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Detection of dioxinlike activity in fish, sediment, and surface wipes using an in vitro bioassay
Chemosphere, Vol.18, Nos.l-6, P r i n t e d in G r e a t B r i t a i n
pp
793-800,
1989
0045-6535/89 $3.00 Perqamon Press plc
+
.OO
DETECTION OF DIOXINLIKE ACTIVITY IN FISH, SEDIMENT, AND SURFACE WIPES USING AN IN VITRO BIOASSAY J.F. Gierthy*, D.W. Lincoln, P. O'Keefe, R. Smith, C. Meyer, D. Hilker and K. Aldous Wadsworth Center for Laboratories and Research New York State Department of Health Albany, New York 12201, U.S.A. ABSTRACT Fish, sediment and surface wipes collected from various sites in New York State were tested for diox/nllke activity using an in vitro bloassay (Flat-Cell Assay). The assay is based on 2,3,7,8-tetrachlorodlbenzo-p-dioxln (TCDD) -induced alterations of post-confluent XBF mouse epithelial cell proliferation and morphology. This may be related to TCDD induction of epithelial differentiation, the cause of chloracne in humans. The bloassay results show qualitative and general quantitative agreement with those generated by gas chromatographlc-mass spectrometric analysis demonstrating the usefulness of the Flat-Cell Assay as a supplement to instrumental analysis. INTRODUCTION 2,3,7,8-Tetrachlorodlbenzo-p-dioxln
(TCDD) is the most biologically active member of a
large group of polyhalogenated aromatics which include polychlorlnated dlbenzodloxlns (PCDDs), dlbenzofurans (PCDFs) and blphenyls (PCBs) (I).
In animals, the wide range of
toxicity associated with these compounds is dependent upon specific structural characteristics (2), and exposure results in a pleotyplc toxic response (3).
One of the most common
clinical manifestations of human exposure is development of acneform lesions which appear to result from hyperkeratoses of the hair folllcles and associated sebaceous glands (3).
This
induced epithelial differentiation has led to the development of an extremely sensitive and relatively specific in vitro bloassay for dloxlnllke compounds (4) referred to here as the Flat-Cell Assay.
It is based on the induction, in epithelial cell cultures, of altered
morphology and cell growth, consistent with TCDD-Induced in vlvo epithelial differentiation
(5). This bioassay functions with extracts of soot (4), sediment (6), and fat (7).
The
current study was undertaken to test the bioassay in fish tissue and surface wipes and to expand testing of sediment samples. METHODS
Samples of fish and sediment from various New York State locations were extracted and cleaned
up t o v a r i o u s
the cleanup (8)
procedures
and comprise
degrees,
resulting
in a number of fractions.
and mass spectrometric
some o r a l l
of organic
(MS) a n a l y t l c a l
extraction,
793
sulfurlc
Complete d~scrlptions
methods are described acid
treatment
and
of
elsewhere
794
neutralization to remove £1sh oil, baslc alumina, carbon, and neutral alumina column proce-
dures.
The various fractions resulting from these sequential procedures were tested in the
Flat-Cell Assay as follows:
briefly, 1 ml of a benzene extract of each sample was solvent-
exchanged to 30 ul of dlmethylsulfoxlde (DMSO) and diluted I:I000 in culture medium (Dulbecco's modified minimal essential medium supplemented with 20% fetal bovine serum).
A series
of tenfold dilutlons was then made in culture medium. The indicator cells, designated XBF, were derived in this laboratory from XB mouse epithelial cells cloned from a mouse teratoma (9).
These cells exhibited a change in
morphology after being grown in medium containing as little as 3-4 ppt TCDD (4,5).
XBF cells
were seeded at a concentration of lO 5 cells/ml medlum/16 mm culture well in 24 well plastic culture plates together with irradiated 3T3 mouse flbroblast cells at 105 cells/ml medlum/16 well.
After 24 hours of incubation (37oc, 5% CO2, 95% humidity) the cultures were refed
with the previously described dilution series.
The cultures were incubated for 14 days, with
three weekly changes of medium containing freshly made dilutions of the sample extracts. cultures were then evaluated for the appearance of the flat-cell morphology.
The
The lowest
concentration of sample capable of inducing the flat-cell effect was taken as the endpolnt and equated to a TCDD standard dilutlon series to determine the TCDD equivalency of the sample as described elsewhere (4,5).
The values are reported here as a range, indicative of
the series of tenfold dilutions of the samples. In initial experiments, the extracts were divided in half and one portion was spiked with TCDD to determine if the sample itself interfered with the assay.
Cytotoxlc effects of
the samples were also monitored to assure that other, nonspeclflc toxic effects would not confound the test.
DMSO and TCDD in DMSO with and without coincidental solvent exchange were
used as solvent control and positive control respectively. RESULTS AND DISCUSSION Fish Samples Studies to determine the minimal chemical cleanup of the fish samples necessary for performing the assay were undertaken using samples which had undergone various stages of cleanup (Table I).
