Assessment of Toxicity of Upper Danube River Sediments Using a Combination of Chemical Fractionation,
the Danio rerio Embryo Assay and the Ames Fluctuation Test
Eric Higley1, Stefanie Grund3, Thomas B.-Seiler2, Urte Lubcke-von Varel6 ,Werner Brack6, Tobias Schulz6, Jan Wölz2, Hanno Zielke2, John Giesy1,5, Henner Hollert2, Markus Hecker4
1. University of Saskatchewan, Saskatoon, SK, 2 RWTH, Aachen, Germany, 3 University of Heidelberg, Heidelberg, Germany, 4 ENTRIX, Inc., Saskatoon, SK, 5 City University of Hong Kong, Hong Kong, China, 6 UFZ Leipzig, Germany.
Sediment Sampling Sites
Introduction
Objectives
Severely contaminated sediments from many rivers and lakes have been shown
to be acutely and chronically toxic to fish and benthic invertebrate species. For
example, sediment samples from the Upper Danube River that were analyzed in
six separate assays were found to have considerable geno-toxic, cytotoxic,
mutagenic, embryo-toxic and estrogenic effects. It has been hypothesized that
decline in fish stocks in the Upper Danube River since the early 1990s may be
associated with this pollution. Here, we report on the results of a study
conducted as a part of a larger study to characterize toxicity of extracts from
sediments of the Danube River by means of Effect Direct Analysis. In this study,
embryo-toxic effects were analyze using the Danio rerio embryo assay, and by
assessing lethal and sub lethal endpoints. In addition, mutagenicity was
assessed using the Ames fluctuation assay. For the sediment samples that
revealed toxicity, fractionation of each sample was performed by separating
compounds according to their polarity, planarity, and the size of the aromatic ring
system. 18 fractions for each sediment sample were tested separately in the
Ames fluctuation assay and Danio rerio embryo assay to assess which group of
chemicals within the sediment sample caused the original toxicity.
1. Assess the toxicity of raw sediment extracts from four locations along the Upper Danube
River using the Zebrafish Embryo Assay and Ames Fluctuation Assay
2. Evaluate which groups of chemicals caused the measured toxicities using new chemical
fractionation techniques that separate the raw sediment extracts into 18 different
chemical fractions.
Results
Mutagenic Effect (# of revertant)
TA98 Bacteria Strain +S9
Lauchert reference
Öpfingen
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
Danio rerio Embryo Assay
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
Lauchert reference
Lauchert
Öpfingen
Sigmaringen
Incubate for 90 minutes with histidine
Viable eggs less
than 1 hour old
are collected
Zebrafish are
breed overnight
Add pH
indicator
media
without
histidine
Place histidine deficient bacteria into 384
well plate without histidine
+
Zebra fish
egg + ISO
water
Incubate for 48 hours
in 96 well plate
Incubate at 37 ° C for 48 hours
After 48 hours, any bacteria that have back
mutated and can produce histidine will live and
grow and turn the media from purple to yellow
Count # of wells
that are yellow
Record
lethal
effects
after 48
hours
*
*
*
12.5
25.0 50.0 100.0 200.0 400.0
Augsburg
Augsburg
2
1
3
Opfingen (3)
(Opf2006)
Lech
Lauchert (tributary), 3 = Opfingen.
according to LFW (2005))
= Sewage treatment plants (> 10,000 residents
***
***
*
50.0 100.0 200.0 400.0
PC
PC
Lauchert reference
Lauchert
Öpfingen
Sigmaringen
**
15.0
*
*
10.0
5.0
0.0
SC
12.5 25.0 50.0 100.0 200.0 400.0
PC
Sediment Equivalent Concentrations (mg/ml)
Figure 2. Dose response of four whole sediment extracts ran in the Ames Fluctuation
Assay with and without the liver enzyme S9 mix and on two different bacteria strains. TA98
bacteria measures frameshift mutations and TA100 bacteria measures base pair
mutations. * indicates significant difference from control.
Figure 4. Mutagenic response of the 18 fractions as measured by the Ames Fluctuation
assay (TA98 and TA100 bacteria with and without S9 bioactivation enzymes).
* indicates significant difference from control
Danio rerio embryo assay on whole extracts
Öpfingen
Sigmaringen
Lauchert
Lauchert Reference
Sediment
sample in
DMSO
Danube River
TA100 Bacteria Strain -S9
*
Sediment Equivalent Concentration (mg/ml)
Bacteria culture
25.0
TA100 Bacteria Strain +S9
SC
Sediment
sample
in DMSO
Lauchert (2)
(Lau2006 and 2004)
Figure 1: Sampling sites along the Upper Danube River; 1 = Sigmaringen, 2 =
***
***
**
70
60
50
40
30
20
10
0
Results and Conclusions
• Mortality of Danio rerio embryos increased in a dose-dependent manner when
exposed to whole sediments collected at Öpfingen and Sigmaringen (Figure 3), but
none of the fractionated samples were toxic. These results indicate that the observed
toxicity was likely due to the combination of groups of chemicals in the whole
sediment samples.
• Toxicity was observed for whole sediments from Sigmaringen, Öpfingen and Lauchert
in the Ames Fluctuation Assay only when TA98 bacteria with S9 were tested (Figure
2). Toxicity was also found in the fractionated samples in both bacterial strains,
although the pattern was inconsistent.
• Fraction 10, which contains PAH’s with five aromatic rings, produced a significant
SC
12.5
25
50
100
mutagenic response in all sediment samples measured only in S9 bio-activated
Sediment equivalents concentration (mg/ml)
samples (Figure 4)
Figure 3. Dose response of four sediment extracts analyzed with the Danio rerio embryo assay • Fraction 15 which contains hydroxyl PAHs produced a significant mutagenic response
in all sediment samples measured only in non bio-activated samples (Figure 4)
Mortality %
Ames Fluctuation Assay
Lauchert
Sigmaringen
***
Mutagenic Effect (# of revertants)
•Crude sediment extracts and all 18 fractions were analyzed for their toxicity using the
Ames fluctuation assay and Danio rerio egg assay
Stuttgart
SWITZERLAND
***
12.5
Ingolstadt
Sediment Equivalent Concentrations (mg/ml)
Mutagenic Effect (# of revertants)
•Samples were extracted and fractionated into different chemical groups using a new
technique by Varel et al., 2008 that uses 3 HPLC columns and separates the sample
into 18 fractions according to their polarity, planarity and the size of their aromatic
system
Danube
Sigmaringen (1)
(Sig2006)
SC
•Sediments were sampled (top 5cm) at four locations along the Upper Danube River
using a Van Veenen grabber in January 2006 (Figure 1)
deficient
Weser
Main
3. Analyze all 18 chemical fractions using the Danio rerio Embryo Assay and Ames
Fluctuation Assay.
Sampling and extraction
+
Elbe
Rhine
Methods
Histidine
Germany