Environmental Toxicology and Chemistry, Vol. 31, No. 5, pp. 1053–1062, 2012
# 2012 SETAC
Printed in the USA
DOI: 10.1002/etc.1777
ENDOCRINE DISRUPTING, MUTAGENIC, AND TERATOGENIC EFFECTS OF UPPER DANUBE
RIVER SEDIMENTS USING EFFECT-DIRECTED ANALYSIS
ERIC HIGLEY,*y STEFANIE GRUND,z PAUL D. JONES,y§ TOBIAS SCHULZE,k THOMAS-B. SEILER,#
URTE LÜBCKE-VON VAREL,k WERNER BRACK,k JAN WÖLZ,# HANNO ZIELKE,# JOHN P. GIESY,yyyzz§§
HENNER HOLLERT,# and MARKUS HECKERy§
yToxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada
zAquatic Ecology and Toxicology Section, Centre for Organismal Studies, University of Heidelberg, Heidelberg, Germany
§School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada
kUFZ-Helmholtz Centre for Environmental Research, Leipzig, Germany
#RWTH Aachen University, Institute for Environmental Research, Department of Ecosystem Analysis, Aachen, Germany
yyCity University of Hong Kong, Hong Kong, China
zzZoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia
§§Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
(Submitted 20 September 2011; Returned for Revision 24 November 2011; Accepted 2 January 2012)
Abstract— Effect-directed analysis (EDA) can be useful in identifying and evaluating potential toxic chemicals in matrixes. Previous
investigations of extracts of sediments from the upper Danube River in Germany revealed acute nonspecific and mechanism-specific
toxicity as determined by several bioassays. In the present study, EDA was used to further characterize these sediments and identify
groups of potentially toxic chemicals. Four extracts of sediments were subjected to a novel fractionation scheme coupled with
identification of chemicals to characterize their ability to disrupt steroidogenesis or cause mutagenic and/or teratogenic effects. All four
whole extracts of sediment caused significant alteration of steroidogenesis and were mutagenic as well as teratogenic. The whole extracts
of sediments were separated into 18 fractions and these fractions were then subjected to the same bioassays as the whole extracts.
Fractions 7 to 15 of all four extracts were consistently more potent in both the Ames fluctuation and H295R assays. Much of this toxicity
could be attributed to polycyclic aromatic hydrocarbons, sterols, and in fraction 7-naphthoic acids. Because the fraction containing
polychlorinated biphenyls, polychlorodibenzodioxin/furan, dichlorodiphenyltrichloroethane, and several organophosphates did not
cause any observable effects on hormone production or a mutagenic response, or were not detected in any of the samples, these
compounds could be eliminated as causative agents for the observed effects. These results demonstrate the value of using EDA, which
uses multiple bioassays and new fractionation techniques to assess toxicity. Furthermore, to our knowledge this is the first study using the
recently developed H295R assay within EDA strategies. Environ. Toxicol. Chem. 2012;31:1053–1062. # 2012 SETAC
Keywords—Fractionation
Steroidogenesis
Mutagen
Polyaromatic hydrocarbon
Polychlorinated biphenyls
great as 26 mg/g dry weight in sediments of the upper Danube
River. Extracts of sediments from the same sites elicited significant aryl hydrocarbon receptor-mediated effects in three
separate cell lines. In addition to dioxin-like compounds, other
chemicals have been reported to occur in these sediments.
Specifically, a variety of compounds that were previously
shown to modulate the endocrine system such as certain pesticides, plasticizers, sterols, and PAHs have all been measured
in sediments [1,2,8,9]. The potential of these sediments to
disrupt endocrine functions has been confirmed in a recent
study that reported in vitro estrogen agonist potency and
modulation of steroidogenesis [9]. However, according to that
study only 2 to 6% of the observed estrogenic effect in the
sediment studied could be attributed to the routinely monitored
estrogenic chemicals estradiol (E2), estrone (E1), ethinylestradiol (EE2), bisphenol A (BPA), or nonylphenol (NP). It has also
been reported that exposure to sediments sampled from the
same locations but at different times in the Danube River
resulted in lesser hatching rates and increased time-to-hatch
in zebrafish (Danio rerio) embryos [1].
Chemicals in environmental matrices such as sediments and
biota occur as mixtures and can include bioactive compounds and
isomers of both natural and anthropogenic origin. Because there
may be no a priori knowledge of the chemicals present in
INTRODUCTION
According to the U.S. Toxic Substances Control Act, more
than 70,000 organic chemicals are currently used in industry.
For some of these chemicals, there is a general lack of toxicity
data, even though there is the possibility of some of these
substances being present at biologically active concentrations in
the environment that might pose a risk to organisms [1–4].
Despite extensive stocking efforts, populations of some fish
in the upper Danube River, Germany, have been reported to be
declining over the last few decades. One concern in the upper
Danube River is the toxicity of sediments to fish [5–7]. To date,
several studies have assessed toxicity of sediments taken from
the upper Danube River and have found several sites where
extracts of these sediments caused significant toxicity. Effects
include genotoxicity, mutagenicity, teratogenicity, or estrogenicity [1–4]. For example, Keiter et al. [2] reported concentrations of 16 priority U.S. Environmental Protection Agency
(U.S. EPA) polycyclic aromatic hydrocarbons (PAHs) to be as
* To whom correspondence may be addressed
(eric.higley@usask.ca).
Published online 13 February 2012 in Wiley Online Library
(wileyonlinelibrary.com).
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Environ. Toxicol. Chem. 31, 2012
samples, analysis of all of the possible chemicals present in an
environmental sample would not only be prohibitively expensive, but because of limits in the available analytical methodologies or authentic standards for some chemicals, not possible.