Duplicate samples were spiked with TCDD.
These studies showed that a
simple organic extraction (hexane-benzene) was inadequate due to the high oll content of the samples.
However, treatment of the samples with sulfuric acid to remove oil, followed by
neutralization, proved to be effective, yielding the same assay results as samples which had undergone the more extensive cleanup procedure for MS analysis.
No interference was noted
with the TCDD-splked samples (data not shown) or in samples spiked to I0 ppm Aroclor 1254 (sample 3, Table I). Thirteen fish samples from various sources were then subjected to this minimal cleanup and tested in the bloassay (Table 2).
The assay predicted the results of MS analysis for
TCDD in all samples which ranged from nondetectable (leas than 2.7 ppt) to 268 ppt TCDD equivalents. The ability to apply the assay to fish after such a minimal cleanup may be a consequence of removal of less potent chemicals such as certain polynuclear aromatic hydrocarbons by blotransformatlon and metabolism.
This study demonstrates that a protocol, using minimal
hexane-benzene and sulfuric acid-neutrallzatlon cleanup, can be established for the detection of dioxinlike activity in fish samples using the Flat-Cell Assay.
795
Application of Flat-Cell Assay (PCA) and Mass Spectrometric (MS) Analysis to Fish Samples Subjected to Progressive Cleanup Procedures
T a b l e I.
Sample No.
Type
Lake trout
Lake trout
3c
FCA TCDD equivalents (ppt)
Fractlon a
Lake trout plus I0 pl~n PCB
1 2 3 4 5
toxic 38.4-384 38.4-384 38.4-384 38.4-384
1
.....
2 3 4 5
NDb ( 1 6 - 3 2 ) HI) ( 1 6 - 3 2 ) ND ( 1 6 - 3 2 ) ND ( 1 6 - 3 2 )
1 2 3 4 5
..... ND (16-32) ND (16-32)
MS TCDD (ppt)
107
(1)
ND ( 1 6 - 3 2 ) ND (16-32)
m) (1)
a
Fraction 1 - extraction; 2 - I+ H2S04, neutralization; 3 - 2+ basic alumina; 4 - 3+ carbon; 5 - 4+ neutral alumina.
b
ND
c
Sample 3 spiked with i0 ppm Aroclor 1254.
-
Not Detected to values in ().
Sediment Samples Preliminary sample fractionation experiments were performed to assess conditions suitable for sediment samples.
A sediment sample (number 17) was subjected to extraction and five
fractionation steps, each of which was monitored by the Flat-Cell Assay (Table 3).
The
results showed that the dloxinllke activity seen in fraction 1 (extract) disappeared by fraction 3.
This indicates the possible presence of certain polynuclear aromatic hydrocar-
bons (PARs) which are weakly active in this assay as compared to TCDD (4) since this progressive cleanup procedure removes PARs by fraction 3.
Based on these results, the minimum
cleanup procedure needed for sediment analysis for detection of dioxinlike compounds involved extraction, multlcolumn and basic alumina.
Experiments were performed on six samples which
were first extracted with an organic solvent (fraction I) followed by multicolumn plus basic alumina treatments (fraction 2). were assumed to be absent.
If neither fraction had activity, then dloxinllke compounds
If activity was present only in fraction I, the activity was at-
tributed to relatively hlgh (compared to TCDD) concentrations of PARs. and 2 had dioxlnllke activity, indicated. quantitation.
If both fractions 1
the presence of a polyhalogenated dloxlnllke compound was
MS analysis was performed on each sample for isomer specific identification and The r e s u l t s
of these experiments
are
sho1~n i n T a b l e 4.
These data
for two of the samples, 21 and 22, no decline in activity was seen with cleanup. indicated the presence of dioxlnllke chemicals in both samples.
show t h a t
This
However, MS analysis for
TCDD showed this compound to be present, at the predicted concentration, only in sample 21.
796
Table 2.
Appllcatlon of Flat-Cell Assay (FCA) to Fish Samples Subjected to Minlmal Cleanup
Procedures and Compared to Mass S p e ct r o m et r i c (MS) Analysls
a
FCA TCDD equlvalents
Type
Sample No.
4
Coho Salmon
5
Brown trout
6
Carp
7
Goldflsh/Carp
8
Spot Tall Shiner
9
Spot Tall Shiner
3.0-30
10.7
10
Spot Tall Shiner
2.7-28
6.8
11
Spot Tall Shiner
53-530
127
12
Spot Tall Shiner
1.9-19
7.8
13
Spot Tall Shiner
6-60
50
14
Spot Tall Shiner
2.1-21
4.9
15
Spot Tall Shiner
12-120
113
16
Spot Tall Shiner
ND (3-6)
ND - Not Detected
Table 3.
