DL-PCBs, and the sum,respectively. These results suggest that the CALUX assay is substantially useful method for the screening ofdioxin-related compounds in fish oils.
Introduction
Fish oils are known sources of nutritionally valuable components such as polyunsaturated fatty acids. The crude
fish oil products are used as feed ingredients for farmed fish and domestic animals, however it has been reported
that fish oils contain relatively high levels of PCDD/DFs, and DL-PCBs compared to other feed ingredients 1, 2.
It is important to routinely monitor these compounds in a vast number of samples to avoid producing feeds withelevated levels of contamination. The regular method for dioxin analysis using HRGC/HRMS would be costly
and time consuming to cover up large number of samples. Meanwhile, the CALUX assay has been widely
employed as a rapid analysis of dioxin-like toxicity in environmental and biological samples including food andfeed 3-5. And while the TEF proposed by WHO (WHO-TEF) 6 was re-evaluated in recent years 7, it has been little
described about not only the results of change of TEQ levels and contributions in substantial samples but also thecorrelation between the re-evaluated TEQ based on chemical analysis and TEQ based on bioanalytial method
such as CALUX assay. We previously carried out the CALUX assay for fish oils used as feed ingredients inJapan, but the number of sample was relatively limited 8. In this study, we expanded the number and collecting
year of samples for analysis, and the contamination levels of PCDD/DFs and DL-PCBs were determined bychemical analysis using HRGC/HRMS and CALUX assay. In the chemical analysis, TEQ were calculated using
the two WHO-TEFs, and the difference in results was noted. TEQ of PCDD/DFs and DL-PCBs obtained by theCALUX assay were compared with WHO-TEQ.
Materials and Methods
Sample collection
A total of 41 fish oil products were collected from several feed manufactories in Japan during 2000-05. Thesesamples were stored at -20℃ until analysis.
Chemical analysis
PCDD/DFs and DL-PCBs were analyzed according to the provisional guideline for analytical method of dioxin
related compounds in feed described by the Ministry of Agriculture, Forestry and Fisheries (MAFF) of Japan 9. A

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10 g of fish oil sample was spiked with 13C-labeled 2,3,7,8-substituted PCDD/DFs and DL-PCBs as internal
standards, and subjected to KOH/EtOH digestion at room temperature for two hours. Then the alkaline solution
was diluted with distilled water, and extracted with hexane three times. The extract was cleaned up by themulti-layer silica gel column, followed by separated to mono-ortho DL-PCBs fraction and non-ortho DL-PCBsand PCDD/DFs fraction using the activated carbon column. The fractionated extracts were concentrated andspiked some congeners of 13C-labeled PCDD/DFs and PCBs as recovery standards. Determination of PCDD/DFsand DL-PCBs were performed by HRGC/HRMS. The average recovery of PCDD/DFs and DL-PCBs were in therange of 50-120%. The limit of quantification (LOQ) of PCDD/DFs, non-ortho DL-PCBs and mono-ortho
DL-PCBs were 0.05-0.2 pg/g, 0.1 pg/g and 0.5 pg/g wet weight (ww) basis, respectively. TEQ were calculatedusing the both conventional 1998 and re-evaluated 2005 WHO-TEFs (Table 1). In case where the congeners
were below those of LOQ, TEQ were calculated as lower bound values, which are equal to zero.
CALUX assay
The assay was carried out as previously described 8. A
1.0-1.3 g of fish oil sample dissolved in hexane was mixed
with about 17 g of 44% (w/w) sulfuric acid-silica gel andshaken followed by left stand for 30 minute. The mixture
was passed through the sulfuric acid-silica gel column andeluted with hexane. The eluent was loaded on XCARBcarbon (Xenobiotic Detection Systems Inc.) column. Thecolumn was washed with hexane to remove remaining
interfering matrix and the bulk of 2-4 ortho substituted
PCBs. Then DL-PCBs fraction was eluted with 15 ml ofhexane/toluene/ethyl acetate (8:1:1), followed by
PCDD/DFs fraction was eluted with 20 ml of toluene. The
fractionated extracts were evaporated and resuspended inhexane. Aliquots of the hexane solution were reconstituted
with 4 μl of DMSO. Immediately prior to dosing cells, thesample extracts and 2,3,7,8-TCDD standard solutions (4 μlof DMSO) was diluted in 400 μl of culture medium.
H1L6.1 mouse hepatoma cells were cultured in 96-well
microplate at 7.5 x 105 cell/well. After 24 hours incubation,cells were exposed in duplicate to the purified sample
extracts and the 9 serial dilutions of 2,3,7,8-TCDD
standard as well as DMSO alone on each plate. Following
20-24 hours of incubation, the luciferase activity wasmeasured using luminometer. Results were expressed inrelative light unit (RLU) and the average RLU value ofDMSO alone was subtracted from all RLU values.
CALUX-TEQ was calculated according to standard curves.
Calculated LOQ of CALUX-TEQ in the sample extracts
depended on sample volume and dilution factor was
1.44-1.97 pg/g ww for PCDD/DFs and 0.868-1.00 pg/g
ww for DL-PCBs. Relative potency (REP) values 10 for
individual congeners of PCDD/DFs and DL-PCBs wereshown in Table 1.