Therefore, approaches have been developed to supplement the
identification and quantification of chemicals by instrumental
analyses with bioanalytical techniques that make use of the
specific properties of certain groups of chemicals to interfere
with specific biological processes. This type of analysis has been
defined as effect-directed analysis (EDA) and is based on a
combination of fractionation procedures, biotesting, and subsequent identification and quantification of individual compounds
or classes of compounds [10–15]. In EDA, matrices containing
mixtures of chemicals, such as extracts of sediments, are first
analyzed using one or a combination of several bioassays that are
responsive to different modes of biological action. If a sample has
been identified as causing significant effects, it is then subjected
to fractionation, which separates the chemicals based on polarity,
molecular size, or other physicochemical properties or combinations [10]. These fractions are then retested in the bioassays in
which they were previously shown to be active. This procedure is
repeated until the activity can be identified and isolated in one or
multiple fractions. These fractions are then subjected to instrumental analyses to identify and quantify individual compounds.
Because the chemical physical characteristics of chemicals in
specific fractions are known, this information can be used to
select the most appropriate analytical techniques to identify and
quantify specific constituents of that fraction. The information
obtained during this process can be used to find the source of the
contaminants, such as industry or agriculture, or decide on
remedial efforts if needed.
Several assays have been developed to evaluate the biological activities of chemicals. Considering the previously
reported mutagenic, teratogenic, and endocrine disrupting
potential of Danube River sediments, in the present study
we focused on endocrine disruption, mutagenicity, and teratogenicity. One cell line that has been shown to be a useful in vitro
model for investigating effects on steroidogenic pathways
and processes including the production of sex steroids is
the human H295R adrenocarcinoma cell line [16–18]. Under
the guidance of the U.S. EPA and the Organization for
Economic Co-operation and Development (OECD), an OECD
H295R steroidogenesis test guideline was developed to address
regulatory needs for the screening of effects of chemicals on
steroidogenesis pathways [19–21]. Thus, standardized protocols and procedures are available for this assay.
Mutagenic and teratogenic effects can be measured using
different types of assays, including the Ames fluctuation assay,
to measure mutagenic effects and the D. rerio embryo assay to
measure teratogenic effects. Danio rerio embryos have a transparent chorion; therefore, lethal and sublethal effects are easily
monitored in the egg [1,22] and have been shown to be sensitive
to toxicants. The Ames fluctuation assay has been shown to be a
useful tool for assessing mutagenic activity in sediments
because it is a rapid and sensitive test that measures the ability
of a sample or chemical to cause back mutations in Salmonella
bacteria [23,24]. In addition, different types of mutations
caused by different chemicals can be determined depending
on the type of Salmonella strain used; the TA100 Salmonella
strain indicates base pair substitutions and the TA 98 Salmonella strain measures frame-shift mutations. Metabolic activation or deactivation of toxicants can also be evaluated by
treating the extract with CYP450-enriched liver microsomes
(S9) to the assay.
E. Higley et al.
The objective of the present study was to use EDA with a
new fractionation technique that uses an online fractionation
procedure with multiple coupled high-performance liquid chromatography columns [13]. This fractionation technique was
used because, compared to other similar fractionation techniques, it decreases loss of material during fractionation and
decreases formation of artifacts. During the first phase of the
present study, extracts of sediments from four sites in the upper
Danube River were analyzed in the following three assays: the
D. rerio embryo assay, the Ames fluctuation assay, and the
H295R assay. During the second phase, each extract was
fractionated into 18 fractions with different physicochemical
properties, and the biological activity of each fraction was
characterized in the D. rerio embryo, the Ames fluctuation,
and H295R assay. To our knowledge, this is the first study using
the recently developed H295R assay within EDA strategies.
MATERIALS AND METHODS
Sampling
In 2006, near-surface samples of bottom sediments were
collected at two locations along the upper Danube River
(Sigmaringen [Sig2006] and Lake Oepfingen [Opf2006]) as
well as at one tributary stream just upstream of its confluence
with the Danube River (Lauchert [Lau2006]). Samples were
collected using a Van Veen grabber or a stainless steel shovel.
Furthermore, an archived Lauchert sediment sample collected
in 2004 (Lau2004) was included as an uncontaminated reference site based on the results of previous studies [2,25]. All
2006 sampling sites were chosen based on a suspected gradient
of sediment contamination at these locations [1,2,25], and
because of their exposure to sewage treatment plants effluents
(Fig. 1).
Soxhlet-extraction and accelerated membrane-assisted clean-up
of sediments
Sediments were pooled, homogenized, lyophilized, and
sieved (mesh size 1.25 mm) immediately after returning to
the laboratory. Dried and sieved sediments were Soxhlet
extracted for 14 h with dichloromethane and acetone (3:1 v/v,
400 ml) according to previously described methods [26].
Extracts were concentrated first by use of rotary evaporation
and then by a gentle stream of nitrogen. Elemental sulphur was
removed by treatment of the extract with copper. Whole
extracts of sediment from each sample were redissolved in
1 ml dimethyl sulfoxide and were used for testing in the D. rerio
embryo assay and the H295R assay. Residues were also redissolved in 1 ml hexane (Hx) and acetone (7:3 v/v) for subsequent
clean-up procedures.