ND a (2.7-5.4) 5.3-53.3
133
5.3-53.3
32
ND (5.3-10.7)
Extract;
PCA TCDD equivalents
Fraction s
2 -
Not Detected
ND (3.0)
ND (2)
Appllcatlon of Flat-Cell Assay (FCA) and Mass Spectrometric Sediment Sample Subjected to Various Cleanup Procedures
Multlcolumn;
0.32-3.2
2
0.32-3.2
3
ND b (0.16-0.32)
4
~
5
ND (0.16-0.32)
6
ND (0.16-0.32)
3 -
to
values
in
().
2+
Basic
(MS) Analysis
(ppb)
1
alumina; 6 - 5+ Silica. ND -
Ii
to values in ().
17
I -
(ppt)
ND (i, 5.3)
26.7-268
Sample No.
b
MS 2,3,7,8-TCDD
(ppt)
to a
MS TCDD (ppb)
(0.16-0.32)
alum/ha; 4 - 3+ Carbon;
~V (0.003)
5 - 4+ Neutral
797
Application of Flat-Cell A s s a y (FCA) a n d Mass S p e c t r o m e t r i c S e d i m e n t S a m p l e s S u b j e c t e d to V a r i o u s C l e a n u p P r o c e d u r e s
T a b l e 4.
S a m p l e No.
FCA TCDD equivalents (ppb)
Fraction a
18 b
19
2O
21
22
23 b
a
Fraction
i - Extract;
b
Method (18) and soll
c
Results
to
MS TCDD (ppb)
I 2
ND (0.055) ND (0.055)
1 2
0.27-2.7 0.027-0.27
1 2
2.7-27 0.3-2.7
0.23
1 2
5.5-55 5.5-55
29, 9.3 c
i 2
27-270 27-270
0.958
1 2
2.7-27 0.055-0.55
0.002
2 - Multlcolumn + basic blank
(MS) A n a l y s i s
0.007
0.045, 0.012 c
alumina.
(23).
o f two d e t e r m i n a t i o n s .
Very little TCDD was seen in sample 22, although it was very active in the Flat-Cell Assay. Further analysis revealed the presence of an unsuspected dloxin analogue, 2,3,7,8-tetrachlorothlanthrene Surface
(6),
which is
used in the chemical
industry
as a chlorination
catalyst.
Wipes
Dry w i p e s a m p l e s Office
Buildlng
a flre
involvlng
collected
from walls
of the 7th and 11th floors
(BSOB), w h i c h was c o n t a m i n a t e d PCBs ( 1 0 ) ,
were analyzed
of the Binghamton State
w i t h PCDD- and P C D F - l a d e n s o o t
f o r FCDDs a n d PCDFs a n d were t e s t e d
resulting for
from
their
ability to induce dioxlnllke activity in the Flat-Cell Assay. The c h e m i c a l
analysis
of these
samples
showed t h a t
2,3,7,8-tetrachlorodlbenzofuran
(TCDF)
concentrations were relatively consistent throughout the samples, with a range of 0.5 to I.i ng/m 2 (data not shown), as were the concentrations of total PCDF congeners and isomers (Table 5).
The r e s u l t s
the ten samples.
show t h a t
dloxtnllke
no f l a t - c e l l
inducing
Field
and the Reagent
Blank,
results tion
indicate
of dioxinlike
activity
S a m p l e s 24 a n d 29 w e r e t o x i c
that
activity
at
flat-cell
the highest
of that
Assay has potential
in extracts
effect)
was d e t e c t e d
concentration
concentration.
Blank (sample 33) exhibited
the Flat-Cell
activity
one tenth
(the at
activity.
the detection
of dry wipe samples of vinyl
in six
walls
of
and showed
S a m p l e 32 w h i c h i s a
no d i o x i n l i k e for
tested
These
and semiquantitao f t h e BSOB.
798
Comparison of Flat-Cell Assay (FCA) and Mass Spectrometry (MS) Analysis of Dry Wipe of Vinyl Wall of t h e Blnghamton S t a t e Office Building
Table 5.
FCA TCDF equlvalents a (ng/m 2)
Sample No.
MS Total PCDF (ng/m 2)
24
< 19-38
18
25
2-19
12
26
4-38
8
27
4-38
5
28
4-38
15
29
< 19-38
15
30
4-38
I0
31
4-38
22
32
ND b (2-4)
ND
33 Blank
ND (2-4)
ND
TCDF equivalents are based on 2,3,7,8-tetrachlorodlbenzofuran flat-cell producing potential st TCDD (4).
(TCDF) having one tenth the
ND = Not Detectable to values in ().
Perhaps more importantly, it also allowed segregation of the two negative samples from those demonstrating dloxlnllke activity. CONCLUSION Dioxinllke activity is defined here by the induction of the flat-cell effect by TCDD, the most biologically active PCDD isomer known.
