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This is to certify that the

thesis entitled

Green Frogs in Southwestern Michigan:
Deformity Survey and Chemical Residues
in Water, Sediment, and Tissue

presented by
Carolyn Ann Dida

has been accepted towards fulﬁllment
of the requirements for

14.8 ' degree in ZOOIQSL

Major professor I

0-7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

 

Date March 3, 2000

 

 

 

 

LEBRARY

Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE ‘ DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

11/00 W.ﬂ6—p.14

 

GREEN FROGS IN SOUTHWESTERN MICHIGAN:
DEFORMITY SURVEY AND CHEMICAL RESIDUES
IN WATER, SEDIMENT, AND TISSUE

By

Carolyn Ann Duda

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Zoology
Institute for Environmental Toxicology

2000

ABSTRACT
GREEN FROGS IN SOUTHWESTERN MICHIGAN:
DEFORMITY SURVEY AND CHEMICAL RESIDUES
IN WATER, SEDIMENT, AND TISSUE
By

Carolyn Ann Duda

In an attempt to explain the etiology of frog deformities and population declines, many
possible causative factors have been examined, including the input of synthetic chemicals
into aquatic systems, where frogs spend much of their lives, including their entire
developmental stages. This study focused on surveying wetlands in southwestern
Michigan that are inﬂuenced by agricultural run-off or urban and industrial inputs for
deformities in green frogs. Of the 1445 green frogs examined in 1998, only 4 (0.28%)
exhibited morphological deformities. Water, sediment and tissue from frog eggs,
tadpoles, juveniles, and adults were analyzed for organochlorine (OC) insecticides,
polychlorinated biphenyls (PCBs), metals, and triazine herbicides by use of traditional
instrumental analysis for most compounds and by Enzyme-Linked Immunosorbent Assay
(ELISA) for triazine analysis. Sample extracts were also assayed for their estrogenic and
dioxin-like activities using recombinant cell lines. Overall, low concentrations were
observed in the frogs and their habitats. No correlation between deformities and

chemicals could be made due to the low deformity rate in the region.

ACKNOWLEDGMENTS

I’d like to thank my advisor, Dr. John Giesy, for helping to make this project possible and
for all his help along the way. Also, thanks are owed to the rest of my committee
members for their advice: Dr. Tom Burton, Dr. Frank D’Itri, Dr. Patrick Muzzall, and Dr.
Matt Zabik. I would also like to thank Dr. Kurunthachalam Kannan, Director of the
Environmental Chemistry Division of the Aquatic Toxicology Laboratory, Cheryl
Summer from the Michigan Department of Environmental Quality, and Dr. Chuck
Mehne from the Kalamazoo region for their long hours Spent helping to plan and
organize this project as well as for their continued advice and involvement in many facets
of this project. Acknowledgements are owed to Dennis Bush, Hillary Flower, Merritt
Gillilland, Karen Glennemeier, Robin Haas, Kevin Henry, K. Kannan, Katie Kemler,
J eong Seong Khim, Chuck Mehne, Patrick Muzzall, Cheryl Summer, and Dan Villeneuve
for their assistance in collecting green frogs on more than one occasion. Thanks are
owed to those individuals from the Department of Zoology and the Aquatic Toxicology
Laboratory who assisted with the chemical analysis and the cell bioassays: Merritt
Gillilland, K. Kannan, Denise Kay, Katie Kemler, Jeong Seong Khim, Stephanie Pastva,
and Dan Villeneuve. Lastly, thanks to the Environmental Geochemistry laboratory at
MSU for use of their laboratory equipment and ICP-MS, especially Dr. Dave Long, Dr.
Lina Patino, Matt Harold and Gary Icopini. This study was supported by the Great Lakes
Protection Fund, project number 73370. On a personal note, I’d like to thank my parents,
Robert and Cheryl Duda, for their support of and interest in my educational and personal

goals throughout the years.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................................... vi
LIST OF FIGURES ................................................................................................... ix
CHAPTER 1:

SURVEY OF GREEN FROG DEF ORMITIES IN SOUTHWESTERN MICHIGAN
Introduction .............................................................................................................. 1
Materials and Methods ................................................................................................ 7
Results and Discussion ............................................................................................. 12
References .............................................................................................................. 15
CHAPTER 2:

ORGANOCHLORINE INSECTICIDES AND PCBS IN WATER AND GREEN
F ROGS FROM SOUTHWESTERN MICHIGAN

Introduction ............................................................................................................ 18
Materials and Methods ............................................................................................. 22
Water and Frog Sampling and Preparation... ...22
F rog Tissue Extraction and Clean- Up ............................................................ 23
Water Extraction and Clean-Up ..................................................................... 25
Instrumental Analysis .................................................................................... 25
Cell Bioassay Analysis ................................................................................... 26
Results ................................................................................................................... 29
Lipids .......................................................................................................... 29
Organochlorine Insecticides .......................................................................... 3O
PCBs ........................................................................................................... 34
T CDD-EQs and EZ-EQs ................................................................................ 35
Discussion .............................................................................................................. 35
Conclusion .............................................................................................................. 42
References .............................................................................................................. 43
CHAPTER 3:
METALS IN SEDIMENT AND GREEN F ROGS FROM SOUTHWESTERN
MICHIGAN
Introduction ............................................................................................................ 48
Materials and Methods ............................................................................................. 53
Results ................................................................................................................... 55
Discussion .............................................................................................................. 60
Conclusion .............................................................................................................. 63
References .............................................................................................................. 64

iv

TABLE OF CONTENTS, CONTINUED

CHAPTER 4:
TRIAZINE HERBICIDE CONCENTRATION IN WATER AND GREEN F ROGS
FROM SOUTHWESTERN MICHIGAN

Introduction ............................................................................................................ 67
Materials and Methods ............................................................................................. 68

Water and Tadpole Sampling and Preparation ................................................. 68

Water Analysis with ELISA ............................................................................ 69

Tadpole Tissue Analysis with ELISA ................................................................ 70

Gas Chromatographic Analysis of Tadpole Tissue ............................................ 71
Results ................................................................................................................... 72
Discussion .............................................................................................................. 73
Conclusion .............................................................................................................. 74
References .............................................................................................................. 76
APPENDICES ........................................................................................................ 79
APPENDIX A ......................................................................................................... 80
APPENDIX B ......................................................................................................... 88
APPENDIX C ......................................................................................................... 93
APPENDIX D ....................................................................................................... 107

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

Table 1 1.

LIST OF TABLES

Sampling location characteristics. Page 10.

Number of green frogs collected, including number of deformities, from
southwestern Michigan, 1998. Page 13.

Prevalence of deformities, and total number of green frogs by location, in
southwestern Michigan. Page 14.

Mass, length, and percent lipid (mean and standard deviation, in parentheses)
of green frog adult, juvenile, tadpole, and egg tissue from southwestern
Michigan, 1998. Page 29.

Concentrations (ng/g, wet wt., mean and standard deviation, in parentheses)
of organochlorine insecticides and total PCBs in green frog tissue from
southwestern Michigan, 1998. Page 32.

Concentration (ng/g, wet wt., mean and standard deviation, in parentheses) of
organochlorine insecticides and total PCBS in green frog tissue from
sampling locations that are signiﬁcantly different at the 95% conﬁdence level
in southwestern Michigan, I998. Page 33.

Samples with signiﬁcant differences in organochlorine residue concentrations
among locations. Underlined locations are not signiﬁcantly different from
one another (0t=0.05). Locations are arranged in order of increasing
concentrations. Page 33.

Samples with signiﬁcant differences in organochlorine concentrations
between life stages. Underlined life stages are not signiﬁcantly different
(0t=0.05) from one another. Life stages are arranged in order of increasing
concentrations. Page 34.

Cell bioassay detection limits. Page 35.

Metal concentrations (pg/g, dry wt., mean and standard deviation, in
parentheses) in sediment and green frog tissue from southwestern Michigan,
1998. Page 58.

Metal concentrations (pg/g, dry wt., mean and standard deviation, in
parentheses) in sediment and green frog tissue from sampling locations that

are signiﬁcantly different at the 95% conﬁdence level in southwestern
Michigan, 1998. Page 59.

vi

Table 12.

Table 13.

Table A.l.

Table A2.

Table A.3.

Table A.4.

Table A.5.

Table A.6.

Table A.7.

Table 3.1.

Table 82.

LIST OF TABLES, CONTINUED

Samples for which metal concentrations varied signiﬁcantly among locations.
Underlined locations are not signiﬁcantly different (a=0.05). Locations are
arranged in order of increasing concentrations, from leﬁ to right. Page 60.

Triazine herbicide concentrations in water and green frog tadpole and
juvenile tissues from southwestern Michigan, 1998. Page 72.

Organochlorine insecticide concentrations (ng/g, wet wt., mean and range, in
parentheses) in adult green frog tissue from southwestern Michigan, 1998.
Page 81.

Organochlorine insecticide concentrations (ng/g, wet wt., mean and range, in
parentheses) in green frog tadpole and juvenile tissue from southwestern
Michigan, 1998. Page 82.

Organochlorine insecticide concentrations (ng/g, wet wt., mean and range, in
parentheses) in green frog egg tissue from southwestern Michigan, 1998.
Page 83.

Signiﬁcant differences of 0C concentrations (a=0.05) between life stages
and between locations and interactions. Shaded values indicate signiﬁcance.
Page 84.

Concentrations (ng/g, wet wt., mean and range, in parentheses) of total PCBs
and of di-, mono-, and non-ortho PCBs in adult green frog tissue from
southwestern Michigan, 1998. Page 85.

Concentrations (ng/g, wet wt., mean and range, in parentheses) of total PCBs
and of di-, mono», and non-ortho PCBs in green frog tadpole tissue from
southwestern Michigan, 1998. Page 86.

Concentrations (ng/g, wet wt., mean and range, in parentheses) of total PCBs
and of di-, mono-, and non-ortho PCBs in green frog egg tissue from
southwestern Michigan, 1998. Page 87.

Metal concentrations (pg/g, dry wt., mean and range, in parentheses) in
sediments from southwestern Michigan, 1998. Page 89.

Metal concentrations (pg/g, dry wt., mean and range, in parentheses) in adult
green frog tissue from southwestern Michigan, 1998. Page 90.

vii

LIST OF TABLES, CONTINUED

Table B.3. Metal concentrations (pg/g, dry wt., mean and range, in parentheses) in green
frog tadpole and juvenile tissue from southwestern Michigan, 1998. Page 91.

Table B.4. Signiﬁcant differences of metal concentrations (a=0.05) between life stages

and between locations and interactions. Shaded values indicate signiﬁcance.
Page 92.

viii

LIST OF FIGURES

Figure 1. Counties of southwestern Michigan from which green frogs were collected,
1998. Page 9.

ix

Chapter 1: Survey of Green Frog Deformities in Southwestern Michigan

Introduction

Amphibians belong to a class of about 4,550 species of caecilians, salamanders, toads,
and frogs (Stebbins and Cohen, 1995). They occupy a dual role in the ecosystem, serving
as predators to many insects and other small animals while acting as prey for a large
number of animals, including reptiles, birds, and mammals. Due to the important role of
amphibians in the ecosystem, a decline in their populations can have deleterious effects
upon many different populations of animals and, thus, upon the many different
ecosystems that they inhabit (Stebbins and Cohen, 1995). Further, amphibians serve as
excellent sentinels for the effects of chemical stressors during development. This is
because they are very sensitive to a variety of factors due to their complex life histories in
both the aquatic and terrestrial environments and the permeability of their skin to water
and electrolytes (Stebbins and Cohen, 1995). Therefore, a decline in amphibian
populations or the expression of developmental abnormalities could be indicative of

environmental changes, including exposure to xenobiotics.

Amphibian populations have been declining in various parts of the world for at least two
decades (Wake, 1991). In February 1990, a workshop sponsored by the National
Research Council’s Board on Biology documented declines over the past 20 years in
certain amphibian populations around the world. These declines include the almost
complete disappearance of boreal toads (Bufo boreas boreas) from their traditional

breeding range in the West Elk Mountains of Colorado (Carey, 1993), and golden toads

(Bufo periglenes) and the harlequin frog (Atelopus varius) from Costa Rica’s Monteverde

Cloud Forest Preserve in the mid-1980s (Pounds and Crump, 1994).

Recently, anatomical deformities of frogs have attracted international attention from both
the scientiﬁc community and the public. In 1995, a group of Minnesota schoolchildren
encountered a large number of northern leopard frogs (Rana pipiens) with hypermorphic
(multiple digits and/or limbs) and hypomorphic (missing digits and/or limbs) limb
abnormalities in a farm wetland in southern Minnesota (Kaiser, 1997). Malfonned frogs
and other amphibians had been studied previously in different regions; however, this
incident brought deformed frogs into the spotlight of many environmental and scientiﬁc

causes.

Deformed mink frogs (Rana septentrionalis) collected from a lake in Minnesota in 1996
and 1997 exhibited numerous limb abnormalities, the most notable being the presence of
more than the normal number of hind limbs (Gardiner and Hoppe, 1999). Out of the 23
frogs examined by skeletal analysis, there were a total of 101 hind limbs. The majority of
these hind limbs were abnormal due to skeletal dysplasia, characterized by triangles,
hypomorphism, split limbs, and supemumary distal elements, and due to skin webbing
(inter-limb, inter-segmental, and intra-segmental webbing). The authors speculated that
the deformities were caused by exposure to degraded products of the insecticide
methoprene that can mimic retinoic acid (Vitamin A), which is known to cause similar

deformities under laboratory conditions (LaClair et a1., 1998).

Populations of some of the 13 endemic frog and toad species in Michigan have declined
in recent years (Michigan Department of Natural Resources (MDNR), 1996), based upon
frog call surveys at known and historical breeding locations. The Blanchard’s cricket
frog (Acrz's crepitans blanchardi) and the boreal chorus frog (Pseudacris triseriata
maculata) are listed as “special concern” (Michigan Natural Features Inventory (MNFI),
1999). In 1996, the northern leopard frog was observed at only nine percent of its
expected breeding sites. Likewise, the green frog (Rana clamitans), wood ﬁog (Rana
sylvatz'ca), and the bullfrog were found at an average of 53, 47, and 4%, respectively, of

their expected breeding sites (MDNR, 1996).

Several hypotheses have been proposed to explain these population declines and
anatomical malformations. Several possible causative agents have been suggested to
cause frog deformities. The proposed mechanisms can be classiﬁed as chemical,
physical, or biological. Speciﬁcally, these hypotheses include the following: 1) chemical
contamination in the water (Burkhart et a1., 1998), 2) acid precipitation (Gannon, 1997),
3) increased levels of ammonia in the water (Jofre and Karasov, 1999), 4) increased
ultraviolet radiation due to the thinning ozone layer (Ankley et al., 1998), and 5) parasites
encysting in the developing limb buds (Sessions and Ruth, 1990). The aforementioned
factors can also contribute to population declines, as well as general diseases, habitat
destruction, climate changes, introduction of predators and competitors, and human

consumption (Wake, 1991).

It is possible that the observed effects are caused by not one, but a combination of factors
acting in combination (Wake, 1991). Furthermore, while the occurrence of deformities
and declines in amphibian populations seem to be global in nature, there is probably no
single universal factor to explain all of the population declines and deformities. For
example, exposure to synthetic chemicals may be affecting some populations whereas
increased UV irradiance may be involved in another region. Most of the proposed
hypotheses suggest that humans are contributing to the demise of amphibians and may be
indicative of environmental changes. However, most studies of amphibian populations
have been conducted relatively recently. Without long-term census data, it will be
difﬁcult to determine whether population declines are due to natural ﬂuctuations or

anthropogenic factors (Pechmann et al., 1991).

Amphibian deformities have been reported since the 1700s (Van Valen, 1974), which
suggests that the deformities currently being observed are a natural phenomenon.
Perhaps the increased attention on deformed frogs is leading to the greater number of
sightings of ﬁ'ogs with developmental abnormalities. Therefore, more research will be

required to fully understand the scope of the amphibian dilemma.

The UV radiation hypothesis has received some support, particularly in regions where the
ozone layer has decreased (Gannon, 1997). A less dense ozone layer allows more
ultraviolet radiation to reach the earth, where it can cause more damage to life (Berner
and Bemer, 1996). Deformed frogs have been found in the northern latitudes, where

increases in UV-B light have been shown to occur in late spring, early summer, and

coinciding with the amphibian breeding season (Ankley et al., 1998). A recent study
produced hind-limb deformities in leopard frog embryos by exposing them to UV-B
radiation under laboratory conditions for more than 24 days (Renner, 1998). However,
this study has also been the center of a great deal of debate, because the laboratory setting
for this study does not duplicate actual environmental settings, as far as physical
parameters in the water column and the amount of UV-B radiation received by the frog
embryos. Another study indicated that treatment of leopard frogs with UV-B light can
induce bilateral and oﬁen symmetrical ectromelia and ectrodactyly (missing limbs and
digits). The authors of this study also admit that these conditions and effects are not

representative of those in nature (Ankley et al., 1998).

The biological (parasite) explanation involves one or more types of parasite contributing
to the deformities through a variety of mechanisms (Johnson et al., 1999; Sessions and
Ruth, 1990). Two major studies have found that naturally occurring trematodes form
cysts in amphibian limb buds, which potentially disrupts limb development and leads to a
range of deformities. Recently, a ﬁmgus known as chytrid skin fungus was found in dead
and dying frogs in Arizona (Milius, 1998; Lips, 1999). This fungus has not been known
to infect vertebrates, but is the same fungus that is considered responsible for recent
amphibian die-offs in Australia and Central America. While this fungus appears to be
more responsible for amphibian deaths than deformities, it is still representative of the
wide variety of parasites that can infect frogs. One possibility is that the fungal infections

are secondary to lesions caused by the chemical or biological etiologies.

Chemicals, primarily pesticides and industrial byproducts, are ubiquitous in the
environment. Even remote areas experience some degree of contamination due to
atmospheric transport of pollutants, such as polychlorinated biphenyls (PCBs), DDT, and
mercury compounds (Wania et al., 1998). Frogs spend the greater part of their
developmental stages in the aquatic environment; hence, they can be directly exposed
during their critical growth period to any chemicals that may be in the water (Stebbins
and Cohen, 1995). Thus, some of these chemicals have the capacity to cause grth
anomalies. For instance, a recent study in the Quebec region found more frogs with
deformed hind limbs in areas where historically pesticides have been used than from
areas that have not been exposed to pesticides (Ouellet et al., 1997). However, no
concentrations of residues were measured. Thus, no correlations or gradients of
deformities and exposure to chemicals could be established. One pesticide shown to
cause life-threatening deformities in leopard frogs is methoprene (Ankley et al., 1998).
Degradation products of methoprene have been suggested to affect the retinoid-signaling
pathway via interaction with one or more retinoid receptors (Ankley et al., 1998). Many
of the compounds currently being studied in relation to the deformity issue have been
characterized as environmental endocrine disruptors, compounds that disrupt the normal

functioning of the endocrine system (Kendall et al., 1998).

To document deformities in southwestern Michigan, an amphibian deformity survey was
initiated in 1998. This deformity survey was a part of a larger study that attempted to
link deformities to mechanical disruption and chemical contamination. The objectives of

this study were to: l) assess the incidence of green frog (Rana clamitans) deformities in

southwestern Michigan; 2) determine concentrations of organochlorine insecticides,
PCBs, metals, and triazine herbicides in green frog adult, juvenile, tadpole, and egg
tissues from the sampling locations; 3) determine the levels of these compounds in the
water and/or sediment inhabited by green frogs; and 4) correlate the incidence of

deformities to chemical concentrations in the frogs and/or their environment.

Materials and Methods

The primary study region was southwestern Michigan (Figure 1). This region was
chosen because of the variety of habitats available. The sampling locations ranged from
agricultural areas, reference locations, and locations with known historical and current
PCB contamination (Table 1). Other regions of Michigan are not as likely to have the
available green frog habitat amidst these different locations. These seven locations were
chosen to give a wide variety of sampling sites with a wide range of potential chemical
inﬂuences. The reference locations (REFl, REF2, and REF 3) are within the Allegan
State Game Area in Allegan County, an area of successional forests that is not currently
and directly impacted by agriculture or urban activity. The agricultural areas (AGRl,
AGR2, and AGR3) are located in St. Joseph and Cass Counties, in a region characterized
by cash crop farming of corn and the usage of triazine herbicides, most notably atrazine.
The Kalamazoo River passes through urbanized and industrialized areas as well as
agricultural areas. The sampling location for this area (KLMZR) is a marsh created by a
nearby dam on the river in Kalamazoo County. Speciﬁc location ranges are given for the

reference and Kalamazoo River sampling locations (Table 1). Speciﬁc locations cannot

be given for the agricultural sampling locations based on request made by the farmers

who own the land.

There were four sampling dates during the summer of 1998 - June 5, June 23, July 22,
and August 26. Sampling for tadpoles took place during the daylight hours, while
sampling for adults occurred between sunset and sunrise. The sampling dates were
chosen based on agricultural applications of triazine herbicides, which would potentially
impact the herbicide concentrations of the agricultural locations. The sampling was
conducted early in the summer after triazine applications, then later in the summer when

less herbicides, if any, were applied (Mehne, personal communication).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Allegan County j l
1
Kalamazoo County L
Cass County \,
St. Joseph County

Figure 1. Counties of southwestern Michigan from which green frogs were collected,

1998.

Table 1. Sampling location characteristics.

 

Site

Characteristics

 

REF]

118ih pond, T.2N. R.14W., section 17. Permanent pond, depth 0-5 ft.,
various grasses border the pond edge, lily pads, submergent vegetation
present. This sampling location is surrounded by mixed hardwood
forest.

 

REF2

Crooked Lake, T.2N. R.15W., section 36. Small lake, depth 0-8 11.,
cattails and various grasses border the lake edge, submergent vegetation
present. This sampling location is surrounded by mixed hardwood
forest.

 

REF3

Swan Creek, T.2N R. 14W., section 20. Marsh in a drainage area, depth
of 0-8 ft., various grasses border the marsh edge, submergent vegetation
is sparse. This sampling location is surrounded by mixed hardwood
forest.

 

KLMZR

Kalamazoo River, T.2N R.14W., section 10. Marsh in a drainage area,
formed from a man-made dam along the Kalamazoo River, depth 0-6
ft., cattails, various grasses, and mixed hardwoods border the marsh
edge, submergent vegetationpresent.

 

AGRl

Temporary pond, depth 0-5 ft., cattails, saw grass, and other various
grasses border the pond edge, submergent vegetation present.
Surrounded by com ﬁelds in an agricultural region.

 

AGR2

Permanent pond, depth 0-3 ﬁ., cattails and various grasses border the
pond edge, submergent vegetation present. Surrounded by com ﬁelds
in an agricultural region.

 

AGR3

 

Drainage ditch, depth 0-3 ft., cattails and various grasses border the
ditch edge, no submergent vegetation. This sampling location is located
in an agricultural region.

The green frog (Rana clamitans) was selected for three reasons. First, the green frog is

one of the most abundant frogs in the Great Lakes region (Harding, 1997); thus, these

frogs are relatively easy to locate and the study will not be negatively impacting a species

with an already small population. Second, green frogs tend to remain in one aquatic

location exhibiting less of a tendency to migrate to upland, dryer areas than other ﬁ'ogs

(Harding, 1997). Therefore, these frogs are more likely to be affected by chemicals in the

body of water where they are caught. Lastly, green frogs are relatively large frogs that

eat a variety of prey from plant material to insects to smaller frogs and snakes (Harding,

1997). Since part of their diet places them at a higher trophic level, residues have the

10

potential to bioaccumulate in their bodies, with the possibility of being passed along

through the eggs to their offspring (Lu, 1991).

The life stages collected were tadpole, juvenile, and adult. These life stages are all
distinct from one another. Tadpoles are found only in the water, and have either no legs
or two developing hind limbs, and a tail. Juveniles are found in the transition area
between land and water, have both hind limbs and forelimbs, and remnants of a tail or tail
stub. Adults lack any form of tail stub and are of breeding size (about three to ﬁve

inches).

Tadpoles, juveniles, and adults were collected by either dip net or hand. Most adults
were collected after sunset. Tadpoles were identiﬁed according to Watermolen and
Gilbertson (1996). All frogs were examined for anatomical deformities, such as
malformations of the tail, eyes, and mouth. Additionally, adults and juveniles were
examined for hypomorphic and hypermorphic limb abnormalities. Possible deformities
were careﬁilly examined to ensure that the deformity was not simply a wound or scar
tissue. For instance, a missing limb may have at ﬁrst appeared to be a hypomorphic limb
abnormality. However, with more careful scrutiny, the skin in the region of where the
limb should be may have scar tissue, which would indicate that this “deformity” was
actually a wound caused by predation. The majority of frogs captured were immediately
released, aﬁer examination, back into the same locations from which they were captured.

Only the minimal number of frogs necessary for future analyses was kept.

ll

Results and Discussion

A total of 1445 green frog adults, juveniles, and tadpoles were examined from all four
sampling dates and all the sampling locations (Table 2). Of these, four were deformed.
One adult had six toes on its right hind leg, one juvenile had a shortened forelimb, one

juvenile had only three legs, and another juvenile had a malformed rear foot (Table 2).

A greater number of frogs from REF 1 were deformed than from the other sampling
locations. More frogs were collected from REFl than from any of the other sampling
locations; therefore, the overall percentage of deformed frogs from this site (0.34%) was
actually less than the overall percentage of deformed frogs from KLMZR (0.38%) (Table
3). The overall deformity rate among the green frogs collected from southwestern
Michigan was 0.28% (Table 3). This percentage is much lower than observed deformity
rates from other regions. For instance, 50% of the leopard frogs caught by the school
children in the Minnesota wetland in 1995 were deformed (North American Reporting
Center for Amphibian Malformations (NARCAM), 2000). Of the 853 anurans observed
at the agricultural locations near Quebec, 12% exhibited physical malformations (Ouellet

etaL,l997)

It is important for the accuracy of the deformity survey to look at all the life stages of the
frogs. For instance, if a tadpole were to have extreme deformities, it may not survive to
adulthood. The same is true for juvenile frogs. If it were missing a leg, then it may be
preyed upon and not survive to adulthood. Therefore, a more accurate deformity count

was conducted by examining frogs of all life stages.