A new accelerated membrane-assisted clean-up (AMAC)
technique was used to purify whole extracts of sediments
according to an optimized protocol described previously
[27]. Briefly, an aliquot of the whole extract of sediment
equivalent to 20 g of sediment was transferred to dialysis
membranes (commercially available polyethylene tubes) and
dialyzed using an ASE 200 device (Dionex). The temperature,
pressure, number, and duration of cycles were chosen as
described previously [13]. After evaporating the extracts to
dryness, the residue was redissolved in dimethyl sulfoxide for in
vitro testing and in 1 ml Hx:dichloromethane (9:1 v/v) to a final
concentration of 25 g sediment equivalents (SEQ)/ml for further
fractionation. Furthermore, because of a limited sample volume, in vitro testing was prioritized and AMAC extracts were
Effect-directed analysis of upper Danube River sediments
Environ. Toxicol. Chem. 31, 2012
1055
Fig. 1. Sampling sites along the upper Danube River. 1 ¼ Sigmaringen; 2 ¼ Lauchert (tributary); 3 ¼ Oepfingen; ¼ Sewage treatment plants (>10,000
residents). Redrawn according to Grund et al. [9].
only used with the D. rerio embryo assay and Ames fluctuation
assay.
the residue was redissolved in dimethyl sulfoxide for subsequent use in bioassays and chemical analysis.
Ames fluctuation assay
Fractionation of extracts of sediments
Fractionation was performed by using a recently developed
automated online fractionation procedure for polycyclic aromatic compounds in extracts of sediment on three coupled,
normal-phase high-performance liquid chromatography columns as described in Luebcke-von Varel et al. [13]. All solvents
used within the fractionation procedure were Suprasolv or
LiChrosolv grade (Merck). Briefly, in the first step, medium
polar and polar compounds are trapped on cyanopropyl silica
with Hx as mobile phase, while nonpolar substances are flushed
to the nitrophenylpropyl silica and porous graphitized carbon
stationary phases. To separate PAHs with more than two
aromatic rings from the more polar PAHs such as nitro- and
keto-PAHs, the cyanopropyl silica-column is switched offline.
Flushing of the nitrophenylpropyl silica and porous graphitized
carbon phases with Hx continues and the remaining chlorinated
diaromatic compounds elute from nitrophenylpropyl silica to
the porous graphitized carbon column. Afterwards, a sequential
fractionated elution from each of the columns was conducted,
starting with the separation of chlorinated diaromatic compounds on porous graphitized carbon in forward and back-flush
mode using Hx and toluene as mobile phases. The nitrophenylpropyl silica phase was then eluted with Hx:dichloromethane (95:5). Finally, the cyanopropyl silica column is eluted
with Hx, dichloromethane and acetonitrile. After passing
through the detector the eluent is collected by the fraction
collector into 18 glass bottles [13]. After fractionation, the
18 fractions of each sample were evaporated to dryness and
All AMAC extracts and fractions were analyzed by using
bacteria strains TA98 (measures frameshift mutations) and
TA100 (measures base pair substitutions) with or without
metabolic enzymes (S9 fraction). Details of the procedure
are explained in Reifferscheid et al. [24]. Briefly, in 24-well
plates, Salmonella were diluted in exposure medium and
exposed to either AMAC extracts, fractions, positive control,
or a 2% dimethyl sulfoxide solvent control. The plates were
incubated for 90 min at 378C. Different positive controls
were used depending on the bacteria strain and if S9 was
used, including 4-nitro-o-phenyldiamine (0.05 mg/ml) for
TA98 without S9, nitrofurantion (0.06 mg/ml) for TA100 without S9, and 2-aminoanthracene (0.025 mg/ml) for both TA98
and TA100 with S9. After 90 min, a bromocresol purple indicator medium was added to all wells. An aliquot of 800 ml from
each well of the 24-well plate was transferred into 16 replicate
wells of a 384-well plate in triplicate for a total of 48 wells per
sample. The plate was incubated at 378C for 48 h to allow any
bacteria to grow that had been back mutated and able to
produce histidine. After 48 h, if a back mutation occurred
and the bacteria reproduced, the media turned from purple to
yellow. The number of yellow wells per replicate group (16
wells per one replicate) were counted and compared to the
solvent control.
Accelerated membrane-assisted clean-up extracts of sediments from Opf2006, Sig2006, Lau2006, or Lau2004 were each
tested at six dilutions (12.5–400 mg SEQ/ml) with a solvent
control and positive control. All dilutions, solvent controls, and
1056
Environ. Toxicol. Chem. 31, 2012
positive controls were conducted in triplicate. All sediment
fractions were investigated at one concentration of 400 mg
SEQ/ml in triplicate.
Danio rerio embryo assay
Fertilized D. rerio eggs were exposed to four or five dilutions
of whole extracts of sediments and AMAC extracts of sediments
from Opf2006, Sig2006, Lau2006, or Lau2004. For a detailed
description of the assay see Hollert et al. [22]. For the fractionated samples, only Opf2006 and Sig2006 extracts were
analyzed because no toxicity was observed in any concentration
of AMAC extract of either Lau2006 or Lau2004. All fractionated samples were investigated at a concentration of 100 mg
SEQ/ml.
In brief, whole extracts of sediments, AMAC extracts of
sediments, or fractions were diluted in water containing
the following compounds (0.294 g/l CaCl2 2 H2O, 1.23 g/l
MgSO4 7 H2O, 0.63 g/l NaHCO3, 0.055 g/l KCl). A total of
10 eggs were added to test tubes containing the diluted extract or
fraction. One egg and 100 ml of test solution were removed and
placed in one well of a 96-well plate. This was repeated for the
other nine eggs in each sample. Whole extracts of sediments and
fractions were analyzed in replicate exposures. After seeding
and dosing, the 96-well plates were covered and placed in an
incubator at 268C for 48 h. At the end of the incubation period,
four endpoints including lack of heartbeat, failure to detach tail,
retarded somite formation, and coagulation of the embryo were
analyzed.
The H295R steroidogenesis assay
Human adrenocortical carcinoma cells, H295R, were
obtained from the American Type Culture Collection (ATCC
CRL-2128) and cultured and exposed under standardized conditions as described previously [16,17,19–21]. Cells were
exposed to extracts or fractions for 48 h in 24-well plates.