As such, the aggregate toxicity of a sample,
whether comprised of high concentrations of a less toxic isomer or low concentrations of highly toxic isomer, is determined and expressed as TCDD equivalents rather than as specific concentrations of isomers and congeners whose biological activity and relevance are unknown. This approach will also take into account possible synergisms and antagonisms which may be associated with complex environmental mixtures such as those found in toxic waste dump sites or in used PCB mixtures found in electrical transformers and capacitors. The Flat-Cell Assay for dloxlnllke activity as well as other bloasssys have potential usefulness in s number of applications.
They can be used as a supplement to the more
expensive hlgh-resolutlon chemical analysis by identifying those samples with no dioxlnllke activity.
These samples could then be removed from the positive samples scheduled for
subsequent chemical analysis for definite identification and quantitatlon of specific congeners and isomers. specific taminated
locations
This appllcatlon would be particularly useful in delineating the
of dloxln
hot spots
in a land area which is suspected
of being con-
or in the mass screening of large numbers of fish or other biota to track the
"/99 source of c o n t a m i n a t i o n , e . g . ,
along a bifurcated stream system.
r e p o r t demonstrate the a b i l i t y
of the F l a t - C e l l
such a s s o o t , s u r f a c e w i p e s , and f i s h , of a c t i v i t y .
The s e m i q u a n t l t a t i v e
r a n k i n g of p o s i t i v e use of t h i s
The d a t a p r e s e n t e d i n t h i s
Assay to d i s c r i m i n a t e between v a r i o u s samples
w i t h r e g a r d t o l a c k of a c t i v i t y
d e t e r m i n a t i o n of d l o x i n l l k e a c t i v i t y
samples for subsequent instrument a n a l y s i s ,
and low or h i g h l e v e l s would a l l o w p r i o r i t y
t h u s making more e f f i c i e n t
llmlted resource.
A n o t h e r u s e of b l o a s s a y s i s t h e d e t e c t i o n of compounds which e x h i b i t d i o x i n l i k e a c t i v i t y but are not the subject of chemical analysis for a particular sample. previously described detection of 2,3,7,8-tetrachlorothianthrene,
An example is the
a sulfur analog of TCDD,
whose activity was detected using the Flat-Cell Assay in a sediment sample from a municipal sanitary sewer. This sample contained very low levels of TCDD, as shown by MS analysis, which could not account for the high activity seen in the bloassay.
This resulted in further
chemical analysis which allowed the detection, quantltatlon and presumable determination of the source of this compound (6). Bioassays may also be used in the examination of suspect compounds, such as PCDD analogs, for dloxlnlike activity.
An example of this is the detection, by chemical analysis, of
1,2,4,5,7,8-hexachloroxanthene
(HCX) at dioxin sites in Missouri.
product of hexachlorophene production. data were available on this compound.
Like TCDD, HCX is a by-
Risk assessment was impossible, since no toxicity Pure HCX was tested using the Flat-Cell Assay and the
results indicate that the dloxinlike activity associated with this compound, if any, was about 10 6 less than TCDD (ii).
Based on these data, the preliminary determination was made
that HCX posed no additional health hazard, since it occurred mainly at dioxin sites. Subsequent animal toxicity studies using guinea pigs also indicated very low toxicity associated with this compound thereby confirming the initial bioassay result (12). Other studies performed in this laboratory, to be published elsewhere, suggest that the Flat-Cell Assay may be useful for the detection and quantitation of aggregate dioxinlike activity in PCB mixtures used as dielectric fluids in electrical power equipment.
This
material is normally subjected to electrical arcing which can result in conversion of PCBs to more toxic PCDFs.
This results in an increase in flat-cell inducing potential which may be
used as an initial estimate of contamination by PCDFs. ACKNOWLEDGEMENTS The authors thank Ms. Victoria Lamberton for preparation of the typescript.
Portions of
this chapter were published in Chlorinated Dloxins and Dibenzofurans in Perspective, copyright Lewis Pub., Inc. (1986), and are reproduced here by permission.
This work was sup-
ported in part by NIEHS grant ES03561 and the New York State Department of Environmental Conservation.
REFERENCES I.
P o l a n d , A. and K n u t s o n , J . C . ,
2.
Safe, S., Chemosphere, 16, 791-802 ( 1 9 8 7 ) .
Ann. Rev. Pharmacol. T o x l c o l . 22, 517-554 (1982).
3.
Klmbrough, R . , CRC C r l t i c a l
4.
Gierthy, J.F.,
and Crane, D., Fund. App1. T o x l c o l . ,
5.
Gierthy, J.F.,
and Crane, D., T o x l c o l . Appl. P h a r m a c o l . , 74, 91-98 ( 1 9 8 4 ) .
Reviews i n T o x l c o l o s ~ , J a n . ,
445-498 (1974).
5, 754-759 (1985).
800
6.
Hilker, D.R., Aldous, K.M., Smith, R.M,, O'Keefe, P.W., Gierthy, J.F., Juruslk, J.,
7.
Ryanj J.J., Lau, B.P.Y., Hardy, J.A., O'Keefe, P., and Gierthy, J.F., Chemosphere, 15,
8.