12

Table 2. Number of green frogs collected, including number of deformities, from
southwestern Michigan, 1998.

 

 

 

 

 

 

 

 

 

 

 

 

 

4Malforrned rear foot

13

Location Adult Juvenile Tadpole
June 5, 1998, N = 154
REF] 17, 1 deforrnedl o 20
REF2 0 0 12
REF3 3 0 20
KLMZR 18 0 20
AGRl 0 0 21
AGR2 0 O 22
AGR3 l 0 0
June 23,1998, N = 417
REF 1 68 8 202
REF 3 7 7 20
KLMZR 11 0 30
AGRl 24 0 O
AGR2 2 0 20
AGR3 14 0 4
July 22, 1998, N = 636
REFl 52 145, 1 deformed: 23o
KLMZR 48 24 29
AGRl 3 0 0
AGR2 3 38 0
AGR3 3 61 0
August 26, 1998, N = 238
REF] 8 30, 1 deformed’ 98
KLMZR 3 47, 1 deformed“ 35
AGR2 10 4 0
AGR3 2 l 0
Total Frogs = 1445
1Six toes on right hind leg
2Shortened forelimb
3Missing hind limb

Table 3. Prevalence of deformities, and total number of green frogs by location, in
southwestern Michigan, 1998.

 

 

 

 

 

 

Location Overall Frog Count Number Deformed Percent Deformed
REF] 878 3 0.34%
REF2 12 0 0
REF 3 57 0 0

Total REF 947 3 0.32%

KLMZR 265 1 0.38%
AGRl 48 0 O
AGR2 99 0 0
AGR3 86 0 0

Total AGR 233 0 0

 

The fact that few deformities were observed during this survey may be indicative of a
relatively stable green frog population in southwestern Michigan and that the residues
measured were either not the causative agent of deformities observed or at least not
occurring at sufﬁcient concentrations at these locations. While some Michigan frog
populations have been reported to have declined (MDNR, 1996), reports of deformities
have been rare. Other reasons for the low deformity rate observed can be due to the
possibility that any deformities caused lethality to the frogs earlier in the season or in

earlier life stages before they could be recorded.

14

References

Ankley, G.T., Tietge, J.E., DeFoe, D.L., Jensen, K.M., Holcombe, G.W., Durhan, E.J.,
Diamond, S.A., 1998. Effects of ultraviolet light and methoprene on survival and
development of Rana pipiens. Environ. T oxicol. Chem. 17, 2530-2542.

Bemer, E.K., Bemer, R.A., 1996. Global Environment: Water, Air, and Geochemical
Cycles. Prentice Hall, Upper Saddle River, New Jersey.

Burkhart, J.G., Helgen, J.C., Fort, D.J., Gallagher, K., Bowers, D., Propst, T.L., Gernes,
M., Magner, J., Shelby M.D., Lucier, G., 1998. Induction of mortality and
malformation in Xenopus laevis embryos by water sources associated with ﬁeld
frog deformities. Environ. Health Perspect. 106, 841-848.

Carey, C., 1993. Hypothesis concerning the causes of the disappearance of boreal toads
from the mountains of Colorado. Conserv. Biol. 7, 355-362.

Gannon, R., 1997. Frogs in peril: Dead and deformed frogs spell trouble for humans.
Popular Science 251, 84-89.

Gardiner, D.M., Hoppe, D.M., 1999. Environmentally induced limb malformations in
mink frogs (Rana septentrionalis). J. Exp. 2001. 284, 207-216.

Harding, J.H., 1997. Amphibians and Reptiles of the Great Lakes Region. The
University of Michigan Press, Ann Arbor, Michigan.

Jofre, M.B., Karasov, W.H., 1999. Direct effect of ammonia on three species of North
American anuran amphibians. Environ. T oxicol. Chem. 18, 1806-1812.

Johnson, P.T.J., Lunde, K.B., Ritchie, E.G., Launer, A.E., 1999. The effect of trematode
infection on amphibian limb development and survivorship. Science 284, 802-

804.

15

Kaiser, J ., 1997. Deformed frogs leap into spotlight at health workshop. Science 278,
2051-2052.

LaClair, J.J., Bantle, J.A., Dumont, J., 1998. Photoproducts and metabolites of a
common insect grth regulator produce developmental deformities in Xenopus.
Environ. Sci. Tech. 32, 1453-1461.

Lips, K.R., 1999. Mass mortality and population declines of anurans at an upland site in
western Panama. Conserv. Biol. 13, 117-125.

Lu, F .C., 1991. Basic Toxicology: Fundamentals, Target Organs, and Risk Assessment
(2“Cl Edn.). Taylor and Francis, Bristol, PA.

Mehne, C., 1998. Personal communication. Animal Clinic, Kalamazoo, Michigan.

Michigan Department of Natural Resources (MDNR) Wildlife Division, 1996. Natural
history information: Michigan frog and toad survey, 12pp.

Michigan Natural Features Inventory (MNFI). Michigan special animals: Endangered,
threatened, special concern, and probably extirpated. [Online] Available
1111 mm. 11 .c. ' 11. '1' 11101111 ' 1111.. ' u , March
1999.

Milius, S., 1998. Fatal skin ﬁmgus found in U. S. frogs. Sci. News 154, 7.

North American Reporting Center for Amphibian Malformations (NARCAM).
Introduction to the malformed amphibian issue. [Online] Available
Wm, March 2000.

Ouellet, M., Bonin, J. Rodrigue, J ., DesGranges, J ., Lair, 8., 1997. Hindlimb deformities
(ectromelia, ectrodactyly) in free-living anurans from agricultural habitats. J.

Wildlife Diseases 33, 95-106.

16

Pechmann, J.H.K., Scott, D.E., Semlitsch, R.D., Caldwell, J.P., Vitt, L.J., Gibbons, J .W.,
1991. Declining amphibian populations: The problem of separating human
impacts from natural ﬂuctuations. Science 253, 892-895.

Pounds, J .A., Crump, M.L., 1994. Amphibian declines and climate disturbance: The case
of the golden toad and the harlequin frog. Conserv. Biol. 8, 72-85.

Renner, R., 1998. Ultraviolet radiation linked to frog deformities. Environ. Sci. Tech.
32, 12A.

Sessions, S.K., Ruth, SB, 1990. Explanation for naturally occurring supemurnerary
limbs in amphibians. J. Exp. Zool. 254, 38-47.

Stebbins, R.C., Cohen, N.W., 1995. A Natural History of Amphibians. Princeton
University Press, Princeton, New Jersey.

Van Valen, L., 1974. A natural model for the origin of some higher taxa. J.
Herpetology. 8, 109-121.

Wake, DB, 1991. Declining amphibian p0pulations. Science 253, 860.

Wania, F ., Pacyna, J .M., Mackay, D., 1998. Global fate of persistent organic pollutants.
T oxicol. Environ. Chem. 66, 81-89.

Watermolen, D.J., Gilbertson, H., 1996. Keys for the identiﬁcation of Wisconsin’s larval
amphibians: Wisconsin endangered resources report #109. Bureau of Endangered

Resources, Wisconsin Department of Natural Resources, Madison, Wisconsin.

17

Chapter 2: Organochlorine Insecticides and PCBs in Water and Green Frogs from
Southwestern Michigan

Introduction

Frog deformities and worldwide amphibian declines have been the focus of a great deal
of scientiﬁc research and debate over the past 15 years. Several hypotheses have been
formulated as to the reasons for these phenomena; however, no deﬁnite causes have been
found (Burkhart et al., 1998). One possible explanation is exposure to chemical residues.
In particular, exposure to chemicals that have the potential to modulate the endocrine

system are of current concern (Ankley et al., 1998).

Organochlorine (OC) insecticides, which were introduced in the mid-twentieth century,
are a group of pesticides that are chlorinated, persistent, and lipophilic (Bunce, 1994).
These compounds include chlordane, which is an insecticide and a fumigant, and lindane
(the y-isomer of hexachlorocyclohexane), which is an insecticide and a scabicide
(V erschueren, 1983). 2,2-bis-p-chloropheny1-1,1,l-trichloroethane (DDT), one of the
most well known of these compounds, was banned in the United States in 1972 because
of its harmful environmental effects (Bunce, 1994). However, it is still in use in some
tropical countries and continues to persist, along with its metabolites DDE and DDD, in

some parts of the environment in both more and less developed countries (Bunce, 1994).

While there are multiple mechanisms of toxic action depending on the species and

compound, most organochlorine compounds are neurotoxic to invertebrates (Wexler,

1998). p, p’-DDT has been shown to cause snout deformities in tadpoles of the common

18

European frog (Rana temporaria) (see Osborn et al., 1981). A follow-up study
concluded that similar effects could be caused by exposure of tadpoles to the steroid
hormone corticosterone (CORT) (Hayes et al., 1997). Hence, it has been hypothesized
that DDT can mimic CORT or act as a more general stressor that can lead to an increase
in endogenous CORT (Hayes et al., 1997). This increase in CORT may then lead to the
observed deformities. Regardless of the mechanism of action, DDT and other
organochlorine insecticides have been implicated as a possible cause of deformities in

frogs.

Like the 0C insecticides, polychlorinated biphenyls (PCBs) are chlorinated hydrocarbons
that are lipophilic, persistent, and have the potential to biomagnify in the food chain
(Bunce, 1994). In total, there are 209 congeners of PCBs, each with a different number
or location of the chlorine substituents. PCBs were ﬁrst detected in wildlife in the mid-
1960s, and were soon discovered to be ubiquitous in the environment. Because of their
toxicity to humans and other animals, and their ability to persist in the environment and
biomagnify up the food chain, PCB production in North America was banned in 1977.
However, due to their recalcitrant properties, great concentrations of PCBs persist in

many regions, primarily in aquatic ecosystem (Bunce, 1994; Zakrzewski, 1997).

The toxicity of each PCB congener is determined by its structure. One of the important
determinants of biological activity is planarity. The planarity of PCB congeners depends
on the number and position of the chlorine atoms. Those congeners with at least two

meta- and two para-chlorine atoms are the most toxic (Safe, 1990). Congeners with

19

chlorine atoms in the ortho-positions are considered to be less toxic because they are less
planar. Planar PCBs are structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). For this reason, planar PCBs elicit similar biological responses as TCDD.
These common responses include teratogenicity, carcinogenicity, embryo lethality, and
suppression of immune responses (Bunce, 1994). One reason for the extreme toxicity of
TCDD is its ability to bind to the aryl-hydrocarbon receptor (Ah-receptor), a cellular
receptor that seems to be present in all vertebrates (Safe, 1986). PCBs and dioxins have
also been shown to exhibit endocrine disrupting effects (Birnbaum, 1995). While the
eggs and tadpoles of some frogs seem to be relatively tolerant to acute exposure of
TCDD (Jung and Walker, 1997), TCDD and coplanar PCBs have been shown to offset

thyroid hormone status of some vertebrates (Brouwer et al., 1989).

Mixtures of PCBs in the environment differ based on the proportion of congeners in the
environmental samples (Bunce, 1994). In order to understand the toxicity of these
mixtures, the World Health Organization has derived speciﬁc toxic equivalency factors
(TEFs) to compare the potential toxicity of environmental PCB mixtures to TCDD (Van
den Berg et al., 1998). TEFs indicate an order of magnitude estimate of the toxicity of a
compound relative to TCDD, based on in-vivo and in-vitro toxicity studies. TEF values,
in combination with chemical residue data, can be used to calculate toxic equivalent
(TEQ) concentrations in various environmental samples, including animal tissues,

sediment, and water (Sanderson and Giesy, 1998).

20

Both DDT and PCBs, along with other synthetic compounds in the environment, have
been demonstrated to be estrogen mimics (Kendall et al., 1998). These weak estrogen
agonists have been postulated to have the potential to offset the neuro-endocrine system.
Speciﬁcally, xeno-estrogens have been suggested as possible causative agents of adverse
effects, including deformities, which have been observed in wildlife (Villeneuve et al.,
1998). Estrogen receptor agonists are compounds that bind to the estrogen receptor (ER),
forming a receptor-ligand complex that elicits a biological response. The endogenous
estrogen l7B-estradiol binds to the ER with the greatest afﬁnity. The ER binding afﬁnity
of other compounds are compared to 17B-estradiol by use of factors called estrogen
equivalents (EQs). The response that is elicited is proportional to the EQ (Villeneuve et

aL,1998)

The primary objectives of this study were to assess the incidence of green frog
deformities in southwestern Michigan, and to correlate those deformities to chemical
residues in the frog tissue. No signiﬁcant deformities were observed among the sampling
locations, as discussed in Chapter 1. Therefore, no correlations between the observed
residues and deformities could be developed. However, the remaining study objectives
remained viable. These objectives involved gathering data on the levels of
organochlorine residues in water and in tissues of green frog eggs, tadpoles, juveniles,

and adults. Concentrations of p,p’-DDT, p,p’-DDE, p,p’-DDD, the a- and y—isomers of

chlordane, the 01-, B-, y-, and 5-isomers of hexachlorocyclohexane (HCH), and the non-

ortho-, mono-ortho-, and di-ortho-substituted PCB congeners were determined in water

21

and tissue. Also, TEQs and EQs of the water and tadpole tissue were determined using

in-vitro cell bioassays.

Materials and Methods

Water and Frog Sampling and Preparation

Tadpoles and adults of the green frog (Rana clamitans) were collected by dip net and
hand from sampling locations in southwestern Michigan (see Chapter 1). All the life
stages from all the locations used in the deformity survey were not analyzed for
organochlorine residues, based on the availability of frogs at each location and the

necessity of those frogs being used in other aspects of the study (Table 4).

Tadpoles and adults were weighed and measured, and the sex of the adults was
determined based on physical features. The most common means of sexing green frogs is
based on the size of the tympanum. In males, the tympanum is larger than the eyes.
Males also may have a yellowish throat, whereas the throat of the females is generally
whitish (Harding, 1997). Tadpoles were pooled into samples of approximately 20 g wet
wt. by location and date. Pooling of samples was not necessary for the adults. Eggs were
collected from the frogs and not from ﬂoating egg masses. In each case where frogs
contained eggs, the eggs were removed prior to extraction. Most of the eggs were pooled
into samples up to a maximum of 20 g by location and date. All frog tissues were stored

at —20°C until analysis.

Four liters of water were collected from each sampling location on the ﬁrst two sampling

22

dates (6/5 and 6/23). Samples were collected into four-liter brown glass solvent bottles
by submerging each bottle into the water until full. To ensure that they were clean,
bottles were washed with water and Liquinox® (Alconox, New York, NY, USA), rinsed
with acetone and hexane, and allowed to dry before use. Water samples were stored at

4°C until analysis.

All glassware, stainless steel homogenizer materials, boiling stones, glass wool, and any
other utensils and surfaces that would contact samples during the analysis were cleaned
by standard procedures to minimize contamination of the sample. Brieﬂy, analytical
items were washed thoroughly with Liquinox® (Alconox, New York, NY, USA) and
water, then rinsed with deionized water. Materials were allowed to air dry before being
rinsed three times with acetone followed by three rinses with hexane. Solvent-cleaned
materials were blown dry with a heat gun and placed on solvent-rinsed aluminum foil.
All solvents used for any part of the analysis or cleaning steps were HPLC grade

(Burdick and Jackson, Muskegon, MI, USA).

F rag Tissue Extraction and Clean- Up

PCBs and OC pesticides were extracted following methods described elsewhere (Kannan
et al., 1995; Khim et al., 1999). The Standard Operating Procedure for the analysis of
organochlorines, such as DDT and PCBs, is presented in Appendix C. Each tissue
sample was thawed at room temperature for 1 h before being prepared for analysis. Each
sample was ground in a stainless steel homogenizer cup by use of an Omni-Mixer

(Sorvall®, PRO Scientiﬁc Inc., Monroe, CT, USA). Sodium sulfate was slowly added

23

during the blending process (about 150 g anhydrous Na2$O4 for a 20 g sample) until the
sample was thoroughly dry. Samples were extracted by Soxhlet extraction with a 3:1
ratio of dichloromethane (DCM) to hexane for at least 12 h. Extracts were concentrated
to 11 ml and a 1 ml subsample was removed to determine the lipid content of the tissues.

Lipid content was measured gravimetrically.

The 10 m1 extracts were further concentrated to 3 ml for fractionation. Florisil (60-100
mesh size; Sigma, St. Louis, MO, USA) was activated at 130°C for at least 12 h. For
each sample, 10 g of F lorisil was suspended in hexane and transferred to a glass
chromatography column. The 3 ml extract was placed on top of the F lorisil and eluted at
a flow rate of approximately 3 ml / min. The ﬁrst fraction (F 1), which contained the least
polar analytes, was eluted from the column with 100 ml of hexane. The moderately polar
compounds were eluted in the second fraction (F2) with 100 ml of 20% DCM in hexane.
The ﬁnal fraction (F3), which was eluted from the column with 100 ml of 50% methanol

in DCM, contained the most hydrophilic compounds.

The volumes of the eluents were reduced by evaporation to 2 ml for F1 and 0.1 ml for F2
and F3. F2 was adjusted to 2 ml with hexane. F3 was taken up to 1 ml with acetonitrile
and set aside for latter analyses of triazine herbicides. F1 and F2 eluents were treated
with 2 ml aliquots of sulfuric acid to destroy interfering compounds. Aﬁer the sulfuric
acid treatments, the hexane extracts were rinsed with deionized water. The eluents were

further concentrated to 1 m1 and placed in GC vials for analysis.

24

Water Extraction and Clean-Up

For each sample, 2 L of water was ﬁltered through glass wool and transferred to a 4 L
separatory funnel. The sample in the separatory funnel was shaken for 15 min with 200
ml DCM and allowed to separate for at least 12 h. The DCM fraction, which contained
the more hydrophobic compounds, was drained into a ﬂask and the procedure was
repeated after a 4 h separation period. The second DCM fraction was added to the same
ﬂask as the ﬁrst and the fractions were concentrated to 0.1 ml. The extracts were
adjusted to 1 ml with acetonitrile and placed into GC vials. In order to be injected into
the GC/ECD, a 0.2 ml aliquant was later removed, concentrated to 0.1 ml, made up to 0.2

ml with hexane, and placed in GC vial inserts inside GC vials.

Instrumental Analysis

PCBs and OC pesticides in the water and tissue samples were quantiﬁed using a gas
chromatograph (Perkin Elmer series 600) equipped with 63Ni electron capture detector
(GC-ECD). A fused silica capillary column coated with DB-l (100%
dirnethylpolysiloxane, 30m x 0.25mm i.d.; J&W Scientiﬁc, Folsom, CA, USA) having a
ﬁlm thickness of 0.25 pm was used. The column oven temperature was programmed
from 85°C (0.5 min hold) to 110°C at a rate of 10°C/min and then to 260°C at a rate of
2°C /min with a ﬁnal holding time of 10 min. Injector and detector temperatures were
kept at 250°C and 350°C, respectively. Helium was used as the carrier gas and nitrogen
was the make up gas. A PCB mixture, containing known concentration of 98 congeners
commonly present in aquatic biota, was used as the PCB standard. This mixture was

prepared from 98 individual PCB standards which were purchased commercially

25

(AccuStandard, New Haven, CT, USA). Concentrations of 98 individually resolved
peaks were summed to obtain total PCB concentrations. OC pesticides were quantiﬁed
from individually resolved peak heights based on the peak heights of standards.
Detection limits for total PCBs and OC pesticides were 1.0 and 0.01 ng/g, wet wt,
respectively. Samples were spiked with PCB and 0C pesticide (CLP-023R, CLP-024R,
AccuStandard) standards and passed through the entire analytical procedure to test for
recoveries (n=3). Recoveries in Florisil F l were 995% and 90i5% for individual PCB
congeners and p, p ’-DDE, respectively (n=3). No other spiked compounds were detected
in F1. Recoveries of remaining OC pesticides in F2 were 105.5% (n=3). No other
spiked compounds were detected in F2. Recoveries of OCs in SRM were between 90 and

105%. Concentrations reported were not adjusted for recovery.

Cell Bioassay Analysis

The Standard Operating Procedures for the following cell bioassays are presented in
Appendix D. MVLN cells are MCF-7 human breast carcinoma cells stably transfected
with a luciferase reporter gene under control of estrogen response elements (EREs) of the
Xenopus vitellogenin A2 gene (Demirpence et al., 1993). H4IIE-luc cells are rat
hepatoma cells which were stably transfected with a luciferase reporter gene under
control of dioxin-responsive enhancers (DREs) (Sanderson et al., 1996). All cells were
cultured in 100-mm disposable petri plates (Corning, Corning, NY, USA) and incubated
in a humidiﬁed 95:5 air : C02 atmosphere. MVLN and H4IIE-luc were maintained at
37°C. MVLN were grown in Dulbecco’s Modiﬁed Eagle Medium with Hams F-12

nutrient mixture (Sigma D-2906, St. Louis, MO, USA) supplemented with 10% fetal

26

bovine serum (FBS; Hyclone, Logan, UT, USA), 27.3 I.U. insulin (Sigma I-1882) / L and
1.0 mM sodium pyruvate (Sigma). H4IIE-luc were cultured in Dulbecco’s Modiﬁed
Eagle Medium (Sigma D-2902) supplemented with 10% FBS (Hyclone). Cells were
passaged when plates became conﬂuent and new cultures were started from frozen stocks

after less than 30 passages.

Detailed methods for the in vitro bioassays used in this study have been described
elsewhere (MVLN assay (Koistinen et al., 1998); H4IIE-luc assay (Sanderson et al.,
1996)). In brief, cells were trypsinized from plates containing 80—100% conﬂuent
monolayers, resuspended in media, and diluted to a concentration of approximately
7.5x104 cells/ml and seeded into the 60 interior wells of 96-well culture ViewPlatesm
(Packard Instruments, Meriden, CT, USA) at 250 pl per well. The 36 exterior wells of

each plate were ﬁlled with 250 pl culture media. Cells were incubated overnight, then

dosed. Test and control wells were dosed with 2.5 ul of the appropriate extract or
solvent. Blank wells received no dose. A minimum of three control wells and three
blank wells were tested on each plate. For initial screening, extracts were tested in three
replicate wells at a single concentration. For dose-response characterization, dilution
series consisting of six concentrations of sample, prepared by 3-fold serial dilution, were

tested (3 replicate wells per concentration).

All bioassay data was collected electronically and imported into a spreadsheet for data

analysis. Relative luminescence units (RLU) were not adjusted for protein. Sample

responses, expressed as mean RLU (across 3 replicate wells), were converted to a

27

percentage of the mean maximum response observed for standard curves generated on the
same day (%-E2-max. and %-TCDD-max. for 17-[3-estradiol and TCDD standards
respectively). This was done to normalize responses for day-to-day variability in
response magnitude. The mean solvent control response (RLU) was subtracted from both
sample and standard responses (RLU) on a plate-by-plate basis, prior to conversion to a
percentage, in order to scale values from 0 to 100%-standard-max. Signiﬁcant responses
were deﬁned as those outside of the 95% conﬁdence interval (expressed in %-standard-

max.) around the mean solvent control response (0%-standard-max.).

The dose-response relationships obtained from bioassay analysis of sample serial
dilutions were generally not complete enough to facilitate formulation of a regression
equation. As a result, relative potency estimates were not possible for most of the
samples analyzed in this study. In cases where a regression equation could be ﬁt to the
dose-response, sample potency (relative to a dioxin or 17-B-estradiol (E2) standard) was
calculated as EC-30 of the standard / EC-30 of the sample. In order for such a point
estimate of relative potency to be valid, the sample and standard dose-responses must be
statistically parallel and have the same maximum achievable response (Putzrath, 1997).
This could not be demonstrated for the samples analyzed in this study. Thus, the relative
potencies calculated should be regarded as approximations, based on assumptions of

equal efﬁcacy and parallel slopes.

28

Results

Lipids

Egg tissue contained greater lipid content (mean range 9.73 - 11.10%) than did either the
adult (mean range 0.50 - 2.63%) or the tadpole tissue (mean range 1.36 - 4.14%) (Table
4).

Table 4. Mass, length, and percent lipid (mean and standard deviation, in parentheses) of
green frog adult, tadpole, and egg tissue from southwestern Michigan, 1998.

 

 

 

 

 

 

 

 

 

Sample location and date Mass (g) Length (cm) Percent lipid (%)
Adult
REF], 6/5, n=3 37.46 (15.71) 68.20 (10.13) 2.63 (1.03)
REF 1, 6/23, n=3 49.46 (0.25) 77.17 (4.80) 0.69 (0.46)
REF 1, 7/22, n=3 34.00 (4.00) 71.00 (5.57) 1.56 (0.49)
KLMZR, 6/5, n=3 32.74 (5.90) 65.52 (4.40) 0.54 (0.33)
KLMZR, 6/23, n=2 25.01 (1.52) 58.58 (1.02) 0.84 (0.12)
AGR], 6/23, n=3 25.52 (3.14) 62.44 (2.26) 0.70 (0.29)
AGR2, 7/22, n=3 24.47 (2.73) 65.67 (2.08) 0.50 (0.28)
AGR3, 7/22, n=2 36.85 (9.69) 70.50 (7.79) 0.87 (0.25)
Juvenile
KLMZR, 8/26, n=2 pools NA NA 1.87 (0.17)
Tadpole
REF], 6/5, n=20 2.60 (0.65) 59.28 (5.82) 1.45“
REF 1, 6/23, n=25 3.43 (0.42) 66.57 (3.77) 3.49*
REF2, 6/5, n=12 5.57 (1.20) 73.54 (10.76) 1.81*
REF3, 6/5, n=20 4.30 (0.81) 72.80 (6.83) 284*
REF3, 6/23, n=20 5.00 (0.82) 73.72 (11.95) 4.14*
KLMZR, 6/5, n=20 3.58 (1.40) 65.44 (10.17) 2.67*
KLMZR, 6/23, n=30 4.37 (1.11) 72.53 (7.60) 2.23“
AGR], 6/5, n=2] 4.38 (1.30) 69.70 (9.10) 1.36*
AGR2, 6/23, n=20 7.84 (2.44) 82.08 (12.49) 3.56*
AGR3, 6/23, n=4 5.52 (1.38) 543842262) 262*
Egg
REF], 6/23, n=2 NA NA 10.97 (2.06)
KLMZR, 6/23, n=1 NA NA 9.73*
AGR], 6/23, n=1 NA NA 10.94*
AGR3, n=1 NA NA 11.10*

 

NA = Not Available

'“No standard deviation is recorded for these samples because they were treated as one pooled sample each,

each pool being from each location and date.