Dimethyl sulfoxide was used as a carrier solvent and did not
exceed 0.1% v/v. For each whole sediment extract, test plates
included five concentrations and a solvent control in quadruplicate. Each of the 18 fractions was dosed at a concentration of
20 mg SEQ/ml in triplicate. Cell viability was evaluated and the
culture medium was stored in an Eppendorf tube at 808C prior
to analysis for hormones. Cell viability was evaluated with the
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide) bioassay [28]. Hormones in culture medium were
extracted and measured by competitive ELISA using the manufacturer’s recommendations (Cayman Chemical Company;
Testosterone [Cat # 582701], 17ß-Estradiol [Cat # 582251]).
Gas chromatography/mass spectrometry analysis
Compounds of interest in the fractions were identified by use
of gas chromatography/mass spectrometry (GC/MS; Agilent
7890A GC interfaced to an Agilent 5975C MSD). Helium
was used as the carrier gas. The GC program for all analyses
consisted of a 4 min hold at 1008C followed by a 48C/min ramp
to 3208C, which was held for 15 min. The injector and GC/MS
transfer line were held at 2858C. Fractions were analyzed for
polychlorinated biphenyls (PCBs), polychlorodibenzodioxin
(PCDD), and common pesticides, including dichlorodiphenyltrichloroethane (DDT) group compounds, the hexachlorohexanes, heptachlor, aldrin-R, heptachlor-epoxide, endosulfan, p,pdichlorodiphenyldichloroethylene, dieldrin, endrin, endosulfan,
endrin-aldehyde, endosulfan-sulfate, methoxychlor, by using
single-ion monitoring (detection limits ¼ 1 ng/ml for PCBs and
E. Higley et al.
2 ng/ml for organic pesticides for each 20 g SEQ/ml fraction).
Two target analytes were monitored for each compound.
Fractions were also screened by using the GC/MS in scan
mode from m/z 50 to 550 to tentatively identify unknown
compounds. Sample spectra at maximum peak height were
compared to the National Institute of Standards and Technology
2008 database to identify compounds. This database contains
spectra of roughly 220,000 compounds. Only a qualitative
assessment of the compounds identified in the MS scan was
performed and is listed in Table 1.
Statistics
All samples were analyzed using the statistical software
SPSS Statistics 17.0. Homogeneity of variance was assessed
with the Levene’s test. Normality of data distributions was
assessed by analysis of a normality plot. A one-way analysis of
variance was used to compare results of whole extracts of
sediment, AMAC extracts and fractions in the Ames fluctuation
assay and H295R assay to the appropriate controls. The parametric post hoc Dunnett’s test was used to compare doses of raw
extract to control. Where normality or homogeneity of variance
tests failed, the nonparametric Kruskal Wallis test was performed, followed by the Mann–Whitney U post hoc, to analyze
differences of all the fractionation samples from controls.
Differences were considered significant at p < 0.05. Median
lethal concentration values for the whole extract of sediment
and AMAC extracts determined by the D. rerio assay were
calculated using the binomial test function of the TOXCALC
software (Michigan State University).
RESULTS
Ames fluctuation assay
Accelerated membrane-assisted clean-up extracts of sediments. With the exception of Lau2004, all AMAC extracts of
sediments resulted in significant genotoxic effects in the Ames
assay (Fig. 2). However, this significant increase in revertants
was observed only with the TA98 strain and after adding
bioactivation enzymes S9 (Fig. 2). The most potent extracts
of sediments were Sig2006 and Opf2006. For these samples,
statistically greater rates of revertants were observed at concentrations as small as 50 or 100 mg SEQ/ml, respectively.
Exposure to the Lau2006 extract caused a significant mutagenic
effect only at the greatest sediment concentration (400 mg
SEQ/ml) tested. The magnitude of the response observed
was approximately three- to fourfold less (5–4 revertants) than
that of the positive control 2-aminoanthracene (16 revertants).
The AMAC extract of Lau2004 sediment did not induce any
revertants at the tested concentrations. Furthermore, none of the
four AMAC extracts caused a significant increase in revertants
in the TA100 bacteria strain either with or without S9 (data not
shown).
Fractionation samples. In general, fractions 8, 10, and
15 produced consistent mutagenic responses among sites. Specifically, significant mutagenic effects were observed in the
TA98 strain with S9 when exposed to Sig2006 fractions 9, 10,
11, 15, and 16 (Fig. 3). Fractions 8, 10, and 11 of sediments
collected at the same site caused significantly greater numbers
of revertants when using the TA100 strain in the presence of S9.
In addition, fractions 3 and 15 caused significantly greater
numbers of revertants when using the TA98 bacterial strain
without S9. No effects occurred in any of the Sig2006 fractions
when using the TA100 bacteria strain without S9.
Sterols or derivatives;
coprostanol
Fatty acids;
sterols or derivatives
15
16
/"
/"
þ
"/"
þ
þ
þ
þ
þ
þ
2
/"
#/"
1
Result of
assay
performedb
Diisooctyl phthalate;
sterols or derivatives
Sterols or derivatives;
long-chain hydrocarbon with
unidentified functionality
Fatty acids; sterols or derivatives
6-ring PAH-like
compounds
Long-chain hydrocarbon with
unidentified functionality
Naphthoic acid;
alkylated PAHs
Chemical measured
by GC/MS
Opfingen
/"
/"
#/"
1
þ
þ
þ
þ
2
Result of
assay
performedb
Fatty acids
(n-hexadecanoic acid;
sterols or derivatives
5- and 6-ring
PAH-like compounds
Long-chain
hydrocarbon with
unidentified
functionality; 6-ring
PAH-like compounds
Sterols or derivatives;
Friedelan-3-one
Sterols or derivatives
5-ring PAH-like compounds
Cyclic
octaatomic sulfur
Phthalate
Phthalate
Phthalate
Phthalate
Naphthoic acid
Chemical measured
by GC/MS
Lauchert
/"
/"
/"
/"
/"
/"
#/-
1
þ
þ
þ
þ
þ
þ
þ
2
Result of
assay
performedb
Fatty acids
Flexol plasticizer 3GO;
phthalate
Long-chain alkanes; 6-ring
PAH-like compounds
Flexol plasticizer 3GO;
phthalate
4-ring PAH- like compound
4- and 5-ring PAH like
compound
5-ring PAH-like compounds
Chemical measured
by GC/MS
Lauchert Reference
"/"
/"
/"
1
þ
þ
þ
þ
þ
þ
2
Result of
assay
performedb
PAH ¼ polycyclic aromatic hydrocarbons.