537-548 (1986). O'Keefe, P., Hilker, D., Meyer, C., Aldous, K., Shane, L., Donnelly, R., and Smith, R.,
9.
Rhelnwald, J.G., and Green, H., Cell, 6, 317-330 (1975).
Hibblns, S.W., Spink, D., and Parillo, R.J., Chemosphere, 14, 1275-1284 (1985).
Chemosphere, 13, 849-860 (1984). I0.
O'Keefe, P.W., Silkworth, J.B., Gierthy, J.F., Smith, R.M., DeCaprlo, A.P., Turner, J.N., Eadon, G., Hilker, D.R., Aldous, K.M., Kamlnsky, L.S., and Collins, D.N., Environmental Health Perspectives, 60, 201-209 (1985).
Ii.
Viswanathan, T.S., Kleopfer, R.D., and Gierthy, J.F., Chemosphere, 16, 869-874 (1987).
12.
DeCaprlo, A.P., Briggs, R.~ Cierthy, J.F., Kim, J.C.S., and Kleopfer, R.D., J. Toxicol. Environ. Health, 20, 241-248 (1987).
pp
793-800,
1989
0045-6535/89 $3.00 Perqamon Press plc
+
.OO
DETECTION OF DIOXINLIKE ACTIVITY IN FISH, SEDIMENT, AND SURFACE WIPES USING AN IN VITRO BIOASSAY J.F. Gierthy*, D.W. Lincoln, P. O'Keefe, R. Smith, C. Meyer, D. Hilker and K. Aldous Wadsworth Center for Laboratories and Research New York State Department of Health Albany, New York 12201, U.S.A. ABSTRACT Fish, sediment and surface wipes collected from various sites in New York State were tested for diox/nllke activity using an in vitro bloassay (Flat-Cell Assay). The assay is based on 2,3,7,8-tetrachlorodlbenzo-p-dioxln (TCDD) -induced alterations of post-confluent XBF mouse epithelial cell proliferation and morphology. This may be related to TCDD induction of epithelial differentiation, the cause of chloracne in humans. The bloassay results show qualitative and general quantitative agreement with those generated by gas chromatographlc-mass spectrometric analysis demonstrating the usefulness of the Flat-Cell Assay as a supplement to instrumental analysis. INTRODUCTION 2,3,7,8-Tetrachlorodlbenzo-p-dioxln
(TCDD) is the most biologically active member of a
large group of polyhalogenated aromatics which include polychlorlnated dlbenzodloxlns (PCDDs), dlbenzofurans (PCDFs) and blphenyls (PCBs) (I).
In animals, the wide range of
toxicity associated with these compounds is dependent upon specific structural characteristics (2), and exposure results in a pleotyplc toxic response (3).
One of the most common
clinical manifestations of human exposure is development of acneform lesions which appear to result from hyperkeratoses of the hair folllcles and associated sebaceous glands (3).
This
induced epithelial differentiation has led to the development of an extremely sensitive and relatively specific in vitro bloassay for dloxlnllke compounds (4) referred to here as the Flat-Cell Assay.
It is based on the induction, in epithelial cell cultures, of altered
morphology and cell growth, consistent with TCDD-Induced in vlvo epithelial differentiation
(5). This bioassay functions with extracts of soot (4), sediment (6), and fat (7).
The
current study was undertaken to test the bioassay in fish tissue and surface wipes and to expand testing of sediment samples. METHODS
Samples of fish and sediment from various New York State locations were extracted and cleaned
up t o v a r i o u s
the cleanup (8)
procedures
and comprise
degrees,
resulting
in a number of fractions.
and mass spectrometric
some o r a l l
of organic
(MS) a n a l y t l c a l
extraction,
793
sulfurlc
Complete d~scrlptions
methods are described acid
treatment
and
of
elsewhere
794
neutralization to remove £1sh oil, baslc alumina, carbon, and neutral alumina column proce-
dures.
The various fractions resulting from these sequential procedures were tested in the
Flat-Cell Assay as follows:
briefly, 1 ml of a benzene extract of each sample was solvent-
exchanged to 30 ul of dlmethylsulfoxlde (DMSO) and diluted I:I000 in culture medium (Dulbecco's modified minimal essential medium supplemented with 20% fetal bovine serum).
A series
of tenfold dilutlons was then made in culture medium. The indicator cells, designated XBF, were derived in this laboratory from XB mouse epithelial cells cloned from a mouse teratoma (9).
These cells exhibited a change in
morphology after being grown in medium containing as little as 3-4 ppt TCDD (4,5).
XBF cells
were seeded at a concentration of lO 5 cells/ml medlum/16 mm culture well in 24 well plastic culture plates together with irradiated 3T3 mouse flbroblast cells at 105 cells/ml medlum/16 well.
After 24 hours of incubation (37oc, 5% CO2, 95% humidity) the cultures were refed
with the previously described dilution series.
