Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in
vitro bioassays
Authors
Edita Mazurová1,4, Luděk Bláha1, Zdeněk Ďuriš2, Ivona Horká2, John P. Giesy3, Rita
Triebskorn4,5
Addresses
1- Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk
University, Brno, Czech Republic
2 - Department of Biology and Ecology, University of Ostrava, Ostrava, Czech Republic
3 - National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan
State University, East Lansing, MI, USA
4 - Animal Physiological Ecology, Eberhard-Karls University of Tübingen, Tübingen,
Germany
5 - Steinbeis-Transfer Centre for Ecotoxicology and Ecophysiology, Rottenburg, Germany
Abstract
Toxicity of compounds presented in sediment contaminated by powdered waste coal has been
examined in a set of experiments. Reproduction parameters of amphipod Gammarus fossarum
and prosobranchian snail Potamopyrgus antipodarum were affected after laboratory
exposures to contaminated coal sediments from Karviná mining area, northeastern Czech
Republic. The exposure to sediment extract has resulted in comparable effects on the
fecundity of P. antipodarum as the exposure to complex sediment sample. High dioxin-like
activity and no estrogen receptor mediated effects were determined in sediment extract using
two in vitro bioassays with H4IIE.luc and MVLN cells. Our findings indicate that compounds
of waste coal origin might evoke endocrine disruptive effects both in vitro and in vivo and
they might represent chronic risks to wildlife and humans.
Key words: coal sediments, sediment biotests, in vitro bioassays, AhR, ER, endocrine
disruption
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X
Introduction
The Ostrava-Karviná region has been severely impacted by anthropogenic activities. The
black-coal mining industry has extensively modified landscape, and, as a consequence of
these activities, secondary flooded ponds with waste coal deposits have also been formed.
These ponds display unique environmental habitats (Ďuriš & Horká, 2001) and it is of general
importance to understand and describe their ecological quality and to assess health risks for
wildlife. In particular, an unusually rich population of the narrow-clawed crayfish
Pontastacus leptodyctylus occurred in some of coal-sediment ponds near Karviná, with a high
frequency of intersex specimens occurrence (Ďuriš & Horká, 2001).
Up to now, toxicity studies in the context of mining activities have been dedicated mostly to
the metal toxicity and ground water contamination. Nevertheless, also some special effects such as alterations of endocrine hormonal control - have also been discussed with relevance to
coal-derived organic compounds (Lindsay et al., 1992). We have studied sediments from the
pond Pilňok that has served for powerded waste coal deposition. The sediments from pond
Pilňok have been examined in both in vitro and in vivo studies with attempt (1) to describe
chronic effects on reproduction in two invertebrate models, and (2) to study receptor mediated
effects (aryl-hydrocarbon receptor, AhR, and estrogen receptor, ER) effects in two in vitro
assays. To better characterize compounds that might contribute to the biological responses,
tests have been performed with the complex sediment, crude organic sediment extract
(containing organic fraction) and the extract treated with sulfuric acid (removal of labile
compounds such as PAHs and leaving dominantly persistent organic pollutants such as
PCDDs/DFs, PCBs, OCPs).
Material and methods
The amphipod Gammarus fossarum (Crustacea) and the prosobranchian snail Potamopyrgus
antipodarum (Mollusca) have been used as in vivo models. Adult specimens of G. fossarum
were sampled from a natural population in Southern Germany and acclimatized for 3 weeks in
the laboratory. Water and sediment from the locality, where animals were collected, have
served as medium, control sediment and as the carrier sediment for the sediment extract. The
90 day exposure to control sediment, complex sediment from the pond Pilňok and the 50%
dilution of the Pilňok sediment has been performed. In each of 3 or 4 replicates, 90 animals
were exposed to 8 L medium and 0.5 kg dry sediment weight. The mortality of G. fossarum
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X
was evaluated and the size and growth of new generation of G. fossarum has been
determined. The sediment biotest with parthenogentic females of P. antipodarum was further
used to examine sediment potency to affect animal fecundity. The snails were obtained from
laboratory culture maintained at the department of Aquatic Ecotoxicology, University in
Frankfurt, Germany (Prof. Jörg Oehlmann). We have tested the effects of the complex Pilňok
sediment (3:1 dilution with the control unpolluted sediment), and the corresponding sediment
extract equivalent (extracts were externally dosed to the control sediment). The test has been
performed in two replicates with a total of 90 animals per sediment variant. The number of
embryos per females was determined in 30 females after 2, 5 and 8 weeks.