a
A summary of results of the Ames fluctuation and H295R assays were also included. Danio rerio embryo assay results are not shown because no significant differences were observed in any fraction compared to control.
b
Bioassays: 1 ¼ H295R (arrows indicate induction (") or inhibition (#) of testosterone and estradiol (T/E2); 2 ¼ significant mutagenicity as measured by the Ames fluctuation assay; F ¼ fraction.
Sterols or derivatives
Flexol plasticizer 3GO;
alkylated PAHs
5-ring PAH-like
compounds
6-ring PAH- like
compounds
Sterol derivatives; 6-ring
PAH-like compounds
Naphthoic acid
Long-chain alkanes
Cyclic octaatomic
sulfur
Chemical measured
by GC/MS
14
13
11
10
8
9
3
4
5
6
7
2
F
Sigmaringen
Sampling site
Table 1. Chemicals detected in fractions of the four sampling sites as measured by gas chromatography/mass spectrometry (GC/MS) in scan modea
Effect-directed analysis of upper Danube River sediments
Environ. Toxicol. Chem. 31, 2012
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Environ. Toxicol. Chem. 31, 2012
E. Higley et al.
whereas fractions 8 to 10 were mutagenic in TA100, both with
S9. Furthermore, Lau2006 fractions 9, 14, and 15 significantly
induced a mutagenic effect in TA98 without S9. No effects
occurred in any of the Lau2004 fractions of sediments in TA100
without S9 and in TA98 with S9. Significant mutagenic effects
were shown in TA100 with S9 when exposed to Lau2004
fractions 4, 8, 9, 10, and 11. In addition, fractions 15 and
17 caused significantly greater numbers of revertants when
using TA98 without S9.
Danio rerio assay
Fig. 2. Mutagenic activity of whole extracts of sediments Sigmaringen
(Sig2006), Oepfingen (Opf2006), Lauchert sampled in 2004 (Lau2004), and
Lauchert sampled in 2006 (Lau2006). Mutagenicy measured by the Ames
fluctuation assay using TA98 bacteria with bioactivation enzymes (S9).
Mutagenic activity is expressed as number of revertants. Values represent the
mean SD. Significant differences reported relative to the solvent control
(SC). Multiple symbols indicate different significant levels: p < 0.05;
p < 0.01;p < 0.001. PC ¼ positive control.
Fraction 10 of the Opf2006 sediment caused significant
mutagenicity in both TA98 and TA100 with S9 (Fig. 3).
Fraction 13 induced significantly greater numbers of revertants
for TA100 with S9. Fractions 15 and 16 induced a significant
mutagenic response for TA98 without S9. No effects were
observed in any of the Opf2006 fractions with TA100 without
S9 (data not shown). The Lau2006 fractions 10, 11, 12, 13, and
15 caused a significantly mutagenic response in TA98 (Fig. 3),
Whole extracts of sediments from Opf2006, Sig2006, or
Lau2006 caused lethality of D. rerio embryos in a dose-dependent manner (data not shown) with Opf2006 and Sig2006
being most toxic with median lethal concentrations of
18.9 mg SEQ/ml and 21.5 mg SEQ/ml, respectively, while
the Lau2006 extract was less toxic by a factor of 3
(Table 2). After clean-up by AMAC, median lethal concentrations of Opf2006 and Sig2006 were enhanced to 75.6 mg
SEQ/ml and 100 mg SEQ/ml, respectively (Table 2), while no
toxicity was observed for Lau2006 and Lau2004. None of the
fractions of sediments from any of the sites caused a significant
increase in mortality compared to the solvent control at the
greatest concentration tested (100 mg SEQ/ml).
The H295R steroidogenesis assay
Whole extracts of sediments. Cytotoxicity was observed at
the greatest concentrations (20 mg SEQ/ml) for all whole
extracts of sediments except for Lau2004 (data not shown).
Cell viability was significantly less when cells were exposed to
20 mg SEQ/ml from Lau2006 or Sig2006 and at concentrations
equal to or greater than 10 mg SEQ/ml at Opf2006. In contrast,
the whole extract of sediments of Lau2004 resulted in a significantly greater growth of cells exposed to 5 or 10 mg SEQ/ml,
and no significant effects on cell viability were observed at any
concentrations tested.
All whole extracts of sediments resulted in significantly
greater E2 production by H295R cells. Sediments from
Lau2006 and Opf2006 caused significant and dose-dependent
greater E2 production at all concentrations tested, whereas
sediments collected from Lau2004 and Sig2006 altered E2
production at concentrations greater than or equal to 2.5 mg
SEQ/ml (Fig. 4). The greatest effects on E2 production were
observed for whole extracts of sediments from Opf2006 and
Sig2006 with maximum E2 production being more than fourfold greater than those in the controls. Concentrations of whole
extracts of sediments required to induce a maximum response
were 2.5 and 5 mg SEQ/ml for Opf2006 and Sig2006, respectively.