The cultures were incubated for 14 days, with
three weekly changes of medium containing freshly made dilutions of the sample extracts. cultures were then evaluated for the appearance of the flat-cell morphology.
The
The lowest
concentration of sample capable of inducing the flat-cell effect was taken as the endpolnt and equated to a TCDD standard dilutlon series to determine the TCDD equivalency of the sample as described elsewhere (4,5).
The values are reported here as a range, indicative of
the series of tenfold dilutions of the samples. In initial experiments, the extracts were divided in half and one portion was spiked with TCDD to determine if the sample itself interfered with the assay.
Cytotoxlc effects of
the samples were also monitored to assure that other, nonspeclflc toxic effects would not confound the test.
DMSO and TCDD in DMSO with and without coincidental solvent exchange were
used as solvent control and positive control respectively. RESULTS AND DISCUSSION Fish Samples Studies to determine the minimal chemical cleanup of the fish samples necessary for performing the assay were undertaken using samples which had undergone various stages of cleanup (Table I).
Duplicate samples were spiked with TCDD.
These studies showed that a
simple organic extraction (hexane-benzene) was inadequate due to the high oll content of the samples.
However, treatment of the samples with sulfuric acid to remove oil, followed by
neutralization, proved to be effective, yielding the same assay results as samples which had undergone the more extensive cleanup procedure for MS analysis.
No interference was noted
with the TCDD-splked samples (data not shown) or in samples spiked to I0 ppm Aroclor 1254 (sample 3, Table I). Thirteen fish samples from various sources were then subjected to this minimal cleanup and tested in the bloassay (Table 2).
The assay predicted the results of MS analysis for
TCDD in all samples which ranged from nondetectable (leas than 2.7 ppt) to 268 ppt TCDD equivalents. The ability to apply the assay to fish after such a minimal cleanup may be a consequence of removal of less potent chemicals such as certain polynuclear aromatic hydrocarbons by blotransformatlon and metabolism.
This study demonstrates that a protocol, using minimal
hexane-benzene and sulfuric acid-neutrallzatlon cleanup, can be established for the detection of dioxinlike activity in fish samples using the Flat-Cell Assay.
795
Application of Flat-Cell Assay (PCA) and Mass Spectrometric (MS) Analysis to Fish Samples Subjected to Progressive Cleanup Procedures
T a b l e I.
Sample No.
Type
Lake trout
Lake trout
3c
FCA TCDD equivalents (ppt)
Fractlon a
Lake trout plus I0 pl~n PCB
1 2 3 4 5
toxic 38.4-384 38.4-384 38.4-384 38.4-384
1
.....
2 3 4 5
NDb ( 1 6 - 3 2 ) HI) ( 1 6 - 3 2 ) ND ( 1 6 - 3 2 ) ND ( 1 6 - 3 2 )
1 2 3 4 5
..... ND (16-32) ND (16-32)
MS TCDD (ppt)
107
(1)
ND ( 1 6 - 3 2 ) ND (16-32)
m) (1)
a
Fraction 1 - extraction; 2 - I+ H2S04, neutralization; 3 - 2+ basic alumina; 4 - 3+ carbon; 5 - 4+ neutral alumina.
b
ND
c
Sample 3 spiked with i0 ppm Aroclor 1254.
-
Not Detected to values in ().
Sediment Samples Preliminary sample fractionation experiments were performed to assess conditions suitable for sediment samples.
A sediment sample (number 17) was subjected to extraction and five
fractionation steps, each of which was monitored by the Flat-Cell Assay (Table 3).
The
results showed that the dloxinllke activity seen in fraction 1 (extract) disappeared by fraction 3.
This indicates the possible presence of certain polynuclear aromatic hydrocar-
bons (PARs) which are weakly active in this assay as compared to TCDD (4) since this progressive cleanup procedure removes PARs by fraction 3.
Based on these results, the minimum
cleanup procedure needed for sediment analysis for detection of dioxinlike compounds involved extraction, multlcolumn and basic alumina.
Experiments were performed on six samples which
were first extracted with an organic solvent (fraction I) followed by multicolumn plus basic alumina treatments (fraction 2). were assumed to be absent.
If neither fraction had activity, then dloxinllke compounds
If activity was present only in fraction I, the activity was at-
tributed to relatively hlgh (compared to TCDD) concentrations of PARs. and 2 had dioxlnllke activity, indicated. quantitation.
If both fractions 1
the presence of a polyhalogenated dloxlnllke compound was
MS analysis was performed on each sample for isomer specific identification and The r e s u l t s
of these experiments
are
sho1~n i n T a b l e 4.
These data
for two of the samples, 21 and 22, no decline in activity was seen with cleanup. indicated the presence of dioxlnllke chemicals in both samples.
show t h a t
This
However, MS analysis for
TCDD showed this compound to be present, at the predicted concentration, only in sample 21.
796
Table 2.