Table 1. WHO-TEFs and CALUX-REP values.
congener
PCDD/DFs
2,3,7,8-TeCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TeCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,4,6,7,8-HpCDF
1,2,3,4,7,8,9-HpCDF
OCDF
DL-PCBs
Non-ortho PCBs
PCB 77
PCB 81
PCB 126
PCB 169
Mono-ortho PCBsPCB 105
PCB 114
PCB 118
PCB 123
PCB 156
PCB 157
PCB 167
PCB 189

WHO-TEF WHO-TEF CALUX abc
1998
2005
REP
1
1
0.1
0.1
0.1
0.01
0.0001
0.1
0.05
0.5
0.1
0.1
0.1
0.1
0.01
0.01
0.0001

1
1
0.1
0.1
0.1
0.01
0.0003
0.1
0.03
0.3
0.1
0.1
0.1
0.1
0.01
0.01
0.0003

1
0.73
0.075
0.098
0.061
0.031
0.00034
0.067
0.14
0.58
0.13
0.14
0.11
0.31
0.024
0.044
0.00160.00010.00010.1
0.01

0.00010.00030.1
0.03

0.00140.00450.038
0.00110.00010.00050.00010.00010.00050.00050.00001
0.00010.000030.000030.000030.000030.000030.000030.000030.000030.000001
0.000140.000001
0.0000003
0.000140.000003
0.0000003
0.0000002

a,bc

WHO-TEF were cited from Van den Berg et al. (1998, 2006)
CALUX-REP were cited from Brown et al. (2001) and provided by XDS Inc.

Results and Discussion
Evaluation by two WHO-TEFs derived from chemical analysis
Mean TEQ calculated by 1998 WHO-TEF were 2.6, 9.9, and 12.5 pg/g wet weight (ww) for PCDD/DFs,DL-PCBs, and the sum, respectively. The mean and range of contribution of DL-PCBs to total TEQ was 79(63-91) %. It has been reported that the changes in 2005-TEF have a limited impact on the total TEQ of somebiological samples with overall decrease in TEQ ranging between 10 and 25% 7. TEQ calculated by re-evaluatedOrganohalogen Compounds Vol 69 (2007)