Table 2. Median lethal concentration (LC50) values for whole and
accelerated membrane-assisted clean-up (AMAC) extracts of sediments in
the Danio rerio assay
Fig. 3. Mutagenic activity of 18 fractions of Sigmaringen (Sig2006),
Oepfingen (Opf2006), Lauchert sampled in 2004 (Lau2004), and Lauchert
sampled in 2006 (Lau2006). Mutagenicity measured by the Ames fluctuation
assay using TA100 and TA98 bacteria with and without bioactivation
enzymes (S9). TA100 without S9 is not shown because no mutagenic effects
were observed in any of the fractions. Furthermore, fractions 1, 2, 5–7, 12, and
18 are not shown because no significant effects were measured at any of the
sites. Mutagenic activity is expressed as number of revertants. Values
represent the mean SD. Significant differences reported relative to the
solvent control (SC). Multiple symbols indicate different significant levels:
p < 0.05; p < 0.01; p < 0.001. PC ¼ positive control.
Site ID
Opf2006
Sig2006
Lau2006
Lau2004
LC50 Whole
extract of sediment
LC50 AMAC
extract of sediment
18.9 mg SEQ/ml
21.5 mg SEQ/ml
58.6 mg SEQ/ml
NA
75.6 mg SEQ/ml
100 mg SEQ/ml
CBD (>100 mg SEQ/ml)
CBD (>100 mg SEQ/ml)
Sig2006 ¼ Sigmaringen; Opf2006 ¼ Oepfingen; Lau2004 ¼ Lauchert collected in 2004, Lau2006 ¼ Lauchert collected in 2006; NA ¼ not available;
CBD ¼ cannot be determined because no lethality was observed in any of
the concentrations.
Effect-directed analysis of upper Danube River sediments
Environ. Toxicol. Chem. 31, 2012
1059
Fig. 4. Effects of the exposure of H295R cells with whole extracts of
sediments Sigmaringen (Sig2006), Oepfingen (Opf2006), Lauchert collected
in 2004 (Lau2004), and Lauchert collected in 2006 (Lau2006) on estradiol
(E2). Cells were treated for 48 h with the indicated concentrations of whole
extracts of raw sediments. Estradiol is expressed as fold changes compared to
solvent controls (SC ¼ 1). Values represent the mean SD. ¼ Significant
differences reported relative to the solvent control. Multiple symbols indicate
different significant levels: p < 0.05; p < 0.01; p < 0.001.
Only exposure to Opf2006 and Lau2004 whole extract of
sediments produced significant changes in production of testosterone (data not shown). Significant increases in testosterone
(T) production were observed for extracts from Opf2006 (1.25,
2.5, and 5 mg SEQ/ml) and Lau2004 (5 and 10 mg SEQ/ml).
The greatest induction of T production occurred after exposure
to 10 mg SEQ/ml of the extract from Opf2006 (1.5-fold from
controls). No significant effects on T production were observed
for Lau2006 and Sig2006 at any concentration and for Opf2006
at the greatest concentration tested.
Fractionation samples. In general, fractions 7, 10, 14, and
15 were the four most potent fractions that significantly changed
E2 or T in at least three of the four sites studied. For example, E2
production was significantly greater in fraction 15 at all four
sites and resulted in threefold greater production of E2, the most
of any fraction at any of the sites. In sample Lau2004, production of E2 was significantly greater when cells were exposed
to fractions 10, 14, and 15. Furthermore, only fraction 15 and
the 1 to 18 combined samples resulted in significantly greater T
production. Fraction 7 of Lau2006 resulted in significantly less
T, but the magnitude of the effect was relatively small. In
contrast, fractions 10, 11, 13, 14, 15, and 16 increased E2
production by up to threefold. Fraction 7 of Opf2006 resulted in
less production of T, but more production of E2 compared to
controls (Fig. 5). Exposure to Opf2006 fraction 18 resulted in a
significantly greater production of T. Estradiol production was
greater than that of the control when cells were exposed to
fractions 14, 15, and 17. Similar to Opf2006, fraction 7 of
Sig2006 resulted in significantly less T production, but greater
E2 production compared to that of the control cells (Fig. 6).
Exposure to Sig2006 fraction 10 resulted in significantly greater
T production compared to controls. Production of E2 was
significantly greater in cells exposed to fractions 9, 10, 13,
and 15 compared to controls.
Fig. 5. Effects of the exposure of H295R cells on 18 fractions from Oepfingen
(Opf2006) sediment samples on testosterone (T) and estradiol (E2). Cells were
treated for 48 h with 20 mg SEQ/ml of each of the 18 fractions in triplicate.
Testosterone and E2 is expressed as fold changes compared to solvent controls
(SC ¼ 1). Values represent the mean SD. Significant differences reported
relative to the solvent control. Multiple symbols indicate different significant
levels: p < 0.05; p < 0.01; p < 0.001.
Chemical residues
Fig. 6. Effects of the exposure of H295R cells on 18 fractions from
Sigmaringen (Sig2006) sediment samples on testosterone (T) and estradiol
(E2). Cells were treated for 48 h with 20 mg SEQ/ml of each of the 18
fractions in triplicate. Testosterone and E2 is expressed as fold changes
compared to solvent controls (SC ¼ 1). Values represent the mean SD.
Significant differences reported relative to the solvent control. Multiple
symbols indicate different significant levels: p < 0.05; p < 0.01;
p < 0.001.
Results of the MS run in scan mode for compounds identification are given in Table 1. Briefly, various PAH-like
compounds, sterols, and naphthoic acids were the predominate
chemicals detected among all samples. Naphthoic acids were
observed in fraction 7 in all samples except Lau2004. Fractions 8
to 13 contained mostly PAHs, which occurred to some extent in
all samples. Sterols or their derivatives were detected primarily in
fractions 13 to 16 of all samples except Lau2004. Of the
compounds quantified, no PCB, PCDD, or DDT group com-
1060
Environ. Toxicol. Chem. 31, 2012
pounds were detected in any of the fractions or samples.