Appllcatlon of Flat-Cell Assay (FCA) to Fish Samples Subjected to Minlmal Cleanup
Procedures and Compared to Mass S p e ct r o m et r i c (MS) Analysls
a
FCA TCDD equlvalents
Type
Sample No.
4
Coho Salmon
5
Brown trout
6
Carp
7
Goldflsh/Carp
8
Spot Tall Shiner
9
Spot Tall Shiner
3.0-30
10.7
10
Spot Tall Shiner
2.7-28
6.8
11
Spot Tall Shiner
53-530
127
12
Spot Tall Shiner
1.9-19
7.8
13
Spot Tall Shiner
6-60
50
14
Spot Tall Shiner
2.1-21
4.9
15
Spot Tall Shiner
12-120
113
16
Spot Tall Shiner
ND (3-6)
ND - Not Detected
Table 3.
ND a (2.7-5.4) 5.3-53.3
133
5.3-53.3
32
ND (5.3-10.7)
Extract;
PCA TCDD equivalents
Fraction s
2 -
Not Detected
ND (3.0)
ND (2)
Appllcatlon of Flat-Cell Assay (FCA) and Mass Spectrometric Sediment Sample Subjected to Various Cleanup Procedures
Multlcolumn;
0.32-3.2
2
0.32-3.2
3
ND b (0.16-0.32)
4
~
5
ND (0.16-0.32)
6
ND (0.16-0.32)
3 -
to
values
in
().
2+
Basic
(MS) Analysis
(ppb)
1
alumina; 6 - 5+ Silica. ND -
Ii
to values in ().
17
I -
(ppt)
ND (i, 5.3)
26.7-268
Sample No.
b
MS 2,3,7,8-TCDD
(ppt)
to a
MS TCDD (ppb)
(0.16-0.32)
alum/ha; 4 - 3+ Carbon;
~V (0.003)
5 - 4+ Neutral
797
Application of Flat-Cell A s s a y (FCA) a n d Mass S p e c t r o m e t r i c S e d i m e n t S a m p l e s S u b j e c t e d to V a r i o u s C l e a n u p P r o c e d u r e s
T a b l e 4.
S a m p l e No.
FCA TCDD equivalents (ppb)
Fraction a
18 b
19
2O
21
22
23 b
a
Fraction
i - Extract;
b
Method (18) and soll
c
Results
to
MS TCDD (ppb)
I 2
ND (0.055) ND (0.055)
1 2
0.27-2.7 0.027-0.27
1 2
2.7-27 0.3-2.7
0.23
1 2
5.5-55 5.5-55
29, 9.3 c
i 2
27-270 27-270
0.958
1 2
2.7-27 0.055-0.55
0.002
2 - Multlcolumn + basic blank
(MS) A n a l y s i s
0.007
0.045, 0.012 c
alumina.
(23).
o f two d e t e r m i n a t i o n s .
Very little TCDD was seen in sample 22, although it was very active in the Flat-Cell Assay. Further analysis revealed the presence of an unsuspected dloxin analogue, 2,3,7,8-tetrachlorothlanthrene Surface
(6),
which is
used in the chemical
industry
as a chlorination
catalyst.
Wipes
Dry w i p e s a m p l e s Office
Buildlng
a flre
involvlng
collected
from walls
of the 7th and 11th floors
(BSOB), w h i c h was c o n t a m i n a t e d PCBs ( 1 0 ) ,
were analyzed
of the Binghamton State
w i t h PCDD- and P C D F - l a d e n s o o t
f o r FCDDs a n d PCDFs a n d were t e s t e d
resulting for
from
their
ability to induce dioxlnllke activity in the Flat-Cell Assay. The c h e m i c a l
analysis
of these
samples
showed t h a t
2,3,7,8-tetrachlorodlbenzofuran
(TCDF)
concentrations were relatively consistent throughout the samples, with a range of 0.5 to I.i ng/m 2 (data not shown), as were the concentrations of total PCDF congeners and isomers (Table 5).
The r e s u l t s
the ten samples.
show t h a t
dloxtnllke
no f l a t - c e l l
inducing
Field
and the Reagent
Blank,
results tion
indicate
of dioxinlike
activity
S a m p l e s 24 a n d 29 w e r e t o x i c
that
activity
at
flat-cell
the highest
of that
Assay has potential
in extracts
effect)
was d e t e c t e d
concentration
concentration.
Blank (sample 33) exhibited
the Flat-Cell
activity
one tenth
(the at
activity.
the detection
of dry wipe samples of vinyl
in six
walls
of
and showed
S a m p l e 32 w h i c h i s a
no d i o x i n l i k e for
tested
These
and semiquantitao f t h e BSOB.
798
Comparison of Flat-Cell Assay (FCA) and Mass Spectrometry (MS) Analysis of Dry Wipe of Vinyl Wall of t h e Blnghamton S t a t e Office Building
Table 5.