29

Organochlorine Insecticides

Concentrations of the isomers of HCH, the isomers of chlordane, and p, p ’-DDT and its
metabolites in all water samples were less than the method detection limit (MDL) of 1.0
ng/L. Raw data of frog tissue concentrations are presented in Appendix A, along with a
table depicting the statistical signiﬁcant differences (a=0.05) between locations and life

stages.

For many of the frog samples, there were no signiﬁcant differences in concentrations
between locations. For individual isomers and metabolites of HCH, chlordane, and DDT,
concentrations of p, p’-DDE and p, p’-DDD were greatest in adults (0.61 ng/g, wet wt.
for each) (Table 5). Concentrations of B-HCH and a-chlordane were the least (at most
0.02 and 0.01 ng/g, wet wt., respectively) in adult tissues (Table 5). There were no
signiﬁcant differences among sampling locations in concentrations of any of the OCs in

adult tissues.

Samples of juveniles contained lesser concentrations of OC insecticides than did the
adults. Most concentrations of individual isomers were less than the MDL (0.01 ng/g,
wet M), with p, p’-DDD being the greatest at 0.09 ng/g, wet wt. (Table 5). There were

no signiﬁcant differences in concentrations of OCs in juvenile tissues among locations.

Some differences in concentrations of residues in tadpole tissue among locations were

observed. Concentrations ranged from less than 0.01 ng/g, wet wt. for p, p’-DDT to 0.29

ng/g, wet wt. for p, p’-DDD (Table 5), but were not statistically different among

30

locations. Residue concentrations that differed among location for tadpole tissue
included y-HCH (lindane), a-chlordane, y-chlordane, and Zchlordane. The greatest
concentrations of lindane and y-chlordane (0.25 and 0.03 ng/g, wet wt., respectively)
were observed at the agricultural location AGR]. The greatest concentration of or-

chlordane (0.14 ng/g, wet wt.) was observed at agricultural location AGR2 (Tables 6 and

7).

Concentrations of OC insecticides in eggs ranged from less than 0.01 ng/g wet wt. for
many of the residues to 5.86 ng/g wet wt. for p, p’-DDE (Table 5). Concentrations of p,
p’-DDT were signiﬁcantly different among locations, with the agricultural location

AGR] having the greatest concentration (1.40 ng/g, wet wt.) (Tables 6 and 7).

When all of the tissue concentrations are analyzed statistically, regardless of location, it is
evident that the egg tissues have the greatest concentrations of many of the OC residues.
These residues are (rt-chlordane, y—chlordane, p, p ’-DDE, p, p’-DDT, ZDDT, and
Zchlordane (Table 8). The concentration of a-HCH was greatest in tadpole tissue (Table

8).

31

Table 5. Concentrations (ng/g, wet wt., mean and standard deviation, in parentheses) of
organochlorine insecticides and total PCBs in green frog tissue from

southwestern Michigan, 1998.

 

 

 

 

 

Insecticide Adult 1 Juvenile2 (n=2) Tadpole2 (n=10) Egg3 (n=5)
Values calculated with 0.00 substituted in for the non-detects
a-HCH 0.02 (0.03) 0.04 (0.04) 0.07 (0.09) 0.00*
B—HCH 0.01 (0.02) 0.00"I 0.08 (0.15) 0.00*
y-HCH 0.06 (0.04) 0.00* **** 0.16 (0.19)
b-HCH 0.03 (0.07) 0.00* 0.15 (0.27) 0.00*
(rt-chlordane 0.01 (0.01) 0.00* **"'* 0.18 (0.30)
y—chlordane 0.04 (0.04) 0.00* **** 0.10 (0.08)
p, p ’-DDT 0.03 (0.07) 0.00* 0.00* ****
p, p '-DDE 0.61 (0.97) 0.01 (0.01) 0.08 (0.14) 5.86 (2.92)
p, p’-DDD 0.61 (0.51) 0.09 (0.01) 0.29 (0.31) 1.71 (2.96)
ZHCH 0.12 (0.12) 0.04 (0.04) 0.33 (0.50) 0.34 (0.37)
Zchlordane 0.05 (0.05) 0.00“ **** 0.29 (0.32)
ZDDT 1.24 (1.19) 0.10 (0.01) 0.37 (0.40) 7.91 (5.32)
lPCBS 19.62 (41.01) 19.12 (5.74) 7.63 (8.35) 79.11 (89.82)
Values calculated with the MDL substituted in for the non-detects
a-HCH 0.03 (0.03) 0.04 (0.04) 0.08 (0.08) 0.01 (0.00)
B-HCH 0.02 (0.01) 0.01 (0.00) 0.08 (0.14) 0.01 (0.00)
y-HCH 0.06 (0.04) 0.01 (0.00) **** 0.16 (0.19)
6—HCH 0.04 (0.07) 0.01 (0.00) 0.16 (0.27) 0.01 (0.00)
a-chlordane 0.01 (0.01) 0.01 (0.00) ***"‘ 0.19 (0.30)
y—chlordane 0.04 (0.04) 0.01 (0.00) **** 0.11 (0.07)
p, p’-DDT 0.03 (0.07) 0.0] (0.00) 0.01 (0.00) ****
p, p ’-DDE 0.61 (0.97) 0.02 (0.00) 0.09 (0.14) 5.86 (2.92)
p, p’-DDD 0.61 (0.51) 0.09 (0.00) 0.29 (0.30) 1.7] (2.96)
ZHCH 0.12 (0.12) 0.04 (0.04) 0.34 (0.50) 0.34 (0.37)
Xchlordane 0.05 (0.05) 0.01 (0.00) **** 0.29 (0.32)
ZDDT 1.24 (1.19) 0.10 (0.01) 0.37 (0.39) 7.98 (5.32)
ZPCBs 19.62 (41.01) 19.12 (5.74) 7.63 (8.35) 79.11 (89.82)

 

Tn=21 for insecticide analysis and n=22 for PCB analysis
2 These were pooled samples
3 Two of the ﬁve samples were pooled
* No standard deviations are presented here because the values were below the MDL and
none could be calculated.
""At the 95% conﬁdence level, there were signiﬁcant differences among locations so a
total mean could not be calculated.

32

Table 6. Concentration (ng/g, wet wt., mean and standard deviation, in parentheses) of
organochlorine insecticides and total PCBs in green frog tissue from sampling
locations that are signiﬁcantly different at the 95% conﬁdence level in
southwestern Michigan, 1998.

 

 

REF 1 REF2 REF3 KLMZR AGRl AGR2 AGR3
Tadpole
y-HCH 0.04 (0.05) 0.01“ 0.01 (0.00) 0.01 (0.00) 0.25* 0.01‘ 0.04“
a-chlordane 0.01 (0.00) 0.04“ 0.02 (0.02) 0.01 (0.00) 0.06“ 0.14* 0.01"
y-chlordane 0.01 (0.00) 0.02* 0.01 (0.01) 0.01 (0.00) 0.03“ 0.01“ 0.01“
Zchlordane 0.01 (0.00) 0.06“ 0.02 (0.03) 0.01 (0.00) 0.09” 0.14“ 001*
Egg
p, p'-DDT 0.01 (0.00) NA NA 0.01 (0.00) 1.40"' NA 0.75"

 

NA = Not analyzed
*No standard deviation is presented because n=1 pool for these samples.

Table 7. Samples with signiﬁcant differences in organochlorine residue concentrations
among locations. Underlined locations are not signiﬁcantly different from one
another (a=0.05). Locations are arranged in order of increasing

 

 

 

 

 

 

 

 

 

 

 

concentrations.
Tissue and Residue Location
Tadpole
y-HCH REF2 REF3 KLMZR AGR2 REF 1 AGR3 AGR]
a-chlordane REF] KLMZR AGR3 REF3 REF2 AGR] AGR2
y—chlordane REF] KLMZR AGR2 AGR3 REF3 REF2 AGR]
Zchlordane REF] KLMZR AGR3 REF3 REF2 AGR] AGR2
Egg
p. p ’-DDT REFl KLMZR AGR3 AGRl

 

33

Table 8. Samples with signiﬁcant differences in organochlorine concentrations between
life stages. Underlined life stages are not signiﬁcantly different (0t=0.05) from
one another. Life stages are arranged in order of increasing concentrations.

 

 

 

 

 

 

 

 

 

 

 

 

Residue Life Stage

a-HCH Adult Egg Juvenile Tadpole
(rt-chlordane Adult Juvenile Tadpole Egg
y-chlordane Juvenile Tadpole Adult Egg
p, p '-DDE Juvenile Tadpole Adult Egg
p, p ’-DDT Juvenile Tadpole Adult Egg
ZDDT Juvenile Tadpole Adult Egg
XChlordane Juvenile Tadpole Adult Egg
ZPCB Tadpole Juvenile Adult Egg
PCBS

Concentrations of PCBs in all water samples were less than the MDL of 1.0 ng/L. PCBs
were detected in tissues of adult, juvenile, tadpole, and eggs of the green frogs. There
were no signiﬁcant differences in concentrations of PCBs among locations. The least
concentration (7.63 ng/g, wet wt.) was observed in tadpoles while the greatest
concentration (79.11 ng/g, wet wt.) was observed in eggs (Table 5). Raw data of frog
tissue concentrations of total PCBs as well as concentrations of the di-, mono-, and non-

ortho PCB congeners are presented in Appendix A.

34

T CDD-EQs and EZ-EQs

Concentrations of TCDD-EQs and E2-EQs were less than the MDL at all locations

 

 

 

(Table 9).

Table 9. Cell bioassay detection limits.

Matrix EQ

Water E2-EQ < 0.2] pg E2/ml

Water TCDD-EQ < 0.41 pg TCDD/ml
Tadpole TCDD-EQ < 41 pg TCDD/m1
Discussion

The differences observed among locations of OC insecticide concentrations for tadpole
and egg tissues can be due to a variety of factors. First, general experimental variation
can be involved. Second, it is possible that the differences among locations observed for
these two life stages are due to the greater lipid content of the eggs and tadpoles. Third,
tadpoles may have a greater exposure to residues in the water and sediment, because they
feed exclusively on aquatic materials and do not leave the aquatic system. Lastly,
perhaps older life stages may have the ability to metabolize and excrete the residues at a

faster rate.

When all of the locations were analyzed together, the egg tissue contained signiﬁcantly
greater concentrations of a-chlordane, y-chlordane, p, p ’-DDE, p, p’-DDT, XDDT, and
Zchlordane than did tadpoles, juveniles, and adults. This may be due to the greater lipid
content of the eggs. OCs tend to accumulate in lipids because of their low polarity and
high octanol-water partitioning coefﬁcient (KW). For instance, the concentrations of

XDDT in adult, juvenile, tadpole, and egg lipid tissue are 119.23, 5.35, 14.12, and 74.65

35

ng/g, respectively. Therefore, because the adults have a lower lipid content than the eggs
(by an average factor of 10.28), ZDDT is actually more concentrated in the lipids of the
adults than in the lipids of the eggs. A similar pattern is followed with the residues of y-
chlordane, p, p ’-DDE, p, p’-DDT, and Zchlordane. Adult green frogs consume a variety
of prey items, including aquatic and terrestrial insects, other invertebrates, smaller frogs,
snakes, and hatchling turtles (Harding, 1997). Therefore, their diet allows them to be
comparatively high on the food chain. Thus, they have the capacity to bioaccumulate
organochlorine residues. Also, adults, being older than the other life stages analyzed,

have had a longer time to accumulate such residues.

Concentrations of OCs in green frogs in the present study were less than those reported in
adult spring peepers (Pseudacris crucifer) collected from Point Pelee National Park in
southern Ontario in April of 1993 (Russell et al., 1995). Historically, DDT had been

applied extensively to this park as mosquito control. ZDDT concentrations reported in

this study were as great as 1,188 pg/kg, wet wt. The authors hypothesize that these high
concentrations are due primarily to those historical applications of DDT. Half of the
amphibian fauna once present at this location has disappeared over the last ﬁfty years.
The authors attributed these disappearances to toxic concentrations of pesticides. A later
study in neighboring locations of southern Ontario near Point Pelee found lesser
concentrations of pesticide residues in adult green frog tissue (Russell et al., 1997).
Concentrations of p,p’-DDE in green frog tissue ranged from 0.58 to 45.02 ug/kg, wet
wt. At these locations, green frogs were the dominant anuran, indicating that at these OC

concentrations the green frog population appeared to not be depleted. The lesser

36

concentrations, in that study, are more similar to the concentrations of p,p’-DDE in this

study.

Concentrations of a-HCH, B-HCH, lindane, p, p’-DDD, p, p’-DDE, heptachlor, dieldrin,
o, p’-DDD, o, p’-DDE, o, p’-DDT, p, p’-DDT, heptachlor epoxide, aldrin, and endrin in
tissues of heron adults, nestlings, and eggs and in the tissues of their primary prey, frogs
(Rana spp.) collected in May and June of 1992 and 1993 from wetlands of Therrnaikos
Gulf, Macedonia, Greece were measured (Albanis et al., 1996). Concentrations of
residues in water ranged from <0.01 to 0.21 ug/L. Few residues were detected in adult
frogs, with the exception of a-HCH, B-HCH, lindane, p, p’-DDD, p, p’-DDE, and
heptachlor. Average concentrations of a-HCH, B-HCH, lindane, p, p’-DDD, and p, p’-
DDE in the frogs were 2.45, 0.56, 3.64, 0.49, and 0.29 rig/kg, dry wt., respectively.
Generally, the wet weight to dry weight ratio is ﬁve. Therefore, these reported values can
be divided by ﬁve to compare to the concentrations from the present study. Overall, the
concentrations of HCH in the frogs in the study in Greece would still be greater by a
factor of about ten on average, while the concentrations of DDD and DDE in Greece

would be less by a factor of about eight.

Twenty 0C pesticides and 39 PCB congeners were determined in mudpuppies and
snapping turtles collected from 1988 to 1992 from the St. Lawrence River in Canada, a
river that is greatly impacted by human activities and synthetic chemicals (Bonin et al.,
1995). Concentrations of DDD, DDE, DDT, a-HCH, lindane, trans-chlordane, and cis-

chlordane in whole mudpuppies were 1.2 to 24.8, 0.3 to 90.0, ND to 8.3, ND to 6.8, ND

37

to 3.3, ND to 7.1, and 0.3 to 13.9 ng/g, wet wt., respectively. Concentrations of PCBs in
whole mudpuppies ranged from 92 to 1082 ng/g, wet wt. These values are slightly

greater than the concentrations observed in the present study.

Concentrations of PCBs and OC pesticides were determined in green frogs and leopard
frogs collected along the north shores of Lake Erie and Lake Ontario in Ontario, Canada
(Gillan et al., 1998). Sediments were also collected in an effort to determine biota-
sediment accumulation factors (BSAFs), as well as to determine the cytotoxicity and
genotoxicity of the sediments. Concentrations of p, p’-DDE in green frogs ranged from
19 ng/g lipid wt. in a large pond in a public park area to 754 ng/g lipid wt. from a small
pond in an agricultural region. Concentrations of p, p’-DDT in green frogs ranged from
ND in a large pond in a public park area to 60 ng/g lipid wt. in a large marsh in a
conservation area. Concentrations of p, p’-DDE in leopard frogs ranged from 60 ng/g
lipid wt. in a shallow creek in a conservation area to 659 ng/g lipid wt. from a large pond
in a provincial region. Concentrations of p, p’-DDT in green frogs ranged from ND in a
few sites to 80 ng/g lipid wt. in a small pond in an agricultural area. In the present study,
the mean percent lipid content of the adult frogs was 1.04%. Therefore, if concentrations
were normalized by lipid weight, the mean concentration of p,p’-DDE in adult green
frogs from southwestern Michigan is 57.86 ng/g, lipid wt., and that of p,p’-DDT is 2.86
ng/g lipid wt. The value for DDE are within the ranges of the green frogs studied in the
Ontario study, and slightly lower than the concentrations in leopard frogs. The value for
DDT is within the concentration ranges for both green and leopard frogs in the Ontario

study.

38

PCB concentrations measured in green frogs in regions of southern Ontario were in the
low pg/kg levels (Russell et al., 1997). PCBs, polychlorinated dibenzo-p-dioxins
(PCDDs), and polychlorinated dibenzo-p-furans (PCDFs) were measured in northern
leopard frogs collected from the lower Fox River and Green Bay regions of Wisconsin in
the summers of 1994 and 1995. The concentrations of total PCBs ranged from 3 to 154
ng/g, wet wt. One PCDD and two PCDFs at concentrations of 6 to 8 pg/g were also
found in the frogs from one site (Huang et al., 1999). The concentration of total PCBs
measured in the present study are comparable to those in Wisconsin. The small
concentrations of TEQs in this study were probably due to the fact that concentrations of

PCBs were also small.

OC pesticides were determined in fat bodies of breeding male green frogs in 1993 and
1994 from orchard wetlands in rural areas of southern Ontario (Harris et al., 1998).
Concentrations of DDD, DDE, and DDT ranged from ND to 0.21 pg/g, 0.05 to 2.81 ug/g,
and ND to 0.46 ug/g, respectively. As calculated previously, the mean lipid
concentrations of p,p’-DDD, p,p’-DDE, and p,p’-DDT of adult green frogs in the present
study were 57.86 ng/g, 57.86 ng/g, and 2.86 ng/g, respectively. Because of the large
range of concentrations in the Ontario study, the values for the current study in

southwestern Michigan are similar, albeit at the very low end.

EROD activity (hepatic ethoxy-resoruﬁn-O—deethylase activity) is a metabolic response

that is mediated by the Ah-receptor upon exposure to dioxins and dioxin-like compounds

39

(Huang et al., 1999). In an aforementioned study (Huang et al., 1999), no signiﬁcant
correlation was found between EROD activity and PCB concentration. This result was
consistent with the fact that the frogs from Wisconsin contained relatively small
concentrations of PCBs (<152 ng/g wet wt.) compared with what was required for
induction in the laboratory (ED50 for EROD is between 700 and 2,300 ng/g wet wt.).
Concentrations of PCBs in green frog tissue from the present study in southwestern
Michigan were lesser than those observed in frog tissue from Wisconsin (<20 ng/g wet
wt. in adults); hence, it would be unlikely that signiﬁcant EROD induction would be

observed in these tissues.

The toxicities of some insecticides to amphibians have been determined. Static bioassays
to determine the relative acute toxicities of pesticides to western chorus frog (Pseudacris
triseriata) and Fowler’s toad (Bufofowleri) tadpoles found that endrin was the most toxic
0C insecticide to the chorus frog tadpoles and the second most toxic insecticide to the
Fowler’s toad tadpoles (Sanders, 1970). Lindarre was the least toxic insecticide to both
frog and toad 4 and 5-wk old tadpoles, with a 96-h TL50 value of 4.4 mg/L for toads and
a 96-h TL50 value of 2.7 mg/L for frogs. Median tolerance limit (TL50 or LC50) values
for toad tadpoles decreased with age (Sanders, 1970). For example, the 96-h T L50 value
for l-wk old Fowler’s toad tadpoles exposed to waterborne exposures of DDT was
measured at 0.75 mg/L, while the 96-h TL50 for 7-wk old tadpoles was measured at 0.03
mg/L. This indicates that DDT might be most toxic to advanced tadpole developmental
stages (Sanders, 1970). Dieldrin was observed to have approximately the same 96-h

TL50 values as DDD in Fowler’s toad tadpoles (0.15 mg/L) but was found to be eight

40

times more toxic than DDT to western chorus frog tadpoles (0.10 mg/L versus 0.80
mg/L) (Sanders, 1970). These values are much greater than those concentrations
measured in the water samples in the present study. Exposure of tadpoles to dieldrin
resulted in structural abnormalities, lesser rates of development, and changes in behavior

(Cooke, 1972).

DDT has been shown to adversely impact the development and behavior of common
European frogs (Cooke, 1970). Uncoordinated activity, weight loss, and restricted
development were observed in tadpoles following 1-h exposures to 0.1, 1.0, and 10.0
mg/L aqueous concentrations of DDT. Aﬁer treatment, tissue concentrations of DDT
ranged from 140 to 180 pg/g (Cooke, 1970). Tadpoles exposed for 24 and 48 h to
aqueous concentrations of DDT (ranging from 0 to 0.5 mg/L) have exhibited hyperactive
behavior either just before or just after developing limb buds (Cooke, 1972). Tissue
concentrations of whole tadpoles with this behavior ranged from 2,000 to 4,000 rig/kg
(Cooke, 1972). Exposures to aqueous DDT at 0.1 mg/L for 2 (1 produced histological
changes in mandibles of some tadpoles (Osborn et al., 1981). This has been attributed to
the disruptive effect DDT has on the development of skin glands in the region above the
upper mandible and to the hyperactivity that DDT causes in tadpoles (Osborn et al.,
1981). Effects of DDT expOsure were more severe for acute exposures than for chronic
exposures even though the two types of exposure resulted in the same concentrations in
tadpoles (Cooke, 1973). The degree of crowding in laboratory populations was positively
related to the severity of sub-lethal effects, including hyperactivity and mandibular

deformities, due to exposure to DDT (Cooke, 1979). Hyperactivity caused by sub-lethal

41

exposure to DDT in tadpoles has been postulated to potentially result in greater risk from
predation (Cooke, 1971). None of these previously observed abnormalities were
observed in the tadpoles of the present study in southwestern Michigan, as would be

expected due to the lower water concentrations of DDT.

Conclusion

The study presented here attempted to link deformities in green frog populations of
southwestern Michigan to organochlorine insecticide and PCB residues in their
environment and tissues. However, only 0.28% of green frogs exhibited obvious
physical abnormalities. Concentrations of DDT and its metabolites, chlordane,
hexachlorocyclohexane, and PCBs were relatively low, in comparison to similar studies,
in the water and in the tissues of eggs, tadpoles, juveniles, and adults. There were few
signiﬁcant differences in organochlorine concentrations between the agricultural,
Kalamazoo River, and reference locations. In other studies, frogs with similar
concentrations of organochlorine residues exhibited no physical deformities. Overall, the
low levels of organochlorine residues in this study can be used as reference background
levels, from which it can be assumed that at or below these measured concentrations, no

signiﬁcant ﬁ'og deformities should be observed.

42

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MCF-7 human breast cancer cells by extracts of pulp mill effluents, sludge, and

sediment exposed to efﬂuents. Environ. Toxicol. Chem. 17, 1499-1507.

45

Osborn, D., Cooke, A.S., Freestone, S., 1981. Histology of a teratogenic effect of DDT
on Rana temporaria tadpoles. Environ. Pollut. (Series A) 25, 305-319.

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Russell, R.W., Gillan, K.A., Haffner, GD, 1997. Polychlorinated biphenyls and
chlorinated pesticides in southern Ontario, Canada, green frogs. Environ. Toxicol.
Chem. 11, 2258-2263.

Russell, R.W., Hecnar, S.J., Haffner, GD, 1995. Organochlorine pesticide residues in
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Safe, S., 1990. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs),
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(TEFs). Crit. Rev. Toxicol. 21, 51-88.

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Pseudacris triseriata and Fowler’s toad Bufo woodhousii fowleri. Copeia 1970,
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0-deethylase induction in H4IIE cells: Implications for their use as bioanalytical

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Chemical Society, Washington DC.

47

Chapter 3: Metals in Sediment and Green Frogs from Southwestern Michigan
Introduction
Frog deformities and worldwide amphibian declines have been the focus of scientiﬁc
research and debate over the past 15 years. Several hypotheses to explain these
phenomena have been proposed; however, no deﬁnite causes have been found (Burkhart
et al., 1998). One possible explanation is exposure to chemical residues. In particular,
exposure to chemicals that have the potential to modulate the endocrine system are of

current concern (Ankley et al., 1998).

Metals of environmental interest include elements which are macro-nutrients or micro-
nutrients in the biosphere and others which have no known biological function and are
generally regarded as toxic (Bunce, 1994). Elements that are biologically essential
include: calcium (Ca), cobalt (Co), copper (Cu), chromium (Cr), iron (Fe), magnesium
(Mg), manganese (Mn), potassium (K), and zinc (Zn). Metals with no established
biological functions include: aluminum (A1), arsenic (As), barium (Ba), cadmium (Cd),
lead (Pb), mercury (Hg), nickel (Ni), scandium (Sc), strontium (Sr), and titanium (Ti)
(Jacobs, 1996). It is important to note that, as Paracelsus states, “No substance is a
poison by itself. It is the dose that deterrrrines the poison” (Lu, 1991). Hence, even the
generally non-toxic metals can be toxic at high enough concentrations in the body, while

the toxic metals may elicit no effects due to extremely low concentrations.

Macrominerals are distinguished from the microminerals by their occurrence in the body.

Macrominerals are generally believed to constitute at least 0.01% of total body weight

48

(Hunt and Groff, 1990). Calcium is a structural component of bones and plays an
integral role in intracellular and hormonal secretion regulation, muscle contraction, blood
clotting, and the activation of some enzyme systems. Like calcium, magnesium is also a
constituent of bone structure. Magnesium is important in nerve impulse transmission,
protein synthesis, and enzyme activation. Another macromineral, potassium, functions as

an electrolyte in the body.