Other chemicals that were quantified and not detected in any
fraction include the hexachlorohexanes, heptachlor, aldrin-R,
heptachlor-epoxide, endosulfan, p,p-dichlorodiphenyldichloroethylene, dieldrin, endrin, endosulfan, endrin-aldehyde, endosulfan-sulfate, and methoxychlor.
DISCUSSION
E. Higley et al.
have been reported for several chemicals. For example, a
synergistic effect with PCBs was observed when exposed to
H4IIE cells in a mixture compared to each PCB separately [30].
In contrast to the results of the D. rerio assay, AMAC
extracts were less potent than the fractions when analyzed in
the Ames fluctuation assay. This is frequently observed in EDA
of mutagens and is probably caused by removal of masking
compounds during fractionation [14].
Whole and AMAC extracts of sediments
Ames fluctuation assay and fractionation
The battery of tests performed in the present study detected
significant mutagenicity and endocrine effects of sediment
extracts. The patterns of effects observed in bioassays in the
present study were similar to those observed in other studies that
have shown that sediments from the Sigmaringen and Lake
Oepfingen locations were generally more potent. For example,
Keiter et al. [2] measured ethoxyresorufin O-deelthylase activity in extracts of 10 upper Danube River sediments and found
that the Opf sediment extract caused the greatest induction of
ethoxyresorufin O-deelthylase activity among all the sediment
extracts studied, and Lau sediments were among the least
potent. The same pattern was observed by Seitz et al. [25]
and Böttcher et al. [5] who measured genotoxicity by use of the
comet assay and the micronucleus test. In the present study,
whole extracts of sediments and AMAC extracts of Opf2006
and Sig2006 exhibited significant potencies in the D. rerio
embryo survival assay, the Ames fluctuation mutagenicity
assay, and the H295R endocrine modulation assay.
The studies by Seitz et al. [25] and Keiter et al. [1] investigated sediments that were collected in 2004. In 2006, new
samples were collected from the same sites. When bio-TEQs
derived from RTL-W1 cells were compared between sites and
years, it was found that the Lau and Sig sediments collected in
2004 were significantly less potent than were extracts of sediments collected in 2006 [29]. In the present study, a similar
trend toward greater potencies in 2006 was observed in the
Ames fluctuation and H295R assays when the results of
the Lau2004 and Lau2006 extracts were compared in both
the AMAC and whole sediment extracts, respectively. It is
unclear why greater toxic potency was observed after these two
years, but it is possible that flood events either deposited new
sediments or eroded the original sediment exposing more
contaminated sediment underneath.
Several rationales can be used to explain the lesser potency
of the AMAC extracts compared to the whole extracts to cause
effects in the D. rerio assay. The AMAC procedure removes
larger lipids and lipid-like matrix components that can interfere
with chemical analysis of environmental samples that is not
removed in the whole extracts of sediments [27]. Furthermore,
the AMAC procedure was shown to have good recoveries of
10 spiked compounds, so it was unlikely that the lesser toxicity
of the AMAC extracts was due to poor extraction efficiency
[27]. However, extraction efficiencies were not determined for
all chemicals. Thus, it cannot be excluded that the lesser toxicity
of AMAC extracts might have been because of removal of some
of the biologically active compounds.
There are two possible explanations for the fact that further
separation of Opf2006 and Sig2006 extracts into 18 fractions
completely eliminated the toxicity of all samples in the D. rerio
assay. One reason for this might be the removal of some active
compounds during the fractionation procedure. Alternatively, it
might be because of the removal of additive or synergistic
interactions between chemicals that were present in the different fractions after separation. Additive and synergistic effects
In general, fractions 8, 10, and 15 produced a consistent
mutagenic response among sites. Specifically, fraction 10 for all
of the sites exhibited greater effects to both TA98 and TA100
with S9 activation, but no mutagenic activity was observed
without S9, which is typical for PAHs, because they need to be
bioactivated to cause an effect [31]. Furthermore, 5-ringed
PAHs were identified in fraction 10 in three of the four sites
(Table 1). According to Keiter et al. [2], between 55 and 88% of
AhR mediated responses observed in upper Danube River
sediments from the same sites was not accounted for by PAHs,
PCBs, and polychlorodibenzodioxin/furan (PCDD/Fs). Of the
AhR-mediated response that could be accounted for, PAHs
accounted for between 10 and 42% of the total toxicological
effects; in all cases, less than 8% of the response was attributed
to PCBs and PCDD/Fs [2]. These results are similar to the
present study, in that we observed no effect in the fractions that
contain PCBs and PCDD/Fs, but saw an effect in fraction 10 that
contains PAHs.
Additionally, in the present study we determined that the
more semipolar and polar compounds were active in all assays
run; these compounds might account for at least some of the
unknown toxicity that was observed by Keiter et al. [2]. For
example, fraction 15, which contains more polar compounds,
produced significant mutagenic effects at all sites in the TA98
bacteria strain without S9. Typical contaminants in polar fractions of sediment extracts are PAHs including nitro-PAHs and
dinitro-PAHs, which have been implicated as possible toxicants
in sediments and would be eluted in fraction 15 [32,33].
Although these compounds were not detected in the GC-MS
scan analysis it is possible that they can exhibit strong mutagenic effects at concentrations below the limits of detection of
the GC-MS analysis, used in the present study [32]. Furthermore, sterols and their derivatives were measured in fraction 15.
However, these are unlikely to have caused the observed effects
because other research that has shown plant sterols to be
antimutagenic and in fact decrease the mutagenic capabilities
of many chemicals [34,35].