FCA TCDF equlvalents a (ng/m 2)
Sample No.
MS Total PCDF (ng/m 2)
24
< 19-38
18
25
2-19
12
26
4-38
8
27
4-38
5
28
4-38
15
29
< 19-38
15
30
4-38
I0
31
4-38
22
32
ND b (2-4)
ND
33 Blank
ND (2-4)
ND
TCDF equivalents are based on 2,3,7,8-tetrachlorodlbenzofuran flat-cell producing potential st TCDD (4).
(TCDF) having one tenth the
ND = Not Detectable to values in ().
Perhaps more importantly, it also allowed segregation of the two negative samples from those demonstrating dloxlnllke activity. CONCLUSION Dioxinllke activity is defined here by the induction of the flat-cell effect by TCDD, the most biologically active PCDD isomer known.
As such, the aggregate toxicity of a sample,
whether comprised of high concentrations of a less toxic isomer or low concentrations of highly toxic isomer, is determined and expressed as TCDD equivalents rather than as specific concentrations of isomers and congeners whose biological activity and relevance are unknown. This approach will also take into account possible synergisms and antagonisms which may be associated with complex environmental mixtures such as those found in toxic waste dump sites or in used PCB mixtures found in electrical transformers and capacitors. The Flat-Cell Assay for dloxlnllke activity as well as other bloasssys have potential usefulness in s number of applications.
They can be used as a supplement to the more
expensive hlgh-resolutlon chemical analysis by identifying those samples with no dioxlnllke activity.
These samples could then be removed from the positive samples scheduled for
subsequent chemical analysis for definite identification and quantitatlon of specific congeners and isomers. specific taminated
locations
This appllcatlon would be particularly useful in delineating the
of dloxln
hot spots
in a land area which is suspected
of being con-
or in the mass screening of large numbers of fish or other biota to track the
"/99 source of c o n t a m i n a t i o n , e . g . ,
along a bifurcated stream system.
r e p o r t demonstrate the a b i l i t y
of the F l a t - C e l l
such a s s o o t , s u r f a c e w i p e s , and f i s h , of a c t i v i t y .
The s e m i q u a n t l t a t i v e
r a n k i n g of p o s i t i v e use of t h i s
The d a t a p r e s e n t e d i n t h i s
Assay to d i s c r i m i n a t e between v a r i o u s samples
w i t h r e g a r d t o l a c k of a c t i v i t y
d e t e r m i n a t i o n of d l o x i n l l k e a c t i v i t y
samples for subsequent instrument a n a l y s i s ,
and low or h i g h l e v e l s would a l l o w p r i o r i t y
t h u s making more e f f i c i e n t
llmlted resource.
A n o t h e r u s e of b l o a s s a y s i s t h e d e t e c t i o n of compounds which e x h i b i t d i o x i n l i k e a c t i v i t y but are not the subject of chemical analysis for a particular sample. previously described detection of 2,3,7,8-tetrachlorothianthrene,
An example is the
a sulfur analog of TCDD,
whose activity was detected using the Flat-Cell Assay in a sediment sample from a municipal sanitary sewer. This sample contained very low levels of TCDD, as shown by MS analysis, which could not account for the high activity seen in the bloassay.
This resulted in further
chemical analysis which allowed the detection, quantltatlon and presumable determination of the source of this compound (6). Bioassays may also be used in the examination of suspect compounds, such as PCDD analogs, for dloxlnlike activity.
An example of this is the detection, by chemical analysis, of
1,2,4,5,7,8-hexachloroxanthene
(HCX) at dioxin sites in Missouri.
product of hexachlorophene production. data were available on this compound.
Like TCDD, HCX is a by-
Risk assessment was impossible, since no toxicity Pure HCX was tested using the Flat-Cell Assay and the
results indicate that the dloxinlike activity associated with this compound, if any, was about 10 6 less than TCDD (ii).
Based on these data, the preliminary determination was made
that HCX posed no additional health hazard, since it occurred mainly at dioxin sites. Subsequent animal toxicity studies using guinea pigs also indicated very low toxicity associated with this compound thereby confirming the initial bioassay result (12). Other studies performed in this laboratory, to be published elsewhere, suggest that the Flat-Cell Assay may be useful for the detection and quantitation of aggregate dioxinlike activity in PCB mixtures used as dielectric fluids in electrical power equipment.
This
material is normally subjected to electrical arcing which can result in conversion of PCBs to more toxic PCDFs.
This results in an increase in flat-cell inducing potential which may be
used as an initial estimate of contamination by PCDFs. ACKNOWLEDGEMENTS The authors thank Ms. Victoria Lamberton for preparation of the typescript.
Portions of
this chapter were published in Chlorinated Dloxins and Dibenzofurans in Perspective, copyright Lewis Pub., Inc. (1986), and are reproduced here by permission.
This work was sup-
ported in part by NIEHS grant ES03561 and the New York State Department of Environmental Conservation.
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