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2005 WHO-TEF in fish oils were somewhat less than the results from conventional 1998-TEF. The mean andrange of the ratio of 2005 to 1998-TEQ in fish oil samples analyzed in this study were 0.83 (0.71-0.98) for
PCDD/DFs, 0.89 (0.81-1.06) for DL-PCBs, and 0.89 (0.80-1.00) for the sum.
The contribution of PCDFs to total 2005-TEQ was marginally decreased, and those of mono-ortho DL-PCBs
were considerably decreased from 15 to 4% (1.9 to 0.4 pg/g ww in actual TEQ) compared to 1998-TEQ. These
results are the outcome of the reduction in TEF values for 2,3,4,7,8-PeCDF and all the mono-ortho DL-PCB
congeners except for PCB 167 in 2005-TEF. In contrast, the non-ortho DL-PCBs contribution was increased
from 64 to 76% (8.0 to 8.4 pg/g ww in actual TEQ). These results suggest that non-ortho DL-PCBs aresubstantially important target with regard to evaluating the risk of dioxin-like compounds in fish-originated feed
and food samples. Among all congeners, the highest contributor to total TEQ derived from both TEFs was PCB
126 in all samples, and the proportion was 63 (52-79) % for 1998-TEQ and 71 (59-80) % for 2005-TEQ.
Although TEQ levels were measurably decreased in most biological samples as a result of changing TEF, thepresence of other polyhalogenated aromatic compounds with possible AhR-activity should be taken into account.
Evaluation by CALUX assay and comparison with WHO-TEQ
Figure 1 shows the result of comparing CALUX-TEQ obtained from a PCDD/DFs fraction and DL-PCBs
fraction with both 1998 and 2005 WHO-TEQ measured by chemical analysis. The mean CALUX-TEQ for
PCDD/DFs was approximately three times higher, while DL-PCBs was approximately two times lower thanthose of WHO-TEQ. Such a tendency has been observed in other investigations that have analyzed
fish-originated samples 11-14. Meanwhile, CALUX-TEQ obtained from the sum of PCDD/DFs and DL-PCBs wasvery close to WHO-TEQ because the overestimation for PCDD/DFs was compensated by the underestimation
for DL-PCBs in the CALUX assay.
To examine the factors affecting the discrepancy found in TEQ measured by bioassay and chemical analysis,
REP-TEQ (theoretical CALUX-TEQ) were calculated by multiplying the concentration of each congener
determined by HRGC/HRMS by its CALUX-REP value instead of WHO-TEF, and compared to WHO-TEQ andCALUX-TEQ (Fig. 1). REP-TEQ of PCDD/DFs was found to be extremely close to WHO-TEQ; it is therefore
speculated that the presence of other AhR agonists, not but the difference between WHO-TEF and CALUX-REP
is responsible for the discrepancy. On the other hand, REP-TEQ of DL-PCBs was lower than WHO-TEQ andsimilar to CALUX-TEQ, thus the discrepancy in the two methods was probably due to the difference between
WHO-TEF and CALUX-REP. The REP value of PCB 126, which accounted for 79 (70-89) % of total 1998
WHO-TEQ in all samples, is 2.6 times less than WHO-TEF, resulting in approximately two times lower
CALUX-TEQ of DL-PCBs than WHO-TEQ.
25
WHO -TEQ (1998)

TEQ (pg/g ww)

20

WHO -TEQ (2005)
REP -TEQ15

CALUX -TEQ

1050

PCDD/DFs

DL-PCBs

PCDD/DFs+ DL-PCBs

Fig. 1. Comparison of mean TEQ levels in 41 fish oil samples obtained by HRGC/HRMS analysis and
CALUX assay. WHO-TEQ and REP-TEQ were calculated using 1998 and 2005 WHO-TEF, andCALUX-REP instead of WHO-TEF, respectively. Error bars are standard deviations.

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Relatively good relationships were observed in TEQ determined by the CALUX assay and HRGC/HRMS
analysis (Fig. 2). The correlation coefficients between CALUX-TEQ and 2005 WHO-TEQ were 0.82, 0.89, and0.90 for PCDD/DFs, DL-PCBs and the sum, respectively. The same result was obtained in 1998 WHO-TEQ,except for the 0.84 of PCDD/DFs. In the case of the sum of PCDD/DFs and DL-PCBs, WHO-TEQ could be
predicted from CALUX-TEQ, as the slope value of linear regression equation for the two methods is nearly 1(y=x). Although care must be taken when the results of the two methods are directly compared, the CALUX
assay in combination with reference chemical analysis could help to control the safety in feed production andcirculation.

2005 WHO-TEQ (pg/g wet wt.)(a) PCDD/DFs
y = 0.3581x - 0.5212
r = 0.82

20(c) PCDD/DFs + DL-PCBs(b) DL-PCBs
y = 2.2616x - 1.4747
r = 0.8930302515y = 0.8277x - 0.4171
r =0.903525202010151510510505005101520005101520253005101520253035

CALUX-TEQ (pg/g wet wt.)

Fig. 2. Correlation between CALUX-TEQ and 2005 WHO-TEQ determined by HRGC/HRMS analysis in 41
fish oil samples. CALUX-TEQ of (a), (b), (c) were obtained from PCDD/DFs fraction, DL-PCBs fraction, andsum of the result of (a) and (b), respectively. Dashed line indicates the bisecting linear correlation (y=x).
Acknowledgements
This work was supported in part by a Grant-in-aid under the Research project for utilizing advance technologies
in agriculture, forestry, and fisheries in Japan.
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