Microminerals constitute less of the total body weight than do macrominerals; however,
there is discrepancy in their actual deﬁnition (Hunt and Groff, 1990). Chromium is
involved in the normal use of blood glucose and the function of insulin, and cobalt is
necessary for the synthesis of Vitamin B12. Copper is required for the proper use of iron
in the body, and is involved in the synthesis of cytochrome oxidase and the development
of connective tissue and blood vessels. Iron is a necessary component of hemoglobin and
myoglobin for oxygen transport and cellular use, as well as being a facilitator in the
transfer of electrons in the electron transport chain. Manganese is needed for normal
brain function, as well as for its role in enzyme systems, collagen formation, bone
growth, urea formation, fatty acid and cholesterol synthesis, and the digestion of proteins.

Zinc is important for energy metabolism and protein synthesis.

While much of the literature lists arsenic and nickel as possessing no biological function,
they may indeed be microminerals (Hunt and Groff, 1990). Arsenic is present throughout
the earth’s crust and is present in varying concentrations in soils. Fallout sources such as

pesticides, smelters, and coal-ﬁred power plants can increase the concentrations of

49

arsenic in certain regions. Arsenic is believed to be necessary for normal growth and for
the usage of iron in many animals. Nickel occurs in ores, and smelting and industrial
uses tend to increase its levels in the environment. While it is known to be a human
carcinogen as well to cause dermal hypersensitivity (Lu, 1991), nickel is possibly

involved in hormonal membrane or enzyme activity.

As mentioned above, there are several metals with no known biological ﬁrnction. Among
these is aluminum, which comprises about 8% of the earth’s crust (Gerhardsson and
Skerfving, 1996). It is widely used as a building material and industrially as an abrasive
and chemical catalyst (Cory-Slechta, 1996). Aluminum has been thought to be a
neurotoxin for over 100 years. In 1898, Dollken described the studies of Siem in 1886, in
which aluminum was shown to elicit nerve and muscle paralysis in frogs, cats, dogs, and
rabbits (summarized by Cory-Slechta, 1996). Another toxic metal, cadmium, is
increasingly used in industry in battery manufacture, metal coating by electroplating, as
well as being a pigment in paints and a stabilizer in plastics (Waalkes and Misra, 1996).
The primary sources of cadmium in the environment include mining, reﬁning, and
smelting operations. Cadmium, a known carcinogen, has a similar chemical nature as
zinc, and as such, displaces zinc in many biological processes. Cadmium can also affect

calcium homeostasis (Waalkes and Misra, 1996).

While mercury constitutes very little of the earth’s crust (about 0.5 ppm) (Cory-Slechta,

1996), it’s toxicity is well studied. Mercury, a component of many batteries, is used in

the manufacture of organomercurials, which are used in agriculture as fungicides. Its

50

electrical applications include mercury as a component of ﬂuorescent and germicidal
lamps in addition to its use as a sealant to exclude air from tungsten light bulb ﬁlaments.
(Bunce, 1994). Mercury is more toxic and lipophilic in its methylated forms. Because of
its increased lipophilicity, it can bioconcentrate and cross the blood-brain barrier, which

leads to its neurotoxic effects (Bunce, 1994).

Like mercury, lead occurs in nature primarily as the sulﬁde (Bunce, 1994). It has been
used as a metal for centuries, most currently its uses include the addition of organolead
compounds to gasoline, the lead-acid storage battery, and as a component of solder. The
similarity in ionic structures between lead and calcium leads to the replacement of
calcium in the body, particularly in bones, with lead. Lead, like mercury, is a neurotoxin

which causes more damage in its organic forms (Bunce, 1994).

Other potentially toxic metals of interest include barium, which occurs in ores of barite,
and is used in a variety of applications, such as alloys in vacuum tubes and spark plugs,
and as a radioactive tracer (Lewis, 1993). It is toxic in its salt form (Lu, 1991).
Scandiurn has no major industrial use, but has been used as an artiﬁcial radioactive
isotope in tracer studies and leak detection (Lewis, 1993). It has been implicated as a
potential carcinogen (Waalkes and Misra, 1996). Strontium is used in alloys (Lewis,
1993). Titanium is used in alloys and as structural constituent in a variety of materials,
including aircraft, missiles, textile machinery, and chemical equipment (Lewis, 1993).

Like scandium, it has been implicated as a carcinogen (Waalkes and Misra, 1996).

51

Some metals have been postulated to be endocrine disruptors, and as such, have the
potential to disrupt the normal development of frogs and other amphibians. The best
known of these is tributyltin (TBT), a suspected androgen (Villeneuve et al., 1998).
TBT, an organotin component of antifouling paints on ships, is a known endocrine
disruptor which causes imposex in gastropods (F ent, 1996). Triphenyltin (TPT) is an
organotin compound that is commonly used in agriculture as a fungicide and can reach
the environment through leaching, runoff, and volatilization. Within the past decade, the
use of organotins has been restricted due to their toxicity to aquatic organisms, and the
effects they have on animals of higher trophic levels. Increased concentrations of TPT
have been shown to decrease survival and growth rates and increase time to
metamorphosis of two tadpole species (Fioramonti et al., 1997). Further, short-term
exposure to TPT has been shown to affect the swimming and feeding behavior of
tadpoles of the European frog (Rana esculenta) (see Semlitsch et al., 1995). Alterations
in these behaviors can prove harmful to the growth, development, and survival of

amphibians.

The primary objectives of this study were to assess the incidence of green frog
deformities in southwestern Michigan, and to correlate those deformities to chemical
residues in the frog tissue. No signiﬁcant deformities were observed among the sampling
locations, as discussed in Chapter 1. Therefore, no correlations between the observed
residues and deformities could be developed. However, the remaining study objectives
remained viable. These objectives involved gathering data on the levels of metals in

sediment and in tissues of green frog tadpoles, juveniles, and adults. Concentrations of

52

Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Ni, Pb, Sc, Sr, Ti, and Zn were
determined. These metals were all analyzed and expressed as total metals, even though
they may be in various physical or chemical forms in tissue (Jacobs, 1996). Many of
these metals, as previously mentioned, are not commonly considered toxic and are
actually necessary to the survival of organisms. However, as a general monitoring study,
the data gathered here may be useful in knowing background metal concentrations in

sediment and green frogs from southwestern Michigan for future reference.

Materials and Methods

Tadpoles and adults of the green frog (Rana clamitans) were collected by dip net and
hand from seven sampling locations in southwestern Michigan (see Chapter 1). Tadpoles
and adults were weighed and measured, and the sex of the adults was determined based
on physical features. The most common means of sexing green frogs is based on the size
of the tympanum. In males, the tympanum is larger than the eyes. Males also may have
a yellowish throat, whereas the throat of the females is generally whitish (Harding, 1997).
Tadpoles were pooled into samples of approximately 20 g wet wt. by location and date.
Pooling of samples was not necessary for the adults. The frogs used for this analysis
were not the same frogs as those used in the organochlorine analysis (Chapter 2). The
livers were the only tissues of adult frogs analyzed for metals. They were removed from
the adults using acid-washed teﬂon forceps and spatulas. The primary reason for
choosing livers is because the liver possesses a larger capacity than most other organs to
bind chemicals such as metals (Lu, 1991). The same is true of the kidneys; however, the

liver is easier to locate and remove from frogs. All frog tissue was stored in acid-washed

53

plastic jars at —20°C until analysis.

Sediment cores were collected from the following sampling locations — REF], REF3,
KLMZR, AGR2, and AGR3. The upper 10 cm was separated and designated the upper
layer while the next 10 cm layer was designated the lower layer. Each layer was air dried
for 48 h. Air drying, rather than drying in an oven, was necessary in order to minimize
Hg volatilization. Dried sediment samples were then ground using pestle and mortar. All

sediment samples were stored in acid-washed plastic jars at —20°C until analysis.

Before a digestion was begun, all vessels and utensils were cleaned. All teﬂon materials,
including liners, lids, spatulas, and autosampler bottles, were acid-washed with nitric acid

and soaked for at least 24 h in a distilled-deionized water (DDW) bath.

Each sample of sediment or frog tissue was weighed (about 0.500 g dry wt.) into a teﬂon
digestion vessel liner that contained 10 ml of nitric acid. The vessels were sealed and
placed in a microwave oven for digestion. Digestion lasted 15 min, followed by at least a
20 min cooling period. After cooling, 90 ml of DDW was added to each sample vessel.
Each sample leachate was ﬁltered through a 0.4 pm Nuclepore ﬁlter into Nalgene"'M
bottles. Half of the leachate was set aside for the ﬁll] metal scan, while the other half was
prepared for mercury analysis. This involved adding gold chloride (10 pL of HAuCl4 /
1.0 mL sample) (Aldrich Chemical Company, Inc., Milwaukee, WI, USA) to each sample

to keep mercury in solution (Gill and Fitzgerald, 1987).

54

For analysis, samples were diluted in order to scan most of the metals in range of the
detector. Sediment samples were diluted 100-fold, and tissue samples were diluted 5-
fold. The analysis was done with a Platform lCP-MS (Micromass, Inc., Manchester,
England). Samples were introduced with a concentric nebulizer that was connected to a

CETAC ASX-SOO auto-sampler.

Results

Raw data of sediment and frog tissue metal concentrations are tabulated in Appendix B,
along with a table depicting statistical p-values for signiﬁcant differences (a=0.05)
between locations and life stages. There were no signiﬁcant differences between the
upper and lower layers of sediments. Therefore, sediment layers were analyzed as

individual samples.

Concentrations of ﬁve of the 20 elements analyzed (Al, Ca, Co, Mg, Sr) in sediments
differed signiﬁcantly among locations when the MDL was substituted for non-detectable
concentrations (Table 10). For those elements that did not differ signiﬁcantly among
locations, the least concentrated were Cd (_<_0.21 ug/g dry wt.), Hg ($0.22 ug/g dry wt.),

Ni ($2.0 ug/g dry wt.), Sc ($2.4 ug/g dry wt), and As ($3.8 ug/g dry wt). The most

concentrated elements in sediment that did not differ signiﬁcantly among locations were

Fe (9.5x102 pg/g dry wt.), Mn (3.6x102 pg/g dry wt.), K (2.3x102 pg/g dry wt.), Ba

(1.0x102 jag/g dry wt.), and Cr (42 ug/g dry wt.) (Table 10).

Of the metals for which concentrations differed signiﬁcantly among locations, Al was

55

most concentrated at the agricultural location AGR2 (9.9x103 pg/g dry wt.) (Tables 9 and
10). This site also exhibited the greatest Co concentration in sediment (6.4 ug/g dry wt.)

(Tables 9 and 10). The greatest concentrations of Ca, Mg, and Sr were observed at the
Kalamazoo River site, KLMZR. These concentrations were 1.7x105, 4.6x103, and 96

ug/g dry wt., respectively (Tables 11 and 12).

Concentrations of four of the elements analyzed (Ba, Ca, Mg, and Ti) in adult livers
differed signiﬁcantly among locations when the MDL was substituted for non-detectable

concentrations (Table 10). Concentrations of elements that did not differ signiﬁcantly
among location included Cd ($0.08 pg/g dry wt.), As ($0.10 ug/g dry wt.), Co ($0.13

pg/g dry wt), and Sc ($0.23 pg/g dry wt) at the low end to K (3.3x103 pg/g dry wt.), Fe

(1.1x103 pg/g dry wt.), Cu (65 pg/g dry wt.), Al (58 rig/g dry wt.), and Zn (27 pg/g dry

wt.) (Table 10).

In adult livers that differed signiﬁcantly among location, the metals Ba, Ca, Mg, and Ti
were most concentrated at the reference location REF 3. The concentrations were 38,
8.6x102, 4.8x102, and 4.1 pg/g dry wt., respectively (Tables 11 and 12). The location
REF3 had a signiﬁcantly higher level of Mg in adult livers than all the other locations

(Table 12).

Juvenile tissues were most concentrated with Ca (2.8x104 pg/g dry wt.), Fe (1.9x104 ug/g

dry wt.), K (4.2x103 pg/g dry wt.), Mg (1.0x103 ug/g dry wt), and Al (3.7x102 ug/g dry

wt). Metals that were least concentrated in juvenile tissues were Cd ($0.03 ug/g dry wt.),

56

Hg ($0.17 ug/g dry wt.), Co ($0.20 pg/g dry wt.), and As ($0.30 ug/g dry wt.) (Table

10).

Tadpole tissues contained the greatest concentrations of Fe (6.5x104 ug/g dry wt.), Ca
(2.3x104 rig/g dry wt.), K (5.8x103 ug/g dry wt.), Mg (1.0x103 jig/g dry wt), and A1
(5.9x102 ug/g dry wt). Metals that were least concentrated in tadpole tissues were Cd
($0.23 ug/g dry wt.), Hg ($0.24 ug/g dry wt.), Co ($0.41 pg/g dry wt.), and Sc ($0.80

pg/g dry wt.) (Table 10).

57

Table 10. Metal concentrations (pg/g, dry wt., mean and standard deviation, in
parentheses) in sediment and green frog tissue from southwestern Michigan,

 

 

 

 

 

 

1998.
Metal Adult‘ Juvenile2 M618 Sediment‘
Values with 0.0 substituted for non-detects
Al 57 (1.2x102) 3.7x102 (3.3x102) 5.9x102 (2.1x102) *m
As 0* 0* 0* 0*
Ba **** 30 (27) 35 (36) 66 (85)
Ca 2.4x102 (3.3x102) 2.8r1104 (1.1x10‘) 2.0x10‘(1.4x10‘) 2.0x10‘ (2.2x10‘)
Cd 0* 0* 0* 0.18 (0.28)
Co 0* 0* 0* 0*
Cr 0* 0* 0* 0*
Cu 65 (30) 1.4 (2.5) 1.8 (3.7) 2.1 (7.3)
Fe 1.1:1103 (2.3x103) 1.9x10“ (2.7x10‘) 6.5x10‘(5.8x10‘) 6.8x10’(1.6x103)
Hg 0*- 0* 0* 0*
K **** 4.3r1103 (6.5x102’ 5.5x103 (3.6x103) 1.5x102 (5.071102)
Mg rm 1.0xlo3 (4.5x103) 9.2x102 (6.5x102) ****
Mn 0* 50 (86) 2.8x102 (4.6x102) 3.6x102 (7.0x102)
Ni 0.61 (2.5) 0* 0* 2.0 (5.6)
Pb 0* 3.9 (3.6) 0* 20 (14)
Sc 0* 0* 0* 0*
Sr 0* 5.8 (10) 4.0 (11) *m
Ti 1.3 (1.5) 17 (8.2) 22 (8.0) 8.2 (14)
Zn 23 (14) 49 (25) 30 (32) 0*
Values with MDL" substituted for non-detects
Al 58 (1.2x102) 3.7x102 (3.3x10T 5.9x102(2.1x102) me
As 0.10 (0.14) 0.30 (0.25) 1.3 (0.82) 3.8 (3.1)
Ba *m 34 (21) 45 (28) 1.0x102 (59)
Ca **** 2.8x10‘ (8.0x102) 2.3x10‘ (2.0x103) *m
Cd 0.08 (0.11) 0.03 (0.04) 0.23 (0.37) 0.21 (0.27)
Co 0.13 (0.17) 0.20 (0.21) 0.41 (0.27) ****
Cr 8.3 (18) 8.8 (5.6) 9.5 (8.4) 41 (61)
Cu 65 (30) 4.0 (2.3) 4.2 (2.7) 13 (7.2)
Fe 1.1x103 (2.3x10’) 1.9x10‘ (2.7x10‘) 6.5x10‘ (5.8x10‘) 9.5x10 (1.5x103)
Hg 0.43 (0.79) 0.17 (0.12) 0.24 (0.17) 0.22 (0.34)
K 3.3r1103 (7.2x102) 4.3x103 (6.5x102) 5.8xlo3 (3.1x103) 2.3x102 (5.6x102)
Mg **** 1.0x103 (4.5x102) 1.1r1103 (5.1x102) *m
Mn 2.9 (4.8) 73 (73) 321102 (4.4x102) 3.6x102 (7.0x102)
Ni 0.9 (2.6) 2.9 (0.01) 2.9 (0.01) 2.0 (5.6)
Pb 0.76 (0.01) 3.9 (3.6) 1.6 (1.5) 24 (9.9)
So 0.23 (0.18) 0.53 (0.23) 0.80 (0.39) 2.4 (0.88)
Sr 0.31 (0.69) 11 (6.4) 13 (8.5) "**
Ti **** 17 (8.2) 22 (8.2) 19 (11)
Zn 27 (7.9) 49 (25) 46 (20) 24 (18)
In=16 livers

2n=3 pooled samples
3n=7 pooled samples

4n=12 samples of top and bottom layers
"' No standard deviations are presented here because the values were below the MDL and none could be

calculated.

"The MDL is a function of sample mass and recovery, and varies from sample to sample. The range of
concentrations is presented in this table by substituting in both 0.0 and the MDLs for non-detected

concentrations.

""At the 95% conﬁdence level, there were signiﬁcant differences among locations so a total mean could

not be calculated.

Table

11. Metal concentrations (pg/g dry wt., mean and standard deviation, in
parentheses)1 in sediment and green frog tissue from sampling locations that
are signiﬁcantly different at the 95% conﬁdence level in southwestern
Michigan, 1998.

 

 

 

 

 

REF] REF3 KLMZR AGRl AGR2 AGR3
Adult
Ba 12 37* 4.2 1.8 16 NS
(17) (5.11) (2.7) (6.7)
Ca 181102 8.6x102“ 1.111102 3.7xlo2 6.0x102 NS
(30) (32) (9.9) (3.1x102)
Mg 2.5xlo2 4.8x102‘ 2.6x102 3.0xlo2 22th2 NS
(55) (26) (28) (25)
Ti 3.3 4.1* 2.8 3.2 2.2 NS
(0.74) (0.28) (0.38) (0.63)
Sediment
A1 3.3:1103 3.1x103 3.7x103 NS 9.9x103 3.9r1103
(1.2x103) (1.4x103) (6.9x102) (2.7x103) (3.0x102)
Ca 1.3x10‘ 1.3x10‘ 1.7x105 NS 3.7x103 6.5x103
(3.5x103) (1.8x102) (2.8x10‘) (2.0x103) (1.5x10’)
Co 0.09 1.9 2.5 NS 6.4 1.5
(0.04) (0.42) (1.0) (2.4) (0.06)
Mg 1.2:1103 1.9r1103 4.6:1103 NS 1.3x103 1.1x103
(2.4x102) (1.8x102) (7.3x102) (6.2x102) (1.8x102)
Sr 21 28 96 NS 13 9.5
(8.9) (12) (21 ) (1.3) (0.7g

 

jValues using the MDL for non-detects

NS = Not sampled

*No standard deviations could be calculated for these because n=1.

59

Table 12. Samples for which metal concentrations varied signiﬁcantly among locations.
Underlined locations are not signiﬁcantly different (a=0.05). Locations are

arranged in order of increasing concentrations, from left to right.

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample, Metal Location

Adult, Ba AGR] KLMZR REF] AGR2 REF 3
Adult, Ca KLMZR REFl AGRl AGR2 REF3
Adult, Mg AGR2 REFl KLMZR AGR] REF3
Adult, Ti AGR2 KLMZR AGR] REF] REF 3
Sediment, Al REF3 REF 1 KLMZR AGR3 AGR2
Sediment, Ca AGR2 AGR3 REF] REF 3 KLMZR
Sediment, Co REF] AGR3 REF3 KLMZR AGR2
Sediment, Mg AGR3 REF] AGR2 REF 3 KLMZR
Sediment, Sr AGR3 AGR2 REF] REF 3 KLMZR

 

 

Discussion

Many of the metals found at relatively low concentrations in sediments, such as Cd, Hg,
Ni, Sc, As, and Sr, are usually not present in most sediments and are generally considered
to be toxic, at high enough concentrations (Bunce, 1994). However, because most were
below the limit of detection, and may even be bound in the soil, which reduces their
bioavailability, they are not considered to be a potential threat to the wildlife, such as

frogs, of the aquatic system. The more concentrated metals, such as Al, Ca, Mg, Fe, Mn,

60

and K are components of biological systems and/or sediments; therefore, the high
concentrations are not alarming. As stated above, the greatest concentrations of Al and
Co were observed in the sediment at AGR2. This may be due to natural variation,
because these concentrations were within acceptable levels. The same is true for the

increased concentrations of Ca, Mg, and Sr at KLMZR.

A similar pattern of metal concentrations is observed in frog tissues. Concentrations of
toxic metals, such as Pb, Cd, As, Co, Hg, Cr, Sc, and Sr, in adult livers were relatively
low. Essential metals, such as Ca, Mg, Al, Fe, and K exhibited the greatest
concentrations in adult livers. There appears to be no pattern of accumulation within
locations based on concentrations in each matrix. For instance, in livers that differed
among locations, the location where the greatest concentrations of Ca and Mg were
observed was REF3, whereas the location where the greatest concentrations of those
elements were observed in sediment was KLMZR. The signiﬁcantly greatest
concentrations of Ba, Ca, Mg, and Ti were observed at REF3; however, this data is based

on only one frog liver, and as such, may not be representative of the entire population.

As with the adult livers, essential metals were most concentrated in the tissues of
juveniles and tadpoles. Ca, Fe, K, Mg, and Al exhibited the greatest concentrations in
each, while Ni, Cd, Hg, Co, As, Sc, Cr, and So were relatively low in each. Pb was much

less concentrated in tadpole tissues and adult livers than in juvenile tissues.

61

Mercury was determined in mudpuppies and snapping turtles collected from 1988 to
1992 from the St. Lawrence River in Canada, a river that is greatly impacted by human
activities and synthetic chemicals (Bonin et al., 1995). Concentrations ranged from <20
to 45 ng/g, wet wt. in mudpuppies and 50 to 180 ng/g, wet wt., in turtle eggs (Bonin et
al., 1995). Unlike the present study, concentrations in this study were not speciﬁcally
measured in liver tissue. Generally, the wet weight to dry weight ratio is ﬁve. Therefore,
these reported values can be multiplied by ﬁve to compare to the concentrations in dry
weight ﬁ'om the present study. The mercury concentration reported in frog livers in the
present study (5430 ng/g, dry wt.) is comparable to the values of those in the St.
Lawrence River study. Even though these species are different from the green frog, these

data indicate that a variety of aquatic organisms are capable of absorbing mercury.

Concentrations of Cd, Pb, Zn, and Cu in soil and wildlife within 30 km of a zinc smelter
site in eastern Pennsylvania were determined seven years after smelting was terminated
in 1980 (Storm et al., 1994). Concentrations of Cd, Pb, Zn, and Cu in green frog tadpole
whole body tissue ranged from 0.3 to 1.5, 2.3 to 5.0, 23.1 to 117.0, and 0.3 to 0.8 mg/kg,
wet wt., respectively. When calculated on a dry weight basis, the concentrations of Cd
and Pb are higher from the smelter study by at least a factor of about 7 each when
compared to the values for tadpole tissue in the present study. Concentrations of Zn and
Cd are more comparable between the two studies, with Zn being at least 2.5 times more
concentrated at the smelter site and Cd levels being similar. Overall, these metal
concentrations were similar for adult red-backed salamander and eastern newt tissue

(Storm et al., 1994).

62

Conclusion

The study presented here attempted to link deformities in green frog populations of
southwestern Michigan to metals in their environment and tissues. However, only 0.28%
of green frogs exhibited obvious physical abnormalities. Concentrations of essential
metals and concentrations of metals that possess no known biological functions were
measured in sediment and tissues of tadpoles, juveniles, and adults. There were few
signiﬁcant differences in metal concentrations between the agricultural, Kalamazoo
River, and reference locations. Overall, the low levels of the known toxic metals can be
used as reference background levels, from which it can be assumed that at or below these

measured concentrations, no signiﬁcant frog deformities should be observed.

63

References

Ankley, G.T., Giesy, J .P., 1998. Endocrine disruptors in wildlife: A weight of evidence
perspective. In Kendall, R., Dickerson, W., Suk, W., Giesy, J.P. (Eds.).
Principles and Processes for Assessing Endocrine Disruption in Wildlife.
SETAC, Pensacola, Florida (1998).

Bonin, J., DesGranges, J.L, Bishop, C.A., Rodrigue, J., Gendron, A., Elliott, J.E., 1995.
Comparative study of contaminants in the mudpuppy (Amphibia) and the common
snapping turtle (Reptilia), St. Lawrence River, Canada. Arch. Environ. Contam.
T oxicol. 28, 184-194.

Bunce, N., 1994. Environmental Chemistry (2"d Edn). Wuerz, Winnipeg, Canada.

Burkhart, J.G., Helgen, J.C., Fort, D.J., Gallagher, K., Bowers, D., Propst, T.L., Gernes,
M., Magner, J., Shelby M.D., Lucier, G., 1998. Induction of mortality and
malformation in Xenopus laevis embryos by water sources associated with ﬁeld
frog deformities. Environ. Health Perspect. 106, 841-848.

Cory-Slechta, D.A., 1996. Comparative neurobehavioral toxicology of heavy metals. In
Chang, L.W., Magos, L., Suzuki, T. (Eds.) Toxicology of Metals. CRC Press,
Boca Raton, Florida.

Fent, K., 1996. Ecotoxicology of organotin compounds. Crit. Rev. T oxicol. 26, 1-117.

Fioramonti, B., Semlitsch, R.D., Reyer H., Fent, K., 1997. Effects of triphenyltin and pH
on the growth and development of Rana lessonae and Rana esculenta tadpoles.

Environ. Toxicol. Chem. 16, 1940-1947.

64

Gerhardsson, L., Skerfving, S., 1996. Concepts on biological markers and biomonitoring
for metal toxicity. In Chang, L.W., Magos, L., Suzuki, T. (Eds.) Toxicology of
Metals. CRC Press, Boca Raton, Florida.

Gill, G.A., Fitzgerald, W.F., 1987. Picomolar mercury measurements in seawater and
other materials using stannous chloride reduction and two-stage gold
amalgamation with gas phase detection. Marine Chemistry 20, 227-243.