The H295R assay and fractionation
Fractionation of the whole extracts of sediments allowed
differentiation of trends in the E2 and T profiles of the
fractions among sites studied, and to determine possible groups
of chemicals that caused the effects observed. For example,
many different types of chemicals, including EE2, estrone, and
plant sterols, possibly found in fraction 15, could account for
greater E2 production by the H295R cells. However, only
sterols or their derivatives were measured in this fraction in
three of the four sites. Furthermore, results from Grund et al. [9]
found that E2, E1, EE2, BPA, and NP only account for between
2 to 6% of the estrogen receptor-mediated activities of nine
upper Danube River sediments, including sediments sampled
from Opf, Lau, and Sig. In fact, more than 94% of the estrogen
receptor-mediated activities were attributed to unknown components [9]. In the present study, fractionation and chemical
Effect-directed analysis of upper Danube River sediments
analysis of the sediments found that sterols could account
for part of the greater E2 production compared to control in
the H295R assay, and it is unlikely that EE2, E1, E2, BPA,
and NP is an issue in these sediments. Plant sterols, in
particular b-sitosterol, have been shown to modulate E2 production in both fish and mammals [36,37]. One study exposed
polecats to b-sitosterol and observed greater plasma E2 and
thyroid hormone concentrations compared to control polecats
[37]. Furthermore, exposure of extracted wood sterols in environmentally relevant concentrations to zebrafish caused a significant change in the sex ratio of zebrafish toward females in
the F2 generation [38].
Research has revealed some PAHs to have endocrine disrupting properties. For example, studies have shown decreased
androstenedione, T, and E2 in flounder ovarian tissue associated
with the exposure to PAHs [8]. Also, it has been reported that
PAHs can act as antiestrogens in breast cancer cell lines [39,40].
Polyaromatic hydrocarbon-like compounds with five aromatic
rings (i.e., benzo[a]pyrene and benzo[a]fluoranthene) were
detected in fraction 10 at all locations except Opf2006
(Table 1). Consequently, Opf2006 fraction 10 was the only
location that did not elicit an effect in the H295R assay. More
research on the effect of five-ring PAHs within the H295R assay
would be needed to verify that they can cause the observed
results.
Fraction 7 was of particular interest because the hormone
profile differed from that observed for any of the other fractions.
Significant decreases in T compared to control were observed in
three of the four sites. Compounds such as naphthoic acidlike compounds were measured in fraction 7 from all three
sites at which changes in E2 or T were measured. These
results correspond with studies that were performed on oil
sands process water. It was found that oil sands process
water induced E2 concentrations and simultaneously inhibited
testosterone production in H295R cells, which is consistent
with our results [41]. Future work would be needed to assess
the effect of naphthoic acid on the H295R assay and to
demonstrate if the observed effect was caused by the naphthoic
acid alone.
Given that exposure to fractions 1 to 6 did not result in any
changes in E2 or T production at any of the sites studied
indicates that chemicals such as many PCBs, polychlorinated
naphthalenes, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 2,3,7,8tetrachlorodibenzofuran that co-elute with these fractions, can
be ruled out as a cause of the observed effects. Other chemicals
that can be found in fractions 1 to 6 could be alkanes or DDT
and its metabolites; therefore, these compounds are also
unlikely to have caused the observed changes in E2 and
testosterone. This was verified with the parallel conducted
chemical analyses during which no PCBs, PCDDs, or DDT
group compounds could be detected in any of the fractions or
samples.
In general, the addition of the response of the fractions
corresponded well with what was measured in the unfractionated whole extracts of sediments. The one exception was
sample Lau2006 where a less than additive effect of the
fractions was observed. This was possibly because of an
interaction between some of the chemicals within the fractions.
Furthermore, most of the greater E2 production observed in the
unfractionated Sig2006 whole extracts of sediments was
accounted for by fractions 7, 9, 10, 13, and 15 and its toxicity
was driven mostly by plant sterols, PAHs, and naphthoic acid.
However, greater E2 production observed in Opf2006 appeared
to be driven primarily by chemicals present in fractions 7, 14,
Environ. Toxicol. Chem. 31, 2012
1061
15, and 17 (mostly naphthoic acid and sterols). For the less
potent extracts of sediments from Lau2004, most of the greater
E2 production was associated with fractions 10, 14, and 15.
CONCLUSIONS
The present study demonstrated EDA is a useful tool for the
identification of candidate chemicals or chemical groups in
complex environmental matrices such as sediments. Fractions 7
to 15 were consistently more toxic in both the Ames fluctuation
and H295R assays among the four extracts and much of this
toxicity was associated with PAHs, nitro-PAHs, dinitro-PAHs,
sterols, and naphthoic acids. Furthermore, chemicals such as
PCBs, 2,3,7,8-tetrachlorodibenzo-p-dioxin, polychlorinated
naphthalenes, DDTs, and several OPs could be excluded as
causative agents for the observed toxicity because the fractions
containing these compounds did not cause any effects for any of
the samples and were not measured in any fraction. Taken as a
whole, these results show the value of using EDA, which uses
multiple bioassays and fractionation techniques to assess the
toxicity of sediments that covers a variety of different biological
endpoints that are caused by unknown chemicals. Furthermore,
to our knowledge this is the first study successfully using the
recently developed H295R assay within EDA strategies.
Acknowledgement—This study was supported by a personal stipend for a
research stay in Aachen to the first author by the Undergraduate Research and
Opportunity Program of the RWTH Aachen University, financed by the
German Excellence Imitative of the German Research Foundation. We thank
M. Murphy and A. Tompsett for their answers to many questions and U.
Basnayaka and A. Toderian for their help in the laboratory. J. Giesy and M.
Hecker were supported by the Canada Research Chair program. J. Giesy was
in part supported through an At-Large Chair Professorship at the Department
of Biology and Chemistry and State Key Laboratory in Marine Pollution, City
University of Hong Kong, the Einstein Professor Program of the Chinese
Academy of Sciences, and the Distinguished Visiting Professor Program of
King Saud University.
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