Jacobs, R.M., 1996. Techniques employed for the assessment of metals in biological
systems. In Chang, L.W., Magos, L., Suzuki, T. (Eds.) Toxicology of Metals.
CRC Press, Boca Raton, Florida.

Lewis, R.J., 1993. Condensed Chemical Dictionary (12th Ed.). Van Nostrand Reinhold
Company, New York, New York.

Lu, RC, 1991. Basic Toxicology: Fundamentals, Target Organs, and Risk Assessment
(2“l Edn.). Taylor and Francis, Bristol, PA.

Semlitsch, R.D., Foglia, M., Mueller, A., Steiner, 1., Fioramonti, B., Fent, K., 1995.
Short-term exposure to triphenyltin affects the swimming and feeding behavior of
tadpoles. Environ. Toxicol. Chem. 14, 1419-1423.

Storm, G.L., Fosmire, G.J., Bellis, ED, 1994. Heavy metals in the environment:
Persistence of metals in soil and selected vertebrates in the vicinity of the
Palmerton zinc smelters. J. Environ. Qual. 23, 508-514.

Villeneuve, D.L., Blankenship, A.L., Giesy, J.P., 1998. Interactions between
environmental xenobiotics and estrogen receptor-mediated responses. In:
Denison, M.S., Helferich, W.G. (Eds.). T oxicant-Receptor Interactions. Taylor

and Francis, Philadelphia, Pennsylvania.

65

Waalkes, M.P., Misra, RR, 1996. Cadmium carcinogenicity and genotoxicity. In
Chang, L.W., Magos, L., Suzuki, T. (Eds.) Toxicology of Metals. CRC Press,
Boca Raton, Florida.

Waalkes, M.P., Misra, RR, 1996. Carcinogenicity and genotoxicity of lead, beryllium,
and other metals. In Chang, L.W., Magos, L., Suzuki, T. (Eds.) Toxicology of

Metals. CRC Press, Boca Raton, Florida.

66

Chapter 4: Triazine Herbicide Concentrations in Water and Green Frogs from
Southwestern Michigan

Introduction

Frog deformities and worldwide amphibian declines have been the focus of a great deal
of scientiﬁc research and debate over the past 15 years. Several hypotheses have been
formulated as to the reasons for these phenomena; however, no deﬁnite causes have been
found (Burkhart et al., 1998). One possible explanation is exposure to chemical residues.
In particular, exposure to chemicals that have the potential to modulate the endocrine

system are of current concern (Ankley et al., 1998).

A class of chemicals that belong to those being scrutinized are the triazine herbicides, most
notably atrazine. Atrazine is primarily used for the control of certain annual broadleaf and
grass plants in regions of com farming, as well as in other agricultural locations (Solomon et
al., 1996). Atrazine is found in many surface and ground waters in North America, and
aquatic ecological effects are a concern (Solomon et al., 1996). Recently, atrazine has been

found to have the potential to be an endocrine disruptor (Sanderson et al., 2000).

Many analytical techniques for triazine herbicides in water and tissue exist. A traditional
means of analysis involves instrumental analysis by gas chromatography (GC) coupled mass
spectrometer detection (MS) (Thurman et al., 1992; Pereira and Rostad, 1990). This method
is sensitive, selective, and quantitative. However, due to the amount of labor and time
involved in this procedure, the use of GC-MS is not always practical. Another method for the

analysis of triazine herbicides has been developed, known as the Enzyme Linked

67

Immunosorbent Assay (ELISA). This method has proven useﬁrl for analyzing this class of
herbicides (Huber, 1985; Thurman et al., 1990), even though it is often not as selective or
speciﬁc as instrumental techniques (Rubio et al., 1991). Comparisons of the two methods by
analyzing atrazine in water and soil have indicated that results from ELISA tests are

comparable to those obtained from GC-MS techniques (Lydy, 1996; Amistadi, 1997).

In this study, concentrations of triazine herbicides in water and green frog tadpole and
juvenile tissue from southwestern Michigan were determined. ELISA analysis was used
for water and GC-MS and ELISA analysis were used for frog tissue. A new method for
the determination of triazines in extracted tissue by ELISA was developed and compared
with the GC-MS method. Because no green frog deformities were observed in the study
area, it is unlikely that the range of concentrations of triazine were sufﬁcient to cause

such effects in southwestern Michigan (Chapter 1).

Materials and Methods

Water and Tadpole Sampling and Preparation

Four liters of water were collected from each sampling location on the ﬁrst two sampling
dates -- 6/5 and 6/23 of 1998. Samples were collected into four-liter brown glass solvent
bottles by submerging each bottle into the water until full. To ensure that they were
clean, bottles were washed with water and Liquinox® (Alconox, New York, NY, USA),
rinsed with acetone and hexane, and allowed to dry before use. Water samples were

stored at 4°C until analysis.

68

Tadpoles of the green frog (Rana clamitans) were collected by methods discussed by dip
net and by hand (see Chapter 1). Tadpoles were weighed, measured, and pooled into
samples of approximately 20 g wet wt. by location and date. Tissues were stored at —

20°C until analysis.

All glassware, stainless steel homogenizer materials, boiling stones, glass wool, and any
other utensils and surfaces that would contact samples during the analysis were cleaned
by standard procedures to minimize contamination of the sample. Brieﬂy, analytical
items were washed thoroughly with Liquinox® (Alconox, New York, NY, USA) and
water, then rinsed with deionized water. Materials were allowed to air dry before being
rinsed three times with acetone followed by three rinses with hexane. Solvent-cleaned
materials were blown dry with a heat gun and placed on solvent-rinsed aluminum foil.
All solvents used for any part of the analysis or cleaning steps were HPLC grade

(Burdick and Jackson, Muskegon, MI, USA).

Water Analysis with ELISA

Prior to analysis, water samples were allowed to heat up to ambient temperature and were
shaken and ﬁltered through glass wool to remove organic particles. The ELISA assay
was a commercial ELISA kit designed speciﬁcally for the analysis of water samples
directly from the ﬁeld (EnviroGardTM Triazine Plate Kit, Strategic Diagnostics
Incorporated, Newark, DE, USA). The instructions given with the kit were followed with
no changes. A spectrophotometer (Cayman Chemical Plate Scanning Spectrophotometer

and Autoreader, Ann Arbor, MI, USA) was used to measure the mass of triazines in the

69

wells of the plate. Absorbance results were normalized based on the blanks and a
calibration graph was made for the standards. Mean normalized sample absorbance
results were compared to the standard calibration graph to obtain the value for the

concentration of triazines in each water sample.

Tadpole Tissue Analysis with ELISA

Extraction methods were similar to those for OCs (Kannan et al., 1995, Khim et al.,
1999). Each tissue sample was thawed at room temperature for 1 h before being prepared
for analysis. Each sample was ground in a stainless steel homogenizer cup by use of an
Omni-Mixer (Sorvall®, PRO Scientiﬁc Inc., Monroe, CT, USA). Sodium sulfate was
slowly added during the blending process (150 g anhydrous NaZSO4 for a 20 g sample)
until the sample was thoroughly dry. Samples were extracted by Soxhlet extraction with
a 3:1 ratio of dichloromethane (DCM) to hexane for at least 12 h. Extracts were
concentrated to 11 ml and a 1 ml subsample was removed to determine the lipid content

of the tissues. Lipid content was measured gravimetrically.

The 10 ml extracts were further concentrated to 3 ml for fractionation. F lorisil (60-100
mesh size; Sigma, St. Louis, MO, USA) was activated at 130°C for at least 12 h. For
each sample, 10 g of Florisil was suspended in hexane and transferred to a glass
chromatography column. The 3 ml extract was placed on top of the F lorisil and eluted at
a ﬂow rate of approximately 3 ml / min. The ﬁrst fraction (F1), which contained the least
polar analytes, was eluted from the column with 100 ml hexane. The moderately polar

compounds were eluted in the second fraction (F2) with 100 ml of 20% DCM in hexane.

70

The ﬁnal fraction (F 3), which was eluted from the column with 100 ml of 50% methanol

in DCM, contained the most hydrophilic compounds.

The volumes of the eluents were reduced by evaporation to 2 ml for F 1 and 0.1 ml for F2
and F 3. F2 was adjusted to 2 ml with hexane. F2 and F3 were the fractions considered
for this study. It was determined by use of a standard that 100% of the atrazine in the
samples would elute in F3. Therefore, F 1 and F2 were set aside for DC analysis. F3 was

diluted to 1 ml with acetonitrile.

In order to ensure that the solvent (acetonitrile) would not adversely impact the results of
the ELISA assay, several series of atrazine standards were prepared in varying ratios of
acetonitrile to water. The calibration regression of atrazine in 5% acetonitrile in water
yielded an RZ-value of 0.9938. Therefore, aliquots of tadpole extracts were diluted to 5%
acetonitrile in water before being dosed into the ELISA plate wells. The same ELISA kit

as that used in the water analysis was used and the methods were the same.

Gas Chromatographic Analysis of Tadpole Tissue

Undiluted extracts were used to verify ELISA results. Samples were injected into a
Hewlett Packard Model 5890 Series 1] Plus Gas Chromatograph Model 5972 with mass
spectrometer (Hewlett Packard) by a Model 7673 autosampler automatic injector

(Hewlett Packard).

7l

Results

Triazine herbicide applications in the study region are composed primarily of atrazine
(Summer, personal communication). Therefore, while values are reported as triazine
concentrations due to the non-speciﬁcity of the ELISA test, it can be assumed that the

majority of the residues are atrazine.

Table 13. Triazine herbicide concentrations in water and green frog tadpole and juvenile
tissues from southwestern Michigan, 1998.

 

 

Location Water Tadpole Juvenile
Concentration, ug/L Concentration, ng/ g Concentration, ng/g
(n=2) wet wt.* wet wt."
REF] 0.063 0.411 0.195
REF2 0.057 0.629 NS
REF3 0.040 0.230 NS
KLMZR 0.034 0.339 0.201
AGR] 0.056 0.442 NS
AGR2 0.214 0.347 NS
AGR3 0.308 0.347 NS

 

*n=2 pools for REF] , REF3, KLMZR, and AGR2
n=1 pool for REF2, AGR], and AGR3

"n=1 pool for REF] and n=2 pools for KLMZR

NS = Not Sampled

The greatest triazine concentrations in water were observed at the agricultural locations
AGR2 and AGR3 (0.214 and 0.308 rig/L), while the least concentrations were found at
the location on the Kalamazoo River, KLMZR, and one of the reference locations, REF3
(0.034 and 0.040 ug/L, respectively) (Table 13). The other agricultural location, AGR],

had the third least triazine concentrations, 0.056 ug/L (Table 13).

As analyzed with the ELISA test, the least triazine concentrations in tadpole tissue were

observed in tadpole were from REF 3 (0.230 ng/g, wet wt.) and KLMZR (0.339 ng/g, wet

72

wt.), similar to the results from the water analysis (Table 13). The greatest
concentrations in tissues were from the reference location REF2 (0.629 ng/g, wet wt.) and
AGR] (0.442 ng/g, wet wt.) (Table 13). The triazine concentrations in tissues of
juveniles were similar to each other, and both locations exhibited juvenile tissues that

contained less triazines than the least tadpole tissue concentrations (Table 13).

The method detection limit (MDL) for the tissue samples analyzed by GC-MS ranged
from 2.15 to 2.83 ng/g, wet wt. Therefore, these detection limits were too high to
compare exactly to the results from the ELISA test. However, these results do not refute

the results from the ELISA test either.

Discussion

The ELISA kit used in this study has been shown to accurately measure concentrations of
triazines in non-extracted water (Strategic Diagnostics, personal communication).
Therefore, it can be assumed that the ELISA kit was able to accurately measure the
triazine concentrations in water. It is logical that the greatest water herbicide
concentration was observed at two of the three agricultural locations due to the known
usage of atrazine in the region. However, the third agricultural location did have one of
the lesser triazine concentrations. This location is known to have a buffer zone built
around the sampling pond to minimize run-off (Mehne, personal communication).
Therefore, this buffer zone may be the reason for the relatively small concentrations in

water at this location.

73

Based on the results of the ELISA analysis, greater triazine concentrations were observed
in the tissue of the tadpoles than in juvenile tissue. This may be due to the greater
potential of the tadpoles to be exposed to the herbicides in the water than the juveniles
since the tadpoles live solely in the water and receive all their nourishment from the

aquatic food chain.

Concentrations of triazines in tissue were not well correlated with water concentrations.
The tadpoles from the agricultural locations contained approximately the same
concentrations as those from the reference locations. These results could represent the
actual concentrations, or they could be a result of error from use of the ELISA, especially
from possible interactions with the solvent in the extracts. More research is needed in
order to be able to justify the use of ELISA tests for tissue extracts. Overall, however, it
is possible that the ELISA is correct, because the results are comparable to those ﬁ'om the

GC-MS in terms of being within the range of possible concentrations.

Conclusion

The study presented here attempted to link deformities in green frog populations of
southwestern Michigan to triazine herbicide residues in their environment and tissues.
However, only 0.28% of green frogs exhibited obvious physical abnormalities. Atrazine,
a triazine herbicide believed to be an endocrine disruptor (Sanderson et al., 2000), is
present in many North American waterways. Concentrations of triazine herbicides,
including atrazine, were measured in water and green frog tadpole and juvenile tissue

using both enzyme-linked immunosorbent assay (ELISA) and gas chromatography with

74

mass spectrometer detection (GC-MS). A method for the determination of triazines in
tissue by ELISA was developed. The ELISA method is a standard method used for the
determination of triazines in water. More testing of this method is necessary before it can
be used as a standard analysis tool for the determination of triazines in tissues. Slightly
higher levels of triazines in water were observed at the agricultural locations. Because no
deformed green frogs were observed at these locations, it can be assumed that the levels

of triazines observed in water do not cause deformities.

75

References

Amistadi, M.K., Hall, J.K., Bogus, E.R., Mumma, R.O., 1997. Comparison of gas
chromatography and immunoassay methods for the detection of atrazine in water
and soil. J. Environ. Sci. Health B32: 845-860.

Ankley, G.T., Giesy, J .P., 1998. Endocrine disruptors in wildlife: A weight of evidence
perspective. In Kendall, R., Dickerson, W., Suk, W., Giesy, J.P. (Eds.).
Principles and Processes for Assessing Endocrine Disruption in Wildlife.
SETAC, Pensacola, Florida (1998).

Burkhart, J.G., Helgen, J.C., Fort, D.J., Gallagher, K., Bowers, D., Propst, T.L., Gernes,
M., Magner, J., Shelby M.D., Lucier, G., 1998. Induction of mortality and
malformation in Xenopus laevis embryos by water sources associated with ﬁeld
frog deformities. Environ. Health Perspect. 106, 841-848.

Huber, S.J., 1985. Improved solid-phase enzyme immunoassay systems in the ppt range
for atrazine in fresh water. Chemosphere 14: 1795-1803.

Kannan, K., Tanabe, S., Tatsukawa, R., 1995. Geographical distribution and
accumulation features of organochlorine residues in ﬁsh in tropical Asia and
Oceania. Environ. Sci. T echnol. 29, 2673-2683.

Khim, J .S., Villeneuve, D.L., Kannan K., Lee, K.T., Snyder, S.A., Koh, C.H., Giesy, J .P.,
1999. Alkylphenols, polycyclic aromatic hydrocarbons, and organochlorines in
sediment from Lake Shihwa, Korea: Instrumental and bioanalytical
characterization. Environ. Toxicol. Chem. 18, 2424-2432.

Lydy, M.J., Carter, D.S., Crawford, CG, 1996. Comparison of gas

chromatography/mass spectrometry and immunoassay techniques of

76

concentrations of atrazine in storm runoff. Arch. Environ. Contam. T oxico]. 31,
378-385.

Mehne, C., 1998. Personal communication. Animal Clinic, Kalamazoo, Michigan.

Pereira N.E., Rostad, CE, 1990. Occurrence, distribution and transport of herbicides
and their degradation products in the lower Mississippi River and its tributaries.
Environ. Sci. Technol. 26: 1400-1406.

Rubio, F .M., ltak, J .A., Scutellaro, A.M., Selisker, M.Y., Herzog, DP, 1991.
Performance characteristics of a novel magnetic particle based ELISA for the
quantitative analysis of atrazine and related triazines in water samples. Food and
Agricu]. Immuno. 3: 1 13-125.

Sanderson, J.T., W. Seinen, Giesy, J.P., Van den Berg, M., 2000. 2-chloro-s-triazine
herbicides induce aromatase (CYP-19) activity in H295R human adrenocortical
carcinoma cells: A novel mechanism for estrogenicity. Toxicolo. Sci. (in press).

Solomon, K.R., Baker, D.B., Richard, R.P., Dixon, D.R., Klain, S.J., LaPoint, T.W.,
Kendall, R.J., Weisskopf, C.P., Giddings, J.M., Giesy, J.P., Hall, L.W., Williams,
W.M., 1996. Ecological risk assessment of atrazine in North American surface
waters. Environ. Toxicol. Chem. 15, 31-74.

Strategic Diagnostics Incorporated, 1998. Personal communication. Newark, Delaware.
(302)456-6789.

Summer, C., 1998. Personal communication. Michigan Department of Environmental
Quality, Lansing, Michigan.

Thurman, B.M., Goolsby, D.A., Meyer, M.J., Mills, M.S., Pomes, M.L., Kolpin, D.W.,

1992. A reconnaissance study of herbicides and their metabolites in surface water

77

of the Midwestern United States using immunoassay and gas

chromatography/mass spectrometry. Environ. Sci. T echno]. 26: 2440-2447.
Thurman, B.M., Meyer, M.T., Pomes, M.L., Perry, C.A., Schwab, A.P., 1990. Enzyme-

Linked Immunoassay compared with gas chromatography/mass spectrometry for

the determination of triazine herbicides in water. Anal. Chem. 62: 2043-2048.

78

APPENDICES

79

APPENDIX A

ORGANOCHLORINE PESTICIDES AND PCBs: RAW DATA AND STATISTICAL
SIGNIFICANCE TABLES

80

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83

Table A.4. Signiﬁcant differences of 0C concentrations (a=0.05) between life stages1
and between locations2 and interactions. Values marked with an asterisk (*)
indicate signiﬁcance.

 

 

 

Residue P-Value (0.00 Substituted for Non- P-Value (0.01 Substituted for '

Detects) MDLs)

Life Stage Location LS x LOC Life Stage Location LS x LOC

(LS) (LOC) (LS) (LOC)

a-HCH 00284" 0.0548 0.7268 0.0281* 0.0714 0.7199
B-HCH 0.0004* 0.0014* <0.0001* 0.0002* 0.0006* <0.0001*
y-HCH 0.0186* 0.2860 0.0381“ 0.0253* 0.3013 0.0401*
8-HCH 0.0085* 0.0221 * 0.0002* 0.0075* 0.0185* 0.0001 *
a-chlordane <0.0001 * 0.0002* <0.0001 * <0.0001 * <0.0001 * <0.0001 *
y-chlordane 0.0007* 0.4419 0.1871 0.0011* 0.51 1 1 0.2091
p,p ’-DDT <0.0001* <0.0001* <0.0001"‘ <0.0001* <0.0001* <0.0001*
p,p ’-DDE <0.0001* 0.4696 0.0005 * <0.0001* 0.4716 0.0005*
p,p ’-DDD <0.0001* 0.0002* <0.0001* <0.0001* 0.0002* <0.0001*
ZHCH 00098" 0.0308* <0.0001* 0.0091* 0.0300* <0.0001*
Echlordane <0.0001* 0.0018* <0.0001* <0.0001* 0.0018“ <0.0001*
ZDDT <0.0001* 0.0552* <0.0001* <0.0001* 0.0556 <0.0001*
ZPCBs 0.0208* 0.2205 0.0823 0.0208* 0.2205 0.0823

 

 

 

n = 39 observations
1Life stages are Adult, Juvenile, Tadpole, and Egg.
2Locations are REFI, REF2, REF3, KLMZR, AGR1,AGR2, AGR3.

84

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86

Table A.7. Concentrations (ng/g, wet wt., mean and rangez, in parentheses) of total PCBs
and of di-, mono-, and non-ortho PCBs in green frog egg tissue from
southwestern Michigan, 1998.

 

 

 

 

 

 

 

 

Site REF], 6/23, KLMZR, 6/23, AGRl, 6/23, AGR3, 6/23,
(n=2) (n=1) (n=1) (n=1)

ZPCBs 25.50 236.93 63.02 44.61
(16.4-34.59)

Di-ortho

138 0.80 6.38 3.04 1.88
(O.65-O.94)

153 10.4 112.69 26.51 25.33

(6.5-14.3)

170 0.54 3.29 1.73 0.72
(0400.68)

180 0.72 6.94 2.90 1.45

(O.64-0.8)

194 0.12 <0.01 0.45 0.16
(0.05019)

Mono-ortho

105 0.17 3.02 3.82 3.24
(<0.01-0.34)

118 4.73 24.24 13.88 8.32
(4.26-5.20)

156 <0.011 0.88 0.50 <0.01

Non-ortho

77 <0.011 <0.01 <0.01 <0.01

126 <0.01‘ <0.01 <0.01 <0.01

169 <0.011 <0.01 <0.01 <0.01

 

INo ranges are presented here because values were below the MDL.
2No ranges are presented for KLMZR, AGR], and AGR2 because n=1 pool.

87

APPENDIX B:

METAL RAW DATA AND STATISTICAL SIGNIFICANCE TABLES

88

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91

Table B.4. Signiﬁcant differences of metal concentrations (a=0.05) between life stages1
and between locations2 and interactions. Values marked with an asterisk (*)
indicate signiﬁcance.

 

 

 

 

 

 

Metals P-Value (0.00 Substituted for Non— P-Value With MDLs for Non-

Detects) Detects

Life Stage Location LS x LOC Life Stage Location LS x LOC

(LS) (LOC) (LS) (LOC)
A1 <0.0001* 0.0004* <0.0001* <0.0001* 0.0004" <0.0001*
As NC NC NC <0.0001 "' 0.2023 0.6006
Ba 0.0383* 0.2767 0.5915 <0.0001“ 0.0562 0.2827
Ca 00002" 0.0633 0.0892 <0.0001* <0.0001“ <0.0001*
Cd 0.0111* 0.0829 0.0871 0.2387* 0.2555 0.2615
Co NC NC NC <0.0001 * 0.0002“ <0.0001“
Cr NC NC NC 0.1097 0.2241 0.5526
Cu <0.0001* 0.4767 0.8086 <0.0001* 0.3969 0.8472
Fe <0.0001* 0.2269 0.0739 <0.0001* 0.2280 0.0736
Hg NC NC NC 0.7418 0.4475 0.6541
K <0.0001* 0.2385 0.0015 <0.0001* 0.5263 0.0085*
Mg <0.0001“ <0.0001 <0.0001 <0.0001* <0.0001“ <0.0001*
Mn 0.1891 0.2754 0.6730 0.0169* 0.1823 0.5462
Ni 0.6494 0.5655 0.7056 0.6852 0.6487 0.6866
Pb <0.0001"‘ 0.1003 0.4748 <0.0001* 0.0033* 0.0217*
Sc NC NC NC <0.0001* 0.2130 0.6760
Sr <0.0001* <0.0001* <0.0001* <0.0001* <0.0001"' <0.0001*
Ti 0.0004* 0.5259 0.5389 <0.0001“ 0.0355“ 0.6720
Zn 0.0004* 0.6787 0.3131 0.0188* 0.6439 0.5823
n = 38

1Life stages are Adult, Juvenile, Tadpole, and Sediment.
2Locations are REFl, REF3, KLMZR, AGRl, AGR2, AGR3.
NC = Not Calculated.

92

APPENDIX C:

STANDARD OPERATING PROCEDURE FOR ORGANOCHLORINE EXTRACTION

AND ANALYSIS

93

Michigan State University
Aquatic Toxicology Laboratory
National Food Safety and Toxicology Center

STANDARD OPERATING PROCEDURE

ANALYSIS OF DDT AND ITS METABOLITES IN BIOLOGICAL AND
ENVIRONMENTAL MATRICES

K. Kannan and J .P. Giesy

1.0 SCOPE AND APPLICATION

This SOP describes the method to determine the concentrations of DDT and it
metabolites in extracts from biota and sediments/soils using fused-silica capillary
columns with electron capture detectors (ECD). Following are the list of target
compounds that can be analyzed by the method described herein. 2,4’-DDD and 2,4’-
DDE can be analyzed by this method, but they fractionate in both the fractions of the
silica gel column, and coelute with some PCN congeners. Since these two compounds
account for only a minor portion of the total DDT concentrations, they are not quantiﬁed
by this method. However, 2,4’-DDT is quantiﬁed because it is a constituent of the
technical DDT preparation accounting for 20% of the total DDTs. Measurement of 2,4’-
DDT can shed light on the chronology of exposure to DDTs. This method is a
modiﬁcation of the US. EPA method 8081A.

 

 

 

Compound CAS Registry No.
4,4'-DDD 72-54-8
4,4'-DDE 72-55'9
4,4'-DDT 50-29-3
2,4’-DDT 789-02-6

 

1.2 This Method can also be used for the determination of PCBs. Therefore, this
method is suitable for those samples that need both PCBs and DDT analysis done in the
same extraction procedure.

1.3 The analyst must select columns, detectors and calibration procedures most
appropriate for the speciﬁc analytes of interest in a study. Matrix-speciﬁc performance
data must be established and the stability of the analytical system and instrument
calibration must be established for each analytical matrix.

1.4 This method is restricted to use by, or under the supervision of, analysts
experienced in the use of gas chromatographs (GC) and skilled in the interpretation of gas

94

chromatograms. Each analyst must demonstrate the ability to generate acceptable results
with this method.

2.0 SUMMARY OF METHOD

2.1 A measured amount of sample (20 g wet tissue for biota or dry wt for soil or
sediments) is extracted using the appropriate matrix-speciﬁc sample extraction technique.

2.2 Solid samples are homogenized with anhydrous sodium sulfate and extracted
with methylene chloride-hexane (3:1) using a Soxhlet extractor (Method 3540 of the
USEPA).

2.3 A variety of cleanup steps may be applied to the extract, depending on the
nature of the matrix interferences and the target analytes. Suggested cleanups include
F lorisil (Method 3620 of the USEPA), silica gel (Method 3630 of the USEPA) and sulfur
(Method 3660 of the USEPA).

2.4 After cleanup, the extract is analyzed by injecting a l-L s ample into a gas
chromatograph with a fused silica capillary column and electron capture detector
(GC/ECD) or a mass selective detector (GC/MSD).

3.0 INTERFERENCES

3.1 Sources of interference in this method can be grouped into three broad
categories.

3.1.1 Contaminated solvents, reagents, or sample processing hardware.

3.1.2 Contaminated GC carrier gas, parts, column surfaces, or detector
surfaces.

3.1.3 Compounds extracted from the sample matrix to which the detector
will respond.

3.1.4 Interferences co-extracted from the samples will vary considerably
from waste to waste. While general cleanup techniques are referenced or provided
as part of this method, unique samples may require additional cleanup approaches
to achieve desired degrees of discrimination and quantitation.

3.2 Interferences by phthalate esters introduced during sample preparation can
pose a major problem in pesticide determinations.

3.2.1 These materials may be removed prior to analysis using Silica Gel
Cleanup.

95

3.2.2 Common ﬂexible plastics contain varying amounts of phthalate esters
which are easily extracted or leached from such materials during laboratory
operations.

3.2.3 Cross-contamination of clean glassware routinely occurs when plastics
are handled during extraction steps, especially when solvent-wetted surfaces are
handled.

3.2.4 Interferences from phthalate esters can best be minimized by avoiding
contact with any plastic materials and checking all solvents and reagents for
phthalate contamination. Exhaustive cleanup of solvents, reagents and glassware
may be required to eliminate background phthalate ester contamination.

3.3 Glassware must be scrupulously cleaned. Clean all glassware as soon as
possible after use by rinsing with the last solvent used. This should be followed by
detergent washing with hot water, and rinses with tap water and organic-free reagent
water. Drain the glassware and dry it in an oven at 130 C for several hours, or rinse
with acetone and hexane prior to use. Store dry glassware in a clean environment.

3.4 The presence of elemental sulfur will result in broad peaks that interfere with
the detection of early-eluting organochlorine pesticides. Sulfur contamination should be
expected with sediment samples.

3.5 Waxes, lipids, and other high molecular weight materials can be removed by
acidic silica gel column chromatography or sulfuric acid treatment.

3.6 Other halogenated pesticides or industrial chemicals may interfere with the
analysis of DDTs. Co-eluting chlorophenols may be eliminated by using silica gel
column chromatography. Polychlorinated biphenyls (PCBs) also may interfere with the
analysis of the organochlorine pesticides. Silica gel column fraction techniques will be
used to separate DDTs from the PCBs. However, p,p’-DDE would appear in PCB
fraction and therefore adequate caution should be exercised in interpreting p,p’-DDE
concentrations.

4.0 APPARATUS AND MATERIALS

4.1 Gas chromatograph: an analytical system complete with gas chromatograph
suitable for on-column and split-splitless injection and all required accessories including
syringes, analytical columns, gases, electron capture detectors (ECD), and
recorder/integrator or data system.

4.2 GC columns

Any one of the following columns will be used. When necessary, conﬁmation of the
target analytes will be made by injecting samples in a column of different polarity.

96

4.2.1 A DB-S MS (5%-phenyl)-methylpolysiloxane) 30 m x 0.25 mm id
fused silica capillary column chemically bonded with at 0.25 urn ﬁlm
thickness (nonpolar column).

4.2.2 A DB-1701 (30 m x 0.25 mm ID) fused silica capillary column
chemically bonded with 14%cyanoporpyl phenyl methylpolysiloxane at 0.25
pm ﬁlm thickness (mid polar).

4.2.3 A DB-l (30 m x 0.25 mm ID) fused silica capillary column chemically
bonded with 100% dimethylpolysiloxane at 0.25 pm ﬁlm thickness (polar).

5.0 REAGENTS

5.1 Reagent grade or pesticide grade chemicals shall be used in all tests. Unless
otherwise indicated, it is intended that all reagents shall conform to speciﬁcations of the
Committee on Analytical Reagents of the American Chemical Society, where such
speciﬁcations are available. Other grades may be used, provided it is ﬁrst ascertained
that the reagent is of sufﬁciently high purity to permit its use without lessening the
accuracy of the determination.

NQIE: Store the standard solutions (stock, composite, calibration, internal,
and surrogate) at 4 C in polytetraﬁuoroethylene (PTFE)-sealed
containers in the dark. When a lot of standards is prepared, it is
recommended that aliquots of that lot be stored in individual small
vials. All stock standard solutions must be replaced after one year or
sooner if routine QC tests indicate a problem.

5.2 Solvents used in the extraction and cleanup procedures include n-hexane,
dichloromethane, acetone, and must be exchanged to hexane or isooctane prior to
analysis. Acetone or toluene may be required for the preparation of some standard
solutions. All solvents should be pesticide quality or equivalent, and each lot of solvent
should be determined to be phthalate free.

5.3 Organic-free reagent water - All references to water in this method refer to
organic-free reagent water.

5.4 Stock standard solutions (1000 mg/L) - May be prepared from pure standard
materials or can be purchased as certiﬁed solutions.

5.4.1 Prepare stock standard solutions by accurately weighing about 0.0100
g of pure compound. Dissolve the compound in isooctane or hexane and dilute to
volume in a 10-mL volumetric ﬂask. If compound purity is 96 percent or greater,
the weight can be used without correction to calculate the concentration of the stock
standard solution. Commercially prepared stock standard solutions can be used at

97

any concentration if they are certiﬁed by the manufacturer or by an independent
source.

5.5 Calibration standards should be prepared at a minimum of ﬁve different

concentrations by dilution of the composite stock standard with isooctane or hexane. The
concentrations should correspond to the expected range of concentrations found in real
samples and should bracket the linear range of the detector.

6.0

5.6 Surrogate standards

The performance of the method should be monitored using surrogate
compounds. Surrogate standards are added to all samples, method blanks, matrix
spikes, and calibration standards. The following compounds are recommended as
possible surrogates.

5.6.1 Tetrachloro-m-xylene has been found to be a useful surrogate for both
the single-column and dual-column conﬁgurations.

PROCEDURE
6.1 Sample extraction

Solid samples (20 g) are homogenized with anhydrous sodium sulfate and
extracted with methylene chloride-hexane (3: 1) using a Soxhlet apparatus. Sodium
sulfate should be cleaned with methylene chloride or baked at 450°C overnight
prior to use. The amount sodium sulfate added to samples varies with the moisture
content of the samples. Generally, for tissues such as ﬁsh muscle or liver, sample
to sodium sulfate ratio is 1:6. After weighing 20 g of homogenized sample, sodium
sulfate is weighed and added to sample in a pestle and mortar or in a homogenizer
cup. Samples are homogenized into ﬁne powder and then transferred into a Soxhlet
apparatus. All the glassware and homogenizer parts that come in contact with
samples will be rinsed with alconox detergent, nonapure water, acetone and hexane
prior to use. Homogenizer parts will be rinsed with detegent, nonapure water,
acetone and hexane between each sample. Surrogate standard (TCMX) will be
spiked at the amount of 50 ng ﬁnal mass. For matrix spike and matrix spike
duplicates, appropriate standards will be spiked at 50 ng ﬁnal mass of individual
DDT compounds. Samples will be extracted for 16 h and the Soxhlet cycle should
be set at 5 cycles/hour (Rheostat set at 5). Condenser should be cleaned with
acetone and hexane and cool water should be turned on prior to use. Extreme care
should be taken to avoid contamination of samples while handling the sample-
weighing and homogenizing. Watch for the ﬁrst two cycles to complete after
turning the Soxhlet on.

6.2. Concentration and Lipid content estimation

98

After 16 h, turn the Soxhlet off. Let the condenser water ﬂow until the
system gets cooled down. Rotary evaporate the solvent at 40°C to approximately 5 to 10
mL. Transfer the extract into a clean graduated test tube. Rinse the round-bottom ﬂask
three times with hexane and transfer the contents into the test tube. Make up the volume
of the solvent to 11 mL. Transfer 1 mL into a pre-weighed aluminum weighing disk
(W1). Dry the solvent and place it in n oven at 80°C for 1 h. Cool it down in a
dessicator for 30 min and take the ﬁnal weight (W2). Don’t touch the aluminum
weighing dishes by hand. Oil from the ﬁngers can affect the results of fat content. Use

clean forceps to handle the weighing dish.

Lipid content (%) = W2-W1 * 10/sample wt (g) * 100

Lipid content is measured only for biological samples and for soil samples it is not
performed. However, for soil/sediments, the volume is made to 10 mL instead of
11 mL and sulfur is removed with activated copper treatment. Activated copper is
prepared by treating/immersing copper granules with concentrated hydrochloric
acid for 30 min with occasional stirring. Acid is decanted safely and copper is
rinsed with nonapure water followed by acetone and hexane rinsing. Aﬁer
activation, copper granules should be shining. Lack of metallic luster means that
the activation is not proper. Transfer active copper granules into the test tube in
which soil/sediment sample extracts are in. Add copper until it stops to turn black.
The amount of copper varied from samples to samples depending on the sulfur
content.

6.3 Extract cleanup

The speciﬁc cleanup procedure used will depend on the nature of the sample to be
analyzed and the data quality objectives for the measurements. For samples of
biological origin multilayer silica gel column chromatography is performed. Silica
gel is packed in a glass column (10 mm id) mounted vertically in the following
order: 2 g silica gel (100-200 mesh, Aldrich), 2 g 40% acidic-silica and 2 g silica.
A thin layer of sodium sulfate is added at the top. Prior to the transfer of samples,
silica gel should cleaned by passing 100 mL of hexane through the column.
Discard the eluate. Then transfer 10 mL of the sample extract and elute the ﬁrst
fraction with 150 mL of hexane. This ﬁaction contains PCBs and p,p’-DDE and a

99

portion of p,p’-DDT. Separation tests need to conducted with every lot of silica gel
to ﬁnd out the volume of hexane needed to elute all the PCBs in fraction 1. Batch to
batch variation in the composition of the silica gel or Florisil or overloading the
column may cause a change in the elution patterns of the DDTs and PCBs. When
compounds are found in two fractions, add the concentrations found in the
fractions, and correct for any additional dilution. This volume can vary from 120 to
160 mL. The second fraction is eluted with 20% dichloromethane and hexane (100
mL) contains p,p’-DDT, p,p’-DDD. Both the ﬁ'actions are collected separately in a
ﬂat bottom ﬂask and rotary evaporated to approximately 1 mL. The extract is
transferred to a test tube and volume is made up to 1 mL accurately. Additional
clean up with sulfuric acid may be needed for lipid rich samples. The 1 mL of the
ﬁnal extract is transferred into a GC vial for instrumental analysis.

6.4 Instrumental analysis

This method allows the analyst to choose between a single-column or a dual-
column conﬁguration in the injector port.

6.4.1 Single-column analysis

A narrow-bore (0.25 mm ID) DB-S or DB-1701 column is used to separate DDT
compounds with greater chromatographic resolution. Use of narrow-bore columns
is suitable for relatively clean samples or for extracts that have been prepared with
one or more of the clean-up options referenced in the method.

Operating conditions vary depending on the sample. Generally, column oven
temperature is programmed from 80°C (1 min hold) to 120°C at a rate of 10°C/min
and then to 260°C at a rate of 2°C/min, with a ﬁnal holding time of 10 min.
Injector and detector temperatures are kept at 250 and 350°C, respectively. Helium
is the carrier gas and nitrogen is the make-up gas. Once established, the same
operating conditions must be used for both calibrations and sample analyses
throughout the project. Always run an acceptable blank prior to running any
standards or samples. Calibration standards will be injected periodically to check
for the instrument calibration status.

6.4.2. Calibration

The procedure for external calibration is used. In most cases, external standard
calibration is preferred because of the sensitivity of the electron capture detector
and the probability of the internal standard being affected by interferences. For
calibration veriﬁcation (each 12-hour shift) all target analytes required in the project
plan must be injected.

100

NQIE:Because of the sensitivity of the electron capture detector, the injection
port and column should always be cleaned prior to performing the
initial calibration.

6.4.3 A l to 2 uL injection volume of each calibration standard is
recommended. Other injection volumes may be employed, provided that the
analyst can demonstrate adequate sensitivity for the compounds of interest.

6.4.4 Calibration factors

When external standard calibration is employed, calculate the calibration
factor for each analyte at each concentration, the mean calibration factor, and the
relative standard deviation (RSD) of the calibration factors, using the formulae
below.

 

6.4.4.1
Calculate CF = Peak Area (or Height) of the Compound in the Standard
the calibration Mass of the Compound Injected (in nanograms)

factor for each
analyte at each concentration as:

6.4.5.Calculate the mean calibration factor for each analyte as:

_ Zen
mean CF = CF = —‘=—'——
n

where n is the number of standards analyzed.

6.4.5.1 Calculate the standard deviation (SD) and the RSD of the
calibration factors for each analyte as:

 

" — 2
gm -CF) SD

SD= RSD==x100
CF

 

n-l

If the RSD for each analyte is 20%, then the response of the instrument is
considered linear and the mean calibration factor can be used to quantitate
sample results. If the RSD is greater than 20%, then linearity through the

101

origin cannot be assumed. The analyst must use a calibration curve or a non-
linear calibration model (e. g., a polynomial equation) for quantitation.

6.4.6 Retention time windows

Absolute retention times are used for compound identiﬁcation.
Retention time windows are crucial to the identiﬁcation of target compounds.
A drift in retention time could be possible depending on the cleanliness of the
sample. The experience of the analyst should weigh heavily in the
interpretation of the chromatograms.

7.0 GAS CHROMATOGRAPHIC ANALYSIS OF SAMPLE EXTRACTS

7.1.1 The same GC operating conditions used for the initial calibration must
be employed for samples analyses.

7.1.2 Verify calibration each 12-hour shift by injecting calibration
veriﬁcation standards prior to conducting any sample analyses. Analysts should
alternate the use of high and low concentration mixtures of single-component
analytes and multi-component analytes for calibration veriﬁcation. A calibration
standard must also be injected at intervals of not less than once every twenty
samples (aﬁer every 10 samples is recommended to minimize the number of
samples requiring re-injection when QC limits are exceeded) and at the end of the
analysis sequence.

7.1.2.1 If the calibration does not meet the i15% limit (either on the
basis of each compound or the average across all compounds), check the
instrument operating conditions, and if necessary, restore them to the original
settings, and inject another aliquot of the calibration veriﬁcation standard. If
the response for the analyte is still not within :L-15%, then a new initial
calibration must be prepared.

7.1.3 Compare the retention time of each analyte in the calibration standard
with the absolute retention time. Each analyte in each standard must fall within its
respective retention time window. If not, the gas chromatographic system must
either be adjusted so that a second analysis of the standard does result in all analytes
falling within their retention time windows, or a new initial calibration must be
performed and new retention time windows established.

7.1.4 Inject a 1 or 2-uL aliquot of the concentrated sample extract. Record
the volume injected to the nearest 0.05 uL and the resulting peak size in area units.

7.1.5 Tentative identiﬁcation of an analyte occurs when a peak from a
sample extract falls within the absolute retention time window. Each tentative
identiﬁcation may be conﬁrmed using either a second GC column of dissimilar
stationary phase or using another technique such as GC/MS.

102

When results are conﬁrmed using a second GC column of dissimilar
stationary phase, the analyst should check the agreement between the quantitative
results on both columns once the identiﬁcation has been conﬁrmed. Unless
otherwise speciﬁed in an approved project plan, the higher result should be
reported, as this is a conservative approach relative to protection of the
environment. If the relative percent difference of the results exceeds 40%, steps
may be taken to address the discrepancy.

7.1.6 When using the external calibration procedure, the quantity of each
component peak in the sample chromatogram, which corresponds to the compounds
used for calibration purposes, is determined as follows. Proper quantitation requires
the appropriate selection of a baseline from which the peak area or height can be
determined.

(Ax)(V.)(D)

C tr ti = —
oncen a on (ﬁg/kg) (CF)(V,)(Ws)

 

where,
Ax = Area (or height) of the peak for the analyte in the sample.

V, = Total volume of the concentrated extract (L).
D = Dilution factor, if the sample or extract was diluted prior to analysis.

If no dilution was made, D = 1. The dilution factor is always
dimensionless.

CF= Mean calibration factor from the initial calibration (area/ng).

V, = Volume of the extract injected (L). The injection volume for
samples and calibration standards must be the same. For
purge-and-trap analysis, V, is not applicable and therefore is set at 1.

Ws = Weight of sample extracted (g). The wet weight or dry weight may be
used, depending upon the speciﬁc application of the data. If units of
kilograms are used for this term, multiply the results by 1000.

Using the units speciﬁed here for these terms will result in a concentration in
units of ng/g, which is equivalent to ug/kg.

7.1.6.1 If the responses exceed the calibration range of the system,
dilute the extract and reanalyze.

103

7.1.6.2 Generally for DDT compounds, there are no coeluting peaks.
If partially overlapping or coeluting peaks are found, change GC columns or
try GC/MS quantitation.

7.1.6.3 Each sample analysis must be bracketed with an acceptable initial
calibration, calibration veriﬁcation standard(s) (each 12-hour analytical shift), or
calibration standards interspersed within the samples.

When a calibration veriﬁcation standard fails to meet the QC criteria, all
samples that will be reinjected after the last standard that last met the QC criteria
must be evaluated to prevent mis-quantitations and possible false negative results,
and re-injection of the sample extracts may be required. More frequent analyses of
standards will minimize the number of sample extracts that would have to be
reinjected if the QC limits are violated for the standard analysis.

However, if the standard analyzed after a group of samples exhibits a response
for an analyte that is above the acceptance limit, i.e., >15%, and the analyte was not
detected in the speciﬁc samples analyzed during the analytical shift, then the
extracts for those samples do not need to be reanalyzed, as the veriﬁcation standard
has demonstrated that the analyte would have been detected were it present. In
contrast, if an analyte above the QC limits was detected in a sample extract, then re-
injection is necessary to ensure accurate quantitation. If an analyte was not detected
in the sample and the standard response is more than 15% below the initial
calibration response, then re-injection is necessary to ensure that the detector
response has not deteriorated to the point that the analyte would not have been
detected even though it was present (i.e., a false negative result).

7.1.7 If the peak response is less than 2.5 times the baseline noise level, the
validity of the quantitative result may be questionable. The analyst should consult
with the source of the sample to determine whether ﬁrrther concentration of the
sample is warranted.

7.2 Validation of GC system qualitative performance

7.2.1 Use the calibration standards analyzed during the sequence to
evaluate retention time stability.

7.2.2 Each subsequent injection of a standard during the 12-hour
analytical shift (i.e., those standards injected every 20 samples, or more
frequently) must be checked against the retention time windows. If any of
these subsequent standards fall outside their absolute retention time windows,
the GC system is out of control. Determine the cause of the problem and
correct it. If the problem cannot be corrected, a new initial calibration must be
performed.

104

7.3 GC/MS conﬁrmation may be used in conjunction with either single-column or
dual-column analysis if the concentration is sufﬁcient for detection by GC/MS.

7.4 Suggested chromatographic system maintenance - When system performance
does not meet the established QC requirements, corrective action is required, and may
include one or more of the following.

GC injector ports can be of critical concern, especially in the analysis of DDT.
Injectors that are contaminated, chemically active, or too hot can cause the
degradation ("breakdown") of the analytes. DDT breakdown to DDD, or DDE.
When such breakdown is observed, clean and deactivate the injector port, break off
at least 30 cm of the column and remount it. Check the injector temperature and
lower it to 205°C, if required.

8.0 QUALITY CONTROL and METHOD PERFORMANCE

The detection limits for individual DDT compounds through the analytical procedure is 1
ng/g, wet wt for biota and 1 ng/g, dry wt, for sediments. Recoveries of DDT compounds
spiked on to sediments and ﬁsh tissues and passed through the whole analytical
procedure ranged from 90-103%. A standard reference material will be run along with
every 20 samples and the observed values will be compared with those of certiﬁed
values. In addition, a method blank, a matrix spike, a duplicate will be done. The
laboratory must evaluate surrogate recovery data from individual samples versus the
surrogate control limits developed by the laboratory. This method has been applied in a
variety of commercial laboratories for environmental and waste matrices.

105

9.0 REFERENCES

l. USEPA. Method 8081A. Analysis of organochlorine pesticides by gas
chromatography. US. Environmental protection Agency, Wahington, DC.

106

APPENDIX D:

STANDARD OPERATING PROCEDURES FOR IN— VITRO CELL BIOASSAYS

107

Michigan State University
National Food Safety and Toxicology Center
Aquatic Toxicology Laboratory

W
H4IIE-luc Bioassay For The Detection Of Ah Receptor Agonists
Version 1 September 14, 1998

Alan Blankenship, Dan Villeneuve, J. Thomas Sanderson,
Sarah Cholger, Katherine Kemler, and John P. Giesy

Supported through:

National Food Safety and Toxicology Center,
Institute for Environmental Toxicology,
and the Department of Zoology

Correspondence to:

Aquatic Toxicology Laboratory
224 National Food Safety and Toxicology Center
Michigan State University
East Lansing, MI 48824-1222 USA
T: (517) 432-6333
F: (517) 432-2310

108

DEFINITIONS AND ACRONYMS

AhR Aryl hydrocarbon receptor

ATL Aquatic Toxicology Laboratory (Michigan Sate University)
DCCF BS Fetal bovine serum that has been charcoal-stripped

EC50 conentration of test agent that causes 50% of maximal response
FBS Fetal bovine serum

H4IIE-luc rat hepatoma cells stably transfected with an AhR-controlled
luciferase reporter gene

construct
PBS Phosphate-buffered saline
RLU Relative luminescent units
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

109

TABLE OF CONTENTS

1.0. PURPOSE ................................................................................................................ 5
2.0. SCOPE AND APPLICATION ............................................................................... 5
3.0. SAFETY CONSIDERATIONS ............................................................................. 7
4.0. EQUIPMENT, MATERIALS, AND REAGENTS .............................................. 7
4.1. Maintenance, preparation and use of H4IIE-luc cells ................................... 7
4.2. Instruments ...................................................................................................... 8
4.3. Supplies and Biochemicals ............................................................................. 9
4.4. Media Preparation ........................................................................................... 10
5.0. METHOD, PROCEDURES, AND REQUIREMENTS ....................................... 11
5.1. Sample Preparation ......................................................................................... 11
5.2. Standards Preparation ..................................................................................... 11
5.3. Plating Cells (Day 1) ....................................................................................... 12
5.4. Dosing Cells (Day 2) ....................................................................................... 13
5.5. Conducting the Bioassay (Day 5) ................................................................... 14
5.6. Protein Detennination ..................................................................................... 16
5.7. Data Analysis .................................................................................................. 17
6.0. RECORDS, DOCUMENTATION, AND QC REQUIREMENTS ................... 17
6.1 Records and Documentation ......................................................................... 17
6.2 QC Requirements and Data Quality Objectives ........................................... 18
7.0 RESPONSIBILITIES ........................................................................................... 18
8.0 REFERENCES ..................................................................................................... 18
ATTACHMENTS

Attachment 201-1: Figure l - TCDD Dose-response Curve with H4IIE-luc Cells
Attachment 201-2: Figure 3 - Viability Assay Sample Data Set (Raw Data)
Attachment 201-3: Figure 4 - Viability Assay Sample Data Set (Calculated Data)
Attachment 201-4: Figure 5 - Luciferase Assay Sample Data Set (Raw Data)
Attachment 201-5: Figure 6 - Luciferase Assay Sample Data Set (Calculated Data)

110

1.0.

2.0

PURPOSE

The H4IIE-luciferase induction assay is an in vitro technique for the identiﬁcation of
aryl hydrocarbon (Ah) receptor-active compounds. The technique uses rat
hepatoma cells (H4IIE-luc) stably transfected with an AhR-controlled
luciferase reporter gene construct were developed at Michigan State
University by Dr. Jac Aarts (Univ. of Wageningen, The Netherlands;
Sanderson et al., 1996). The assay is also referred to as the chemical
activated luciferase gene expression (CALUX) system (Murk et al., 1996).
These cells express ﬁreﬂy luciferase in response to Ah receptor agonists. Luciferase
activity is measured conveniently and with high sensitivity as light emission using a
plate-scanning luminometer. Luciferase induction potential is assessed by comparison
of the response to that of 2,3,7,8-tetrachlorodibenzo-p—dioxin (T CDD), the most potent
agonist for the mammalian Ah receptor.

SCOPE AND APPLICATION

Ah receptor agonists include polyhalogenated aromatic hydrocarbons (PHAHs),
such as polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and
dibenzofurans (PCDFs) which are persistent environmental contaminants found in
all parts of the world. A number of these compounds cause a variety of adverse
effects in laboratory studies on rats and mice (reviewed by Poland and Knutson
1982). These include hepatotoxicity, certain types of cancer, thymic atrophy and
other immunotoxicities, a wasting syndrome, reproductive toxicities, terata and the
induction of enzymes and porphyrins. A number of these toxicities have also been
observed in wildlife in areas with elevated levels of PHAHs, particularly in ﬁsh-
eating birds in the Great Lakes (reviewed by Gilbertson et a1. 1991; Giesy et al.
1994a; 1994b).

Interest exists in assessing the risk posed by these PHAHs to ﬁsh and wildlife,
which may also reﬂect the risk to humans. One aspect of risk assessment is the use
of bioanalytical assays to detect and determine the toxicity of complex mixtures of
these chemicals in extracts of environmental compartments such as soil, water and
biota. Quantitative instrumental analysis of complex mixtures of these compounds
is a difﬁcult and expensive task. Furthermore, demonstrating the presence of one or
many of these compounds in samples provides only limited information on their
biological potency, particularly when present in a complex mixture with many
potential interactions.

In order to develop a suitable bioassay, an understanding of the mechanism of
action for the compounds is required. In the case of a number of PHAHs,
considerable knowledge of the mechanism by which they cause their toxicities has
been acquired. As previously mentioned, PHAHs are persistent agonists for the Ah
receptor (Poland and Knutson 1982). Binding of agonist to receptor results in an
activated receptor-ligand complex that translocates to the nucleus. Here it interacts
with speciﬁc sequences on the DNA, termed dioxin—responsive elements (DREs;

lll

also called XREs or AhREs), in order to alter gene transcription (reviewed by
Whitlock 1990; Okey et al. 1994). A rapid and sensitive response that is under
direct regulation of the Ah receptor is the induction of cytochrome P-450 1A
isoenzymes (CYP1A1 and 1A2) and their associated ethoxyresoruﬁn O-deethylase
(EROD) activity (N ebert and Gonzalez 1987). Good correlations exist between the
Ah receptor-binding afﬁnity of persistent PHAHs and their EROD-inducing
potency in vitro, and, dependent on the endpoint, their toxic potential in vivo (Safe
1986; 1990). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), in particular, binds
tightly to the Ah receptor, and is a potent inducer of EROD activity and highly
toxic. For the above reasons, the capacity of single compounds or complex
environmental mixtures to induce EROD activity is considered to be a reasonable
measure of their toxic potential. This mechanistic knowledge has been applied in
vivo in biological monitoring of ﬁsh and birds, and in vitro in the use of bioassays
for the screening of environmental extracts for Ah-active components. The H4IIE
rat hepatoma cell bioassay (Tillitt et al. 1991), is widely used for this purpose. In
this assay, EROD-inducing potencies (EDso values) of single compounds and
environmental samples are determined from complete dose-response curves and
compared to that of TCDD in order to express the biological potency of the tested
samples in TCDD-equivalents (TCDD-EQs). The bioassay integrates potential
non-additive interactions among Ah receptor agonist and between Ah receptor
agonists and other compounds by measuring a ﬁnal receptor-mediated response
(Giesy et al. 1994a).

For the same purpose as the currently used wild-type H4IIE bioassay
(H4IIE-wt), a recombinant H4IIE cell line (H4IIE-luc) has been developed that
exhibits Ah receptor-mediated luciferase expression (Aarts et al. 1995). This cell
line has been stably transfected with a luciferase reporter gene under
transcriptional control of several DREs from mouse (Aarts et al. 1993; Denison et
al. 1993). These DRE sequences are highly conserved among species, unlike the
Ah receptor which can exhibit greatly different ligand-binding properties among
species. A preliminary report using luciferase-transfected mouse Hepa1c1c7
hepatoma cells indicated that these cells are more sensitive to Ah receptor
agonists than the wild-type cells (Aarts et al. 1993; Sanderson et al., 1996). It
has been suggested that luciferase-transfected cell lines would have more
favorable properties than their respective wild-types, such as greater selectivity,
sensitivity and dynamic range. This has been postulated because the Ah receptor-
mediated expression of luciferase, being foreign to the cell, is probably not
affected by post-transcriptional and -translational events which inﬂuence
CYP1A1 expression. Furthermore, the recombinant cells would not be dependent
on a ﬁrnctional CYP1A1 gene or protein for responsiveness, although the Ab
receptor-mediated pathway would still need to be present. Another theoretical
consideration is that luciferase is assayed on the basis of light production for
which extremely sensitive detectors exist; also, the turnover number or molecular
activity of luciferase is so high that it allows the detection of very few molecules
of the enzyme, relative to CYP1A1. Further, recombinant cells are readily

112

3.0.

4.0.

amenable to further improvements in responsiveness by genetic engineering of the
reporter gene construct.

The threshold dose (i.e., detection limit) and EDso (i.e., effective dose to elicit
50% of the maximal response) for luciferase induction in H4IIE-1uc cells were
approximately 0.1 and 1.2 pg/well, respectively, as determined from 41 separate
standard TCDD curves analyzed in 1997 (Figure 1). Coefﬁcients of variation
(standard deviation/mean x 100) for the assay were under 10% at all
concentrations tested. For a sample size of 20 g tissue and a ﬁnal extract volume
of 0.25 ml, the H4IIE-luc assay will detect 1 part per trillion (ppt; pg/g, wet
weight) TCDD-equivalents. With a sample size of 5 g tissue, 4 pg/g (wet
weight) TCDD-equivalents will be detected.

SAFETY CONSIDERATIONS

TCDD and many related compounds have been found to be carcinogenic. In addition,
the ethidium homodirner used in the cell viability assay is a powerful mutagen. Care
should be taken to minimize exposure. According to institutional guidelines (refer to
the Safety Manual for the Aquatic Toxicology Laboratory at Michigan State
University), medium should be collected in a liquid trap for disposal as hazardous
waste.

EQUIPMENT, MATERIALS, AND REAGENTS

4.1. WWW

The assay uses a stably transfected cell line developed by Dr. Jac Aarts at
Michigan State University (Sanderson et al., 1996). Brieﬂy, rat hepatoma
cells [American Type Culture Collection (ATCC) catalog #CRL 1548)] were
stably-transfected with an inducible reporter plasmid, pGudLucl.1. This
plasmid contains the ﬁreﬂy luciferase gene under PHAH-inducible control of
four DREs. Exposure of these cells to Ah receptor-active chemicals results in
induction of luciferase activity in a time-, dose-, and AhR-dependent manner.

4.1.1. Maintain adherant cells in continuous culture in 100 mm tissue
culture plates (Coming #25020-100, Cambridge, MA; 1-800-492-
1110), 75 cm2 ﬂasks (Corning #25113-75) or any appropriate vessel
at a maximum density of 80-90% conﬂuence in 10% Full Medium
(See Media Preparation). Incubator conditions are 37 C, 5% C02
humidiﬁed atmosphere.

4.1.2. Subculture cells 1:6 every week (depending upon density)
maintaining a minimum cell density of 15-25%.

113

4.2. Insmrments

4.2. 1. Dynatech ML3000 Luminometer
(Dynatech Technical Support: (800) 336-4543; Chantilly, VA)

4.2.2. Cytoﬂuor 2300/2350 Fluorescence Measurement System
(Millipore Technical Support: (800) 645-5476; Bedford, MA)

NOTE: All users of the Dynatech ML3000 Luminometer and the Cytoﬂuor 2300 must read
and be familiar with the Operators Manual before using the instrument. A working
knowledge of Microsoft Windows is also necessary. For both the luminometer and
ﬂuorescence measurement system, instrument use is recorded in a log book (located next to
the instrument). For each use, the person, date, number of samples, type of samples, and
results of proﬁciency standards are recorded. In addition, any abnormal operation of the
instrument is recorded. Calibration is performed by analyzing a positive control or sample
with known activity each day that the assay is run. If the positive control exceeds i 20% of
the on-going average (as determined by a comparison to a proﬁciency curve maintained in
the instrument log book), the positive control will be rerun. If exceedance is conﬁrmed with
the second analysis, the manufacturer will be contacted as a corrective action so that the
instrument can be recalibrated.

4.2.3. Pipets

a.

Eppendorf Repeat Pipetter (Brinkmann Instruments #22 26 000-
6; Westbury, NY) with sterile, Biopur 12.5 ml combitips
(Brinkmann Instruments #22 49 520-8) for dispensing cells into
the 96-well plate and for changing media. Calibration checked
weekly by weight of water check and the results are entered in
calibration log book. Adjustments made by MSU Biochemistry
Instrument Shop when accuracy exceeds manufacturer’s
speciﬁcations of i 0.3%.

Rainin Pipetman Pipets (0.5 - 10 u] capacity, Rainin #P-10; 20 -
200 pl, Rainin #P-200 ; 100 - 1000 ul, Rainin #P-lOOO;
Wobum, MA) for making sample dilutions, dosing cells, etc.
Calibration checked weekly by weight of water check and the
results are entered in calibration log book. Adjustments made by
MSU Biochemistry Instrument Shop when accuracy exceeds
manufacturer’s speciﬁcations of i 2.5% for P-10, 1.0 % for P-
200, and 3.0% for P-1000.

Brinkrnann Eppendorf Multichannel Pipetter 30-300 pl
(Brinkrnann Instruments #22 45 120-1) for washing cells and
adding reagents for viability and Luclite reagents. Calibration
checked weekly by weight of water check and the results are
entered in calibration log book. Adjustments made by MSU

114

Biochemistry Instrument Shop when accuracy exceeds
manufacturer’s speciﬁcations of i 1.5%.

d. Drummond Pipet-Aid F iller/Dispenser (Drummond #4-000-111-
TC, available from Fisher #13-681-15D) for dispensing volumes
greater than 1 ml, such as for changing media on cells.

NOTE: For all pipettors, except the Drummond Pipet-A id, calibration test results are
recorded weekly, along with the name of the person and any abnormal operation of the

instrument.

4.3. Suppliesandﬂioshcmicals

4.3.1.

4.3.2.

4.3.3.

96-Well ViewPlatesTM (Packard Instruments #6005181; Meriden,
CT); white 96-well plates, sterile, tissue culture treated, with lids and
self-adhesive sticker for bottom of plates

Cost: $297/50 plates;
Ordering: (800) 856-0734
Technical support: (800) 323-1891

Cell viability assay reagents; sold either as a kit from Molecular
Probes (#L-3224; Eugene, OR) or as individual components:

Calcein AM (Molecular Probes #C-3100);
MW = 994.87; made up as 4000x (2 mM) stock (50 ug/ 12.56 ul
DMSO)

Ethidium homodimer I (Molecular Probes #E 1169)
MW = 857; made up as 2000x (1 mM) stock in DMSO

Ordering: (800) 438-2209
Technical support: (541) 465-8353

LucLiteTM Kit (Packard Instruments # 6016911 - 1000 assay kit;
Meriden, CT). Make fresh on same day as assay. Dissolve one
bottle of lyophilized reagent with 10ml buffer (supplied) for every
133 assays (individual wells) to be analyzed.

Cost: 3420/1333 assays using 75 til/assay (or 1000 assays if using
the manufacturer’s suggested volume of 100 ul/assay. Preliminary
studies showed equivalent responses at both 75 ul and 100 pl).
Ordering: (800) 856-0734

Technical support: (800) 323-1891

115

4.3.4.

4.3.5.

4.4.1.

4.4.2.

10x Trypsin-EDTA solution (Sigma #T-4174, St. Louis, M0) for
dissociation of cells from plates. From a 10x concentrated solution,
a 1x working solution is prepared using sterile PBS as the diluent.

Dulbecco’s phosphate-buffered saline (PBS) with Ca2+ and Mg2+

First, make up 10 L of Cay-free and Mg2+- free Dulbecco’s PBS:

2.0 g
2.0 g
80.0 g
11.5 g
10.0 L

KCl
KH2P04
NaCl
NazHPO4
H20a

“distilled/deionized or Nanopure biological grade

To each 1 L of PBS, add 0.1 g anhydrous CaClz and 0.1 g MgClz o
6H20 Keep at room temperature (good for at least 2 - 4 weeks).

4.4. MW

10% Full Medium

Dulbecco’s Modiﬁed Eagle’s Medium (Sigma #D-2902) mm
mm (known to be estrogenic and with unknown inﬂuence on
H4IIE cells), and sodium bicarbonate. Prepare as instructed by the
manufacturer, adjust the pH to ~7.3, and then add :

10% fetal bovine serum (Hyclone deﬁned FBS # SH30070.03;
Logan, UT; 1-800-492-5663)

10% DCCFBS Medium

Prepared as for the full medium, except the fetal bovine serum is
replaced with dextran/charcoal-stripped fetal bovine serum (available
from Hyclone #SH30068.03).

ll6

5.0 METHOD, PROCEDURES, AND REQUIREMENTS

5.1.

5.2.

Sample Preparation

Two types of samples may be assayed: pure compounds and mixtures derived
from environmental or tissue samples. For pure compounds, sample preparation
consists of dissolving the material in an appropriate solvent. The solvent of
choice is isooctane (because of its low toxicity and for direct comparison to
TCDD, which is dissolved in isooctane). However, if the material is insoluble in
isooctane, the following solvents can be tried:, ethanol, acetone, p-dioxane,
acetonitrile, and methanol. A stock solution should be prepared at 5 mM for
compounds of unknown activity and stored in amber glass vials at -20°C.
However, it may be necessary to test concentrations near the limit of solubility
of the compound. Ideally, a volume of 1-2 ml at the highest possible
concentration should be obtained for standards and samples. This volume is
sufﬁcient for splitting samples for instrumental analysis and for preparation of
serial dilutions.

Samples derived from environmental matrices such as tissue, water, or sediment
should be extracted and concentrated according to appropriate protocols (in
accordance with SOP #211 - Extraction and Analysis of PCBs and Non-ortho
Coplanar PCBs in Biological Matrices). The volume of sample should be
recorded before re-dissolution in the assay solvent so that a dilution or
concentration factor can be calculated. To prepare dilutions, 0.5 ml of isooctane
(assuming that this is the solvent of choice) is added to ﬁve appropriately labeled
2 ml amber GC glass vials with teﬂon lined caps. Then 0.5 ml of the 1x extract
(original extract that is undiluted) is added to the ﬁrst vial (labeled 0.5x), the lid
enclosure tightened, and then the sample is vortexed well. Then 0.5 ml of the
0.5x stock is added to the next vial (labeled 0.25x), the lid enclosure tightened,
and then the sample is vortexed well. These steps are repeated until all dilutions
are prepared. The sample dilutions will be 100, 50, 25, 12.5, 6, and 3% of the
extract. If less than 1 ml of original 1x extract is available, the above mentioned
volumes should be proportionately reduced. After dissolution in the assay
solvent, the samples should be stored at -20°C.

Standards Preparation

A large range of standards should be prepared to deliver a ﬁnal concentration of
TCDD between 0.03 - 100 pg/well in a volume of 1.25 pl (i.e., make 200x stock
solutions). Generally, 6 concentrations will achieve a full dose response curve
(ﬁnal = 0.1, 0.3, l, 3, 10, 3O pg/well) for TCDD. Ideally, standards should be
dissolved in the same solvent as the samples, but this is not always possible. In
this case, be sure to conduct assays with both solvent controls and compare them
to blanks (see Dosing Cells). A stock of TCDD in isooctane is maintained in
Room 181, URCF, in a 1 liter volumetric ﬂask. The concentration is written on
the ﬂask and is tested for purity and accuracy by GC/MS and GC/ECD by

117

comparison to commercially available certiﬁed standards. To make dilutions,
prepare the following stock concentrations (assuming a stock concentration of

 

 

10000 ng/ml):
ﬁnal mass. stock
in wells concentration
(pg/1.25 111) (ng/ml)
12500 10000
10000 8000
1000 800
100 80

The following six concentrations are
used for the bioassay:

30 24
10 8
3 2.4
1 0.8

0.3 0.24

0.1 0.08

 

NOTE: Adherence to these procedures will insure consistent response of cells. Timing and
cell density are critical. An assay may be completed in 5 days.

5.3. Plating Cells (Day 1)

Prior to conﬂuence, write down the passage number of the cells, ,aspirate media
from cell culture dishes, wash the cells with sterile PBS without Ca and Mg
and trypsinize the cells with 3 ml of 1x sterile trysin-EDTA solution for 5
minutes at 37°C. Add trysinized cell suspension to 27 ml of “10% Full
Medium” and determine the number of cells/ml with a hemacytometer (for more
information on cell counting, refer to “Cell Culture: A Manual of Basic
Techniques”, by Dr. Ian F reshney, 1996) . Dilute the cell solution to a
concentration of 60,000 cells/ml with media. Add 0.25 ml of cell suspension to
each well (15,000 cells) of a 96-well ViewPlateT" using an Eppendorf“ repeat
pipettor. Care must be taken that the cell suspension is turiforrn each time that
the pipettor is reﬁlled. This is done by gently inverting the tube or bottle of cell
suspension end-over-end several times immediately prior to reﬁlling the pipettor.
1f the outer 36 wells are not being used for the experiment (recommended), ﬁll
them with either sterile media or PBS to maintain humidity consistently across
the plate. Use of the outer wells is not recommended because of an edge effect
caused by inconsistent growth of cells in these wells.

NOTE: cell number per well is one of the largest contributors to variation in the data - so

take plenty of time to do this step properly. Cell passage number should be noted to monitor
long-term changes in the responsiveness and growth characteristics of the cells.

118

5.4. Dosing Cells (Day 2)

Dose cells 24 hours after plating and continue exposures for 3 days. First,
examine cells to ensure consistent plating. Aspirate media (attach p-lO tips to
the suction line to minimize cell scraping), and replace with 0.25 ml of
DCCF BS media that is prewarrned to 37°C.

NOTE: the use of cold media should be avoided because it may “shock" or stress the cells
and it may cause precipitation of test agents that have poor water solubility).

A typical plate design is shown in Figure 2. Use at least three replicates per
treatment. Note that with TCDD, only two replicates are used for a typical plate
design. Each control and TCDD concentration are averaged for all plates within
a given experiment. Use a negative control with no treatment (blank) and a
solvent control treated with pure solvent only. Use at least ﬁve concentrations of
each compound or extracts tested. Prepare 200x chemical stocks in the
appropriate solvent (see Sample Preparation). In general, it is best to dose cells
directly in the well; however, for water miscible solvents, the test agent can be
added to a sterile glass vial containing 2 ml of media, mixed, and then added to
each well. Each well shall receive 1.25 pl of sample or standard or solvent
control as noted in the plate design below.

Earlier versions of this assay determined that cross-talk can occur when high
activity samples are directly adjacent to low activity samples. Therefore, as a
corrective action, the plate design shown in Figure 2 was developed. Note that
two blank columns border samples, so that there is no cross-talking between
samples and TCDD and between samples and controls.

119

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Row/ 2 3 6 7 8 10 ll 12
Col
A
B 0.1 pg 0.1 pg C1 C1 C1 solvent
TCDD TCDD Conc. Conc. Conc. control
1 l 1
C 0.3 pg 0.3 pg C1 C1 C1 solvent
TCDD TCDD Conc. Conc. Conc. control
2 2 2
D 1 pg 1 pg C1 C1 C1 solvent
TCDD TCDD Conc. Conc. Conc. control
3 3 3
E 3 pg 3 pg C1 C1 C1 blank
TCDD TCDD Conc. Conc. Conc.
4 4 4
F lOpg lOpg C1 C1 C1 blank
TCDD TCDD Conc. Conc. Conc.
5 5 5
G 30 pg 30 pg C1 C1 C1 blank
TCDD TCDD Cone. Cone. Conc.
6 6 6
H

 

 

Figure 2. A typical plate design for determination of Ah receptor agonist activity in H4HE-
luc cells. Note that blanks, solvent controls, and a standard curve for TCDD can be
analyzed on the same plate with one test sample (labeled C1, at six different concentrations).
Two blank columns are recommended between samples and either TCDD or controls to
prevent any possible cross—contamination.

120

5.5. Conducting the Bioassay (Day 5)

5.5.1. Preparation steps (prior to assay)

Inspect plates visually with and without microscope - check
degree of conﬂuence, homogeneity from well-to-well, and any
signs of cytotoxicity or altered morphology

Set up cytoﬂuor for viability assay:

set 1: excitation = B emission = B sensitivity = 3
set 2: excitation = C emission = E sensitivity = 4

NOTE: sensitivity can be adjusted if values are too high (9999) or low (not different

from blanks)

Preparation of viability assay reagent (refer to Supplies and
Biochemicals Section):

Each plate will need 1.8 ml or 3 m1, depending on whether 36
or 60 wells are used per plate, respectively plus a little extra (2
m1). Dilute the appropriate amounts of calcein and ethidium
with the appropriate volume of media without FBS as shown
below:

 

Number of
plates

OO\IO\M-§UJNt—'

Volume of viability assay reagent needed
[total volume (ml); calcein stock (pl); Ethidium stock
(PDL

using 36 wells using 60 wells

3.8 ml; 0.95 p1; 1.9 pl 5 ml; 1.25 pl; 2.5 pl
5.6 ml; 1.4 pl; 2.8 pl 8 ml; 2 pl; 4 pl
7.4 ml; 1.85 p1; 3.7 pl 11 ml; 2.75 pl; 5.5 pl
9.2 ml; 2.3 pl; 4.6 pl 14 ml; 3.5 pl; 7 pl
11 ml; 2.75 pl; 5.5 pl 17 ml; 4.25 pl; 8.5 pl
12.8 ml; 3.2 pl; 6.4 pl 20 ml; 5 pl; 10 pl
14.6 ml; 3.65 pl; 7.3 pl 23 ml; 5.75 pl; 11.5 pl
16.4 ml; 4.] pl; 8.2 pl 26 ml; 6.5 pl; 13 pl

CAUTION: ethidium homodimer is a powerful mutagen - handle with care and throw
contaminated tips, etc., into biohazard bags

121

Set up luminometer:

Mode = Cycle; Pause = 2 sec;

Data = All; Mix = Off;

Gain = High; Temp. = 30°C;

Cycle = 1-3; AID reads = 20 (number of times
that each well is analyzed per cycle)

Set up vacuum aspirator (attach p-lO tips to each suction line to
minimize scraping surface area and only use the plate washer
to aspirate and not to dispense PBS)

Prepare LucLite substrate solution and luciferase positive
control (must be used on the same day as prepared)

1) Reconstitute lyophilized substrate solution by adding 10
ml of Assay buffer solution A (provided with kit) to one
vial of lyophilized substrate (each vial is enough for 133
assays if 75 pl is used). Agitate gently until a
homogeneous solution is formed (a slight turbidity is
acceptable). Equilibrate to room temperature before use.

NOTE: if more than one vial is reconstituted, combine and mix to prevent any variation
from the substrate between plates.

2) Reconstitute the lyophilized luciferase positive control
with 200 pl of distilled (or nanopure water). Each vial
contains sufﬁcient luciferase for 20 controls.

5.5.2. Cell Viability Assay Procedure (process one plate at a time)

remove plate from incubator and aspirate media, then rinse 1
time with PBS

Add 50 pl of PBS with Ca2+ and Mg2+ to all wells using a 8-
channel pipet

Add 50 pl of viability assay reagent to all wells using a 8-
channel pipet

Incubate at room temperature for 10 minutes and then scan
plate in the Cytoﬂuor instrument

122

e. Export/print data (check that values are appropriate, otherwise
adjust sensitivity and rescan). Password protect data ﬁles with
a project-speciﬁc password. Data analysis are discussed in
Section E along with an example raw data ﬁle and a Microsoft
Excel spreadsheet version of a sample data analysis.

1'. Aspirate viability reagent/PBS, and rinse 1 time with PBS
using vacuum
aspirator

g. Seal the bottom of the ViewPlates with self-adhesive TopSeal
(or tape very well). This increases the signal detection in the
luminometer.

h. Add 75 p1 of PBS with Ca2+ and Mg2+ (or media without serum
or phenol red) to all wells using an 8-channel pipet

i. Working under SUBDUED light conditions, add 75 pl/well of
reconstituted LucLite substrate solution, cover with foil, and
agitate gently.

j. Wait twenty minutes before counting the plate to allow full
signal generation.

k. Measure luminescence on the Dynatech ML3000 luminometer

I. Save, print, and copy data to disk. Password protect data ﬁles
with a project-speciﬁc password. Data analysis are discussed
in Section E along with an example raw data ﬁle and a
Microsoft Excel spreadsheet version of a sample data analysis.

5.6. mm

Protein determination is readily accomplished in the same plates with same cell
lysates after measuring luminescence. A ﬂuorescamine-based protein assay
(Sanderson et al., 1996) or Micro-BCA Assay (available from Pierce (800) 874-
3723) can be used. Degree of conﬂuency of wells was verified
microscopically, and in most cases this made normalization to protein
unnecessary; therefore, luciferase activity is reported as either relative
light units (RLU) or percent of solvent control.

123

5.7. DataAnalxsis
5.7.1. Miahilluﬂara

Average the three measurements for calcein AM and ethidium. Divide
the average calcein AM ﬂuorescence for each sample by its ethidium
homodimer ﬂuorescence to obtain a live to dead ratio. Graph the
average calcein AM ﬂuorescence and standard deviation for the
negative control, solvent control, and each concentration tested.
Examine the calcein AM data visually. If the blank has greater
viability than the other treatments, the solvent may be toxic to the cells.
If viability decreases with increasing concentration of the test
substance, the test substance may be toxic to the cells. In either of
these cases, the luciferase data must be regarded with great suspicion.
If the solvent is toxic, try a different solvent or a lower concentration of
solvent. If the test substance is toxic, try extracting the toxic
component (e. g., removing sulfur compounds from sediment extracts),
or conclude that cytotoxicity is likely to preclude any dioxin-like
effects.

5.7.2. Lusifsrasellata

Calculate averages and standard deviations for each treatment.
Graph the change in response and its standard deviation against
concentration. Relative potencies should be calculated through
conversion of the data to probit values or any other appropriate
transformation that linearizes the sigmoidal dose-response curve.
For a complete description and decision tree for data analysis
methods, refer to the Standard Operating Procedures entitled,
“Estimation of Relative Potencies Based on In Vitro Bioassay
Results”. To convert data to probit values, the concentration
producing maximal induction is set at 100% and the responses of all
other concentrations are converted to a percentage of this maximal
level. A lookup table function can be used in a spreadsheet program
such as Excel or Lotus 1,2,3 to convert percentages to probit values
(contact the Aquatic Toxicology Laboratory for a copy of this table
on disk).

6.0- RECORDS, DOCUMENTATION, AND QC REQUIREMENTS
6.1 Records and Documentation
The primary analyst shall document any anomalies and/or deviation from the
speciﬁed method in a bound, serially numbered, laboratory notebook with tear-

out carbon copies. All electronic ﬁles and hardcopies will be kept at the
Aquatic Toxicology Laboratory at Michigan State University and a duplicate

124

6.2

copy will be kept in the Archive Room of Dr. John Giesy (Dept. of Zoology,
Michigan State University). This information will also be recorded in the data
package and listed in the Case Narrative Form (in accordance with SOP 802).
The primary analyst will sign and date any forms as the analyst.

The technical reviewer will record any problems noted during the technical
review. The technical reviewer will return the items to the analyst for
corrections prior to inclusion into the data package. The technical reviewer will
sign and date all forms as the reviewer.

QC Requirements and Data Quality Objectives

The threshold dose and EC50 for luciferase induction in H4IIE-1uc cells were
approximately 0.1 and 1.2 pg/well, respectively, as determined from 41
separate standard TCDD curves analyzed in 1997 (Figure 1). Coefﬁcients of
variation (standard deviation/mean x 100) for the assay were under 10% at all
concentrations tested for any single day of experiments. A proﬁciency curve
is maintained for the EC50 and threshold doses for TCDD standard curves
(every plate has a standard curve). If for a particular plate, the ECso or
threshold for the TCDD standard exceeds :1; 20 % of the proﬁciency curve,
then the sample on the plate in question will be reanalyzed. For a sample size
of 20 g tissue and a ﬁnal extract volume of 0.25 ml, the H4IIE-luc assay will
detect 1 part per trillion (ppt; pg/g, wet weight) TCDD-equivalents. With a
sample size of 5 g tissue, 4 pg/g (wet weight) TCDD-equivalents will be
detected.

7.0. RESPONSIBILITIES

The primary analyst will complete the analysis as speciﬁed in this SOP and
provide documentation of raw data and any anomalies and provide data to the
data analyst who will perform data calculations in accordance with SOP 202.

The technical reviewer will determine if data quality objectives were met, notify
the analyst if any problems were found.

8.0. REFERENCES

Aarts, J.M.M.J.G., Denison, M.S., De Haan, L.H.J., Schalk, J.A.C., Cox, M.A. and

Brouwer, A. 1993. Ah receptor-mediated luciferase expression: a tool for
monitoring dioxin-like toxicity. Short paper, Dioxin ’93, 361-3 64.

Aarts, J.M.M.J.G., Denison, M.S., Cox, M.A., Schalk, M.A.C., Garrison, P.A., Tullis, K.,

De Haan, L.H.J., and Brouwer, A. 1995. Species-speciﬁc antagonism of Ah
receptor action by 2,2’,5,5’-tetrachloro- and 2,2’,3,3’,4,4’-hexachlorobiphenyl. Eur.
J. Pharmacol. in press.

125

Aquatic Toxicology Laboratory/Michigan State University, Laboratory Quality Control
Plan (LQCP), September 1998.

Aquatic Toxicology Laboratory/Michigan State University, Safety Manual, September
1998.

Denison, M.S., El-Fouly, M.H., Aarts, J.M.M.J.G., Brouwer, A., Richter, C. and Giesy,
JP. 1993. Production of a novel recombinant cell line bioassay system for the
detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Short paper,
Dioxin ’93, 365-368.

Giesy, J.P., Ludwig, JP. and Tillitt, DE. 19943. Dioxins, dibenzofurans, PCBs and
Colonial ﬁsh-eating water birds. In: Dioxins and Health, Chapter A. Schechter, ed.,
Plenum Press, N.Y..

Giesy, J .P., Ludwig, J .P. and Tillitt, D.E. 1994b. Deforrnities in birds of the Great Lakes
region: assigning causality. Environ. Sci. Toxicol. 28, 128A-l35A.

Gilbertson, M., Kubiak, T. and Ludwig, J. 1991. Great Lakes embryo mortality, edema,
and deformities syndrome (GLEMEDS) in colonial ﬁsh-eating birds: similarity to
chick-edema disease. J. T oxicol. Environ. Health, 33, 455-520.

Murk, A.J., Jonas, A., Brouwer, A., Leonards, P.E.G. and Denison, MS. 1996.
Application of the CALUX (chemical activated luciferase gene expression) assay
for measuring TCDD-equivalents in sediment, pore water, and blood plasma
samples. Organohalogen Compounds 27, 291-296.

Okey, A.E., Riddick, D.S. and Harper, RA. 1994. The Ah receptor: mediator of the
toxicitiy of 2,3,7,8-tetrachlorodibenzo—p-dioxin (TCDD) and related compounds.
T oxicol. Lett. 7_Q, 1-22.

Nebert, D.W. and Gonzalez, F .J . 1987. P450 genes: structure, evolution, and regulation.
Ann. Rev. Biochem., 56, 945-993.

Poland, A. and Knutson, J .C. 1982. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related
halogenated aromatic hydrocarbons: examination of the mechanism of toxicity.
Annu. Rev. Pharmacol. T oxicol., 22, 517-544.

Program Manager Rocky Mountain Arsenal, Chemical Quality Assurance Plan (CQAP),
April 1996.

Safe, SH. 1986. Comparative toxicology and mechanism of action of polychlorinated
dibenzo-p-dioxins and dibenzofurans. Ann. Rev. Pharmacol. T oxicol., 2.6, 371-399.

Safe, SH. 1990. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs) and
dibenzofurans (PCDFs) and related compounds: environmental & mechanistic
considerations which support the development of toxic equivalency factors (TEFs).
CRC Crit. Rev. T oxicol., 21, 51-71.

Sanderson, J.T., J. M. M. J. G. Aarts, A. Brouwer, K. L. Froese, M. S. Denison and J. P.
Giesy. 1996. Comparison of ah receptor-mediated luciferase and ethoxyresoruﬁn o-
deethylase induction in h4iie cells: implications for their use as bioanalytical tools
for the detection of polyhalogenated aromatic hydrocarbons. W
Phannaggl. 137(2):316-325.

Scatchard, G. 1949. The attraction of proteins for small molecules and ions. Ann. NY.

Acad. Sci, 51, 660-672.

Tillitt, D.E., Giesy, JP. and Ankley, GT. 1991. Characterization of the H4IIE rat
hepatoma cell bioassay as a tool for assessing toxic potency of planar halogenated
hydrocarbons in environmental samples. Environ. Sci. T echnol. 25, 87-92.

126

Whitlock, J.P.Jr. 1990. Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzo-p-
dioxin action. Ann. Rev. Pharmacol. T oxicol., 3_Q, 251-277.

127

W

MCF-‘l ERE-Luciferase Cell Bioassay For The Detection Of
Estogen Receptor Agonists And Antagonists

Version 2.0 July, 1997

Alan Blankenship, Dan Villeneuve,
Vincent J. Kramer, and John P. Giesy

Supported through:

National Food Safety and Toxicology Center,
Institute for Environmental Toxicology,
Department of Fisheries and Wildlife, and the Department of Zoology

Correspondence to:

Aquatic Toxicology Laboratory
201 Pesticide Research Center
Michigan State University
East Lansing, MI 48824-1222 USA
T: (517) 353-9195
F: (517) 353-5598

128

I. Introduction

The luciferase induction assay is an in vitro technique for the identiﬁcation of
estrogenic and anti-estrogenic compounds. The technique uses human breast carcinoma
MCF—7 cells stably transfected with an estrogen receptor-controlled luciferase
reporter gene construct (MCF-7 ERE-Luc; obtained from Dr. Michel Pons, Institut
National de la Sante et de la Recherche Medicale, 60 Rue de Navacelles, 34090
Montpelier, Phone: 67043 7.00; Pons et al., 1990). These cells express ﬁreﬂy luciferase
in response to estrogen agonists. Luciferase activity is measured conveniently and with high
sensitivity as light emission using a plate-scanning luminometer. Estrogen antagonists can
be detected by measuring a decrease in luciferase production (decrease in light) after
administration of the test compound in the presence or absence of l7-B—estradiol (E2). The
technique has been modiﬁed from the published procedure to run in 96-well plates, allong
the analysis of a large number of samples. Luciferase induction potential is assessed by
comparison of the response to that of standard compounds: E2 for agonists and ICI 182,780
for antagonists.

11. Materials

A.EmmﬁmmdmeCEZERELuuclls

The assay uses a stably transfected cell line donated by Dr. Michel Pons. Brieﬂy, human
breast carcinoma MCF-7 cells [American Type Culture Collection (ATCC) catalog
#HTB-22) were stably-transfected with a luciferase reporter gene plasmid consisting of
the XJaeyis vitellogenin promoter region containing 4 estrogen responsive elements and
the herpes simplex thymidine kinase promoter upstream of the ﬁreﬂy luciferase reporter
gene (Pons, et al. 1990). The cells produce luciferase in response to treatment with E2.

1. Maintain adherant cells in continuous culture in 100 mm tissue culture
plates (Corning #25020-100, Cambridge, MA; 1-800-492-1110), 75 cm2
ﬂasks (Corning #25113-75) or any appropriate vessel at a maximum density
of 80-90% conﬂuence in 10% Full Medium (See Media Preparation).
Incubator conditions are 37 C, 5% CO2 humidiﬁed atmosphere.

NOTE: MCF-7 cells are a transformed human cell line that must be handled
in a laminar ﬂow hood, preferably NSF Type 11 B2 containment. All items
contacting the cells should be considered infectious material and sterilized
before ﬁnal disposal. Personnel should follow all institutional safety
precautions including safety glasses, gloves and lab coat.

2. Subculture cells 1:2 every 3 to 4 days (depending upon density)
maintaining a minimum cell density of 20-30%.

129

BMW

1. Dynatech ML3000 Luminometer
(Dynatech Technical Support: (800) 336-4543; Chantilly, VA)

2. Cytoﬂuor 2300/2350 Fluorescence Measurement System
(Millipore Technical Support: (800) 645-5476; Bedford, MA)

NOTE: All users of the Dynatech ML3000 Luminometer and the
Cytoﬂuor 2300 must read and be familiar with the Operators Manual before using the
instrument. A working knowledge of Microsoft Windows is also necessary.

05 l' ”3.1.!

1. 96-Well ViewPlates” (Packard Instruments #6005181; Meriden, CT);
white 96-well plates, sterile, tissue culture treated, with lids and self-
adhesivesticker for bottom of plates

Cost: $297/50 plates;

Ordering: (800) 856-0734

Technical support: (800) 323-1891

2. Cell viability assay reagents; sold either as a kit from Molecular Probes
(#L-3224; Eugene, OR) or as individual components:

Calcein AM (Molecular Probes #C-3100);
MW = 994.87; made up as 4000x (2 mM) stock
(50 pg/ 12.56 pl DMSO)

Ethidium homodimer 1 (Molecular Probes #E 1169)
MW = 857; made up as 2000x (1 mM) stock in DMSO

Ordering: (800) 43 8-2209
Technical support: (541) 465-8353

3. LucLiteTM Kit (Packard Instruments # 6016911 - 1000 assay kit;
Meriden, CT) Make fresh on same day as assay. Dissolve one bottle of
lyophilized reagent with 10 ml buffer (supplied) for every 133 assays
(individual wells) to be analyzed.

Cost: $420/ 1333 assays using 75 pl/assay (or 1000 assays if
using the manufacturer’s suggested volume of 100
pl/assay. Preliminary studies showed equivalent responses
at both 75 pl and 100 pl).

Ordering: (800) 856-0734

Technical support: (800) 323-1891

130

4. Dulbecco’s phosphate-buffered saline (PBS) with Ca2+ and Mg2+
First, make up 10 L of Ca2+-free and Mg2+- free Dulbecco’s PBS:

2.0 g KCl

2.0 g KH2PO4
80.0 g NaCl
1 1.5 g Na2HPO4
10.0 L H2Oa

adistilled/deionized or Nanopure biological grade

1 L of PBS, add 0.1 g anhydrous CaCl2 and 0.1 g MgCl2 - 6H20
Keep at room temperature (good for at least 2 - 4 weeks).

D. MediaPrsnaration
1. 10% Full Medium

Dulbecco’s Modiﬁed Eagle’s Medium with Ham’s F-12 Nutrient
Mixure (1:1 mix; Sigma #D-2906) Him—W, sodium
bicarbonate, and with 15 mM HEPES buffer and L-glutamine.
Prepare as instructed by the manufacturer, adjust the pH to ~7.3, and
then add :

10% fetal bovine serum (Hyclone deﬁned FBS #
SH30070.03; Logan, UT; 1-800-492-5663)

1 mM sodium pyruvate

1 mg/ml bovine insulin (Sigma #1-1882)

2. 10% DCCFBS Medium
Prepared as for the full medium, except the fetal bovine serum is
replaced with dextran/charcoal-stripped fetal bovine serum
(available from Hyclone #SH30068.03).
III. Hazards and Precautions
E2 and many other estrogenic compounds have been found to be carcinogenic. In addition,
the ethidium homodimer used in the cell viability assay is a powerful mutagen. Care should

be taken to minimize exposure. According to institutional guidelines, medium should be
collected in a liquid trap for disposal as hazardous waste.

131

IV. Sample and Standards Preparation
A. Sample Preparation

Two types of samples may be assayed: pure compounds and mixtures derived from
environmental or tissue samples. For pure compounds, sample preparation consists of
dissolving the material in an appropriate solvent. The solvent of choice is ethanol (because
of its water miscibility and low toxicity). However, if the material is insoluble in ethanol,
the following solvents can be tried: isooctane, acetone, p-dioxane, acetonitrile, and
methanol. A stock solution should be prepared at 5 mM for compounds of unknown
activity and stored in amber glass vials at -20°C. However, it may be necessary to test
concentrations near the limit of solubility of the compound.

Samples derived from environmental matrices such as tissue, water, or sediment should be
extracted and concentrated according to appropriate protocols. The volume of sample
should be recorded before re-dissolution in the assay solvent so that a dilution or
concentration factor can be calculated. After dissolution in the assay solvent, the sample
should be stored at 40%.

B. Standards Preparation

A large range of standards should be prepared to deliver a ﬁnal concentration of l7-B-
estradiol (E2) between 0.15 - 500 pM (200x stocks in ethanol between 30 pM - 100 nM).
Generally, 8 concentrations will achieve a full dose response curve (ﬁnal = 0.15, 0.5, 1.5, 5,
15, 50, 150, 500 pM) for E2. Ideally, standards should be dissolved in the same solvent as
the samples, but this is not always possible. In this case, be sure to conduct assays with both
solvent controls and compare them to blanks (see Dosing Cells).

ICI 182,370 is a potent anti-estrogen in this cell line (Wakeling and Bowler, 1992).
Construct an 8 point concentration range to cover the entire response range of these cells
as follows: 10 pM, 1 pM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM.

V. Procedure

NOTE: Adherence to these procedures will insure consistent response of cells.
Timing and cell density are critical. An assay may be completed in a 5 day week.

A.Elatingﬂellamarcu

Prior to conﬂuence, trypsinize the cells and determine the number of cells/n11. Dilute the
cell solution to a concentration of 60,000 cells/ml with media. Add 0.25 ml of cell
suspension to each well (15,000 cells) of a 96-well ViewPlateTM using an Eppendorf“A repeat
pipettor. Care must be taken that the cell suspension is uniform each time that the pipettor is
reﬁlled. This is done by gently inverting the tube or bottle of cell suspension end-over-end
several times immediately prior to reﬁlling the pipettor. If the outer 36 wells are not being
used for the experiment (recommended), ﬁll them with either sterile media or PBS to

132

maintain humidity consistently across the plate. Use of the outer wells is not recommended
because of an edge effect caused by inconsistent growth of cells in these wells.

NOTE: cell number per well is one of the largest contributors to variation in the data - so
take plenty of time to do this step properly.

amangﬁellsmaxz)

Dose the cells 24 hours after plating (exposures continue for 3 days). First, examine the
cells to ensure consistent plating. Aspirate the media (I attach p-10 pipet tips to the suction
line to minimize cell scraping), and replace with 0.25 ml of 10% DCCFBS media that has
been prewanned to 37°C.

NOTE: the use of cold media should be avoided because it may “shock” or stress the cells
and it may cause precipitation of test agents that have poor water solubility).

A typical plate design is shown in Figure 1. Use at least three replicates per treatment. Use
a negative control with no treatment (blank) and a solvent control treated with pure solvent
only. Use at least ﬁve concentrations of each compound or extracts tested. Prepare 200x
chemical stocks in the appropriate solvent (see Sample Preparation). In general, it is best to
dose the cells directly in the well; however, for water miscible solvents, the test agent can
be added to a sterile glass vial containing 2 ml of media, mixed, and then added to each
well.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Row/ 1 2 3 4 5 6 7 8 9 10 11 12
Col
A
B 0.5 0.5 pM 0.5 pM C1 C1 C1 C2 C2 C2 solvent
pM E2 E2 E2 Conc. Conc. Conc. Conc. Conc. Conc. control
1 l 1 l 1 l
C 1.5 1.5 pM 1.5 pM C1 C1 C1 C2 C2 C2 solvent
pM E2 E2 E2 Conc. Conc. Cone. Conc. Conc. Conc. control
2 2 2 2 2 2
D 5pM 5pM 5pM C1 C1 C1 C2 C2 C2 solvent
E2 E2 E2 Conc. Conc. Conc. Conc. Conc. Conc. control
3 3 3 3 3 3
E 15 15pM 15pM C1 C1 C1 C2 C2 C2 blank
pM E2 E2 E2 Conc. Conc. Conc. Conc. Conc. Conc.
4 4 4 4 4 4
F 50 50 pM 50 pM C1 C1 C1 C2 C2 C2 blank
pM E2 E2 E2 Conc.5 Conc.5 Conc.5 Conc.5 Conc.5 Conc.
5
G 150 150 150 C1 C1 C1 C2 C2 C2 blank
pM E2 pM E2 pM E2 Conc. Conc. Cone. Conc. Conc. Conc.
6 6 6 6 6 6
H

 

Figure l. A typical plate design for determination of estrogen agonist activity in MCF-7 ERE-Luc cells. Note
that blanks, solvent controls, and a standard curve for E2 can be analyzed on the same plate with two test
samples (labeled Cl and C2, both at six different concentrations).

133

 

CConductingtheﬂioassaxiDarLil

1. Preparation steps (prior to assay)

Inspect plates visually with and without microscope - check
degree of conﬂuence, homogeneity from well-to-well, and any
signs of cytotoxicity or altered morphology

Set up cytoﬂuor for viability assay:
set 1: excitation = B emission = B sensitivity = 3
set 2: excitation = C emission = E sensitivity = 4

NOTE: sensitivities can be adjusted if values are too high (9999) or too
low (not different from blanks)

0.

Prepare viability assay reagent:

CAUTION: ethidium homodimer is a powerful mutagen - handle
with care and throw contaminated tips, etc., into biohazard bags

Each plate will need 3 ml or 5 ml, depending on whether 60 or 96
wells are used per plate, respectively plus a little extra (2 ml)
Dilute the appropriate amounts of calcein and ethidium with the
appropriate volume of media without F BS as shown below:

134

 

 

 

 

Number of Volume of viability assay reagent needed
plates [total volume (ml); calcein stock (pl); Ethidium stock
011)]
using 60 wells using 96 wells
1 5 ml; 1.25 pl; 2.5 pl 7 ml; 1.75 pl; 3.5 pl
2 8ml; 2p]; 4p] 12 ml; 3pl; 6p]
3 11 ml; 2.75 pl; 5.5 pl 17 ml; 4.25 pl; 8.5 pl
4 14 ml; 3.5 pl; 7 pl 22 ml; 5.5 pl; 11 pl
5 17 ml; 4.25 pl; 8.5 pl 27 ml; 6.75 pl; 13.5 pl
6 20 ml; 5 pl; 10 pl 32 ml; 8 pl; 16 pl
7 23 ml; 5.75 pl; 11.5 pl 37 ml; 9.25 pl; 18.5 pl
8 26 ml; 6.5 pl; 13 pl 42 ml; 10.5 pl; 21 pl
(1. Set up luminometer:
Mode = Cycle; Pause = 2 sec;
Data = All; Mix = Off;
Gain = High; Temp. = 30°C;
Cycle = 1-3; AID reads = 20 (number of times
that each well is
analyzed per cycle)

e. Set up vacuum aspirator (attach p-lO tips to each suction line to
minimize scraping surface area and only use the plate washer to
aspirate and not to dispense PBS)

f. Prepare LucLite substrate solution and luciferase positive control

(must be used on the same day as prepared)

1)

2)

Reconstitute lyophilized substrate solution by adding 10 ml of
Assay buffer solution A (provided with kit) to one vial of
lyophilized substrate (each vial is enough for 133 assays if 75
p1 is used). Agitate gently until a homogeneous solution is
formed (a slight turbidity is acceptable). Equilibrate to room

temperature before use.

NOTE: if more than one vial is reconstituted, combine and mix
to prevent any variation from the substrate between plates.

Reconstitute the lyophilized luciferase positive control with
200 p1 of distilled (or nanopure water). Each vial contains

sufﬁcient luciferase for 20 controls.

135

Cell Viability Assay Procedure (process one plate at a time)

remove plate from incubator and aspirate media, then rinse 1 time
with PBS

Add 50 pl of PBS with Ca2+ and Mg“ to all wells using a 8-channel
pipet

Add 50 pl of viability assay reagent to all wells using a 8-channel
pipet

Incubate at room temperature for 10 minutes and then scan plate in
the Cytoﬂuor instrument

Export] print data (check that values are appropriate, otherwise
adjust sensitivity and rescan)

Aspirate viability reagent/PBS, and rinse 1 time with PBS using
vacuum
aspirator

Seal the bottom of the ViewPlates with self-adhesive TopSeal (or

tape very well). This increases the signal detection in the
luminometer.

Add 75 pl of PBS with Ca2+ and Mg2+ (or media without serum or
phenol red) to all wells using an 8-channel pipet

Working under SUBDUED light conditions, add 75 pl/well of
reconstituted substrate solution and agitate gently.

Wait ﬁve minutes before counting the plate to allow full signal
generation.

Measure lurrrinescence on the Dynatech ML3000 luminometer

Save, print, and copy data to disk

DE'D ..

Protein determination is readily accomplished in the same plates with same cell lysates after
measuring luminescence. A ﬂuorescamine-based protein assay (Sanderson et al., 1996) or
Micro-BCA Assay (available from Pierce (800) 874-3723) can be used. It is our experience,
however, that normalizing to protein concentration prevents proper analysis of the dose-
response luminescent data because estrogen-mediated luciferase induction and estrogen-

136

induced protein synthesis cocorrelate together. Degree of conﬂuency of wells was
veriﬁed microscopically, which was found to make normalization to protein
unnecessary; therefore, luciferase activity is reported as either relative light units
(RLU) or percent of solvent control.

137

R.DataAnalxsis

Wm - Average the three measurements for and ethidium. Divide the average
ethidium homodimer ﬂuorescence for each sample by its calcein AM ﬂuorescence to obtain
a dead to live ratio. Graph the average calcein AM ﬂuorescence and standard deviation for
the negative control, solvent control, and each concentration tested. Examine the calcein
AM data visually. If the blank has greater viability than the other treatments, the solvent
may be toxic to the cells. If viability decreases with increasing concentration of the test
substance, the test substance may be toxic to the cells. In either of these cases, the luciferase
data must be regarded with great suspicion. If the solvent is toxic, try a different solvent or a
lower concentration of solvent. If the test substance is toxic, try extracting the toxic
component (e. g., removing sulfur compounds from sediment extracts), or conclude that
cytotoxicity is likely to preclude any estrogenic/antiestrogenic effects.

Lngifaraseﬂata - Calculate averages and standard deviations for each treatment. Graph
the change in response and its standard deviation against concentration. Relative
potencies should be calculated through conversion of the data to probit values or any
other appropriate transformation that linearizes the sigmoidal dose-response curve. To
convert data to probit values, the concentration producing maximal induction is set at
100% and the responses of all other concentrations are converted to a percentage of this
maximal level. A lookup table function can be used in a spreadsheet program such as
Excel or Lotus 1,2,3 to convert percentages to probit values (contact the Aquatic
Toxicology Laboratory for a copy of this table on disk).

Alternatively, the dose-response data may be transformed in the same way as receptor-
binding data to produce analogies of Woolf and Scatchard plots. Due to the limitations of
the underlying assumptions of these analyses, care should be taken to include only data
points that are on the linear rising part of the dose-response curves, when plotted on a log-
linear scale. - At low concentrations of ligand the assumption that ﬁ'ee ligand
concentration is equal to total concetration does not hold. At high concentrations of
ligand, the response starts to level of and decrease (receptor saturation is assumed). The
ideal range is probably ﬁom the ED2o-ED30. - The EDso and maximum response
(ERODm) values generated using Scatchard/Woolf plots are always compared with the
visually determined values for conﬁrmation. It is not necessary to have the complete dose-
response curve in order to determine EDso and ERODum values, although the
determinations become less reliable. Do not accept EDso values which are higher than the
highest tested dose. If a full dose-response curve cannot be attained, increase the number
of concentrations on the linear rising part of the curve in order to have sufﬁcient data points
to perform a reliable Scatchard/Woolf analysis. A preferred approach is to concentrate the
sample.

138

VII. References

Pons, M.D., D. Gagne, J.C. Nicolas, and M. Mehtali. 1990. A new cellular model
of response to estrogens: A bioluminescent test to characterize (anti)estrogen
molecules. Biotechnigues 9:450—457.

 

Sanderson, J.T., J. M. M. J. G. Aarts, A. Brouwer, K. L. Froese, M. S. Denison and J. P.
Giesy. 1996. Comparison of ah receptor-mediated luciferase and ethoxyresoruﬁn o-
deethylase induction in h4iie cells: implications for their use as bioanalytical tools
for the detection of polyhalogenated aromatic hydrocarbons. Tampa],
Pharmacol. 137(2):316-325.

Wakeling, A., and]. Bowler. 1992. ICI 182,780, A new antioestrogen with clinical
potential. 1. Steroid Biochem. Molec. Biol. 43(1-3):173-177.

139

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