In vitro dioxin-like effects mediated by aryl hydrocarbon receptor (AhR) and estrogen
receptor (ER)-dependent actitivites were tested in H4IIE-luc and MVLN cell lines,
respectively as described previously (Janošek, J., Hilscherová, K., Bláha, L., and Holoubek, I.
(2006). Environmental xenobiotics and nuclear receptors-Interactions, effects and in vitro
assessment. Toxicology in Vitro 20, 18-37.). Activities of AhR and ER are determined by
transcriptional reporter gene assay using luciferase enzyme in both cell lines (assessment of
luminiscence using lucifering and ATP). The effects of 24h exposures to dilution series of the
crude and sulfuric acid-treated sediment extracts were compared with the potencies of
reference compounds - 2,3,7,8-tetrachloro-dibenzo-p-dioxin, TCDD for AhR- and 17βestradiol for ER-mediated activities.
Results and discussion
The laboratory experiment with G. fossarum has shown that sediment from pond Pilňok has a
strong influence on reproduction (Fig. 1). The overall mortality of adults was high and
surviving fractions were about 40% in control, 31% in diluted Pilňok sample and 20% of
adults in complex undiluted Pilňok sample. Regardless to this observation, the most obvious
effect was enhanced fecundity in Pilňok sediment variants, and highly significant difference
when compared to the control was observed (χ2 (df=1), p<0.001). Moreover, the maturation of
juveniles seemed to be accelerated in the presence of the both Pilňok samples as juveniles
reached maturity only in these experimental variants. The crustaceans possess complicated
endocrine hormonal control and the signalling pathway towards the gonad control is triggered
from neural centres (thoracic ganglia and X-organ). Also hormonal modulation of moulting
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X
hormone and methyl farnesoate (crustaceans’ juvenoid hormone) are involved in the control
of gonad function (Withers, 1992). It has also been shown that sex steroid hormones are
presented in crustaceans (Verslycke et al., 2002) and testosterone (vertebrate male sexual
hormone) was detected in vitro as competitive ligand for the ecdysteroid receptor in Kc cells
assay (Mu & LeBlanc, 2002). Although we cannot conclude what hormonal pathway was
altered in G. fossarum by exposure to Pilňok sediment, our findings clearly demonstrate that
compounds present in the sediment had estrogenic effects on adults as well as on juveniles by
accelerating their maturation. That is also interesting regarding the fact that also a population
of another higher crustacean species, the narrow-clawed crayfish P. leptodactylus, inhabiting
the Pilňok pond up to 2003-04, was unusually rich and dense (Ďuriš & Horká, 2001). It
should be eligible to examine the sediment impact to crayfish fecundity and development.
35
*
number of specimens
30
*
25
20
15
F
10
M
5
juv
0
C
50% Pilňok
100% Pilňok
Fig. 1. Number of survived females (F), males (M) and new born juveniles (juv) of
Gammarus fossarum in three month exposure to control sediment (C), 50% diluted Pilňok
sediment (50% Pilňok) and real Pilňok sediment sample (100% Pilňok). Shown data (mean
and standard deviation) obtained from 4 replicates except for 50% Pilňok, where triplicates
were done. Significance of χ2 (df=1) at p<0.001 marked with star.
The positive effect on fecundity was also observed in snail P. antipodarum. The females
exposed to both complex sediment sample and the organic extract produced significantly
more embryos than females from control variants after 5 weeks exposure (t-test, p<0.05;
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X
Fig.2). However, this effect wasn’t observed after prolonged exposure (8 weeks). The
sediment test with P. antipodarum has been originally developed for studies of estrogenic
potency of real sediment samples (Duft et al., 2003). Similar temporal variability, as observed
in our study, was previously published by (Jobling et al., 2003). The decrease in embryo
number after a long-term exposure might be attributed to the depletion of food and energy
reserves during prolonged exposures. The consistence in responses to both complex sediment
and the sediment extract is a very noteworthy and important observation. This finding
indicates that the effects on reproduction are caused mostly by the organic compounds present
in the sediment extract, and that the bioavailability of these compounds in sediment extract is
well comparable with the real sediment situation. Other compounds present in real sediment
that could negatively affect reproduction were for example cadmium or zinc (Tillmann,
1999). However, the effects of these metals were negligible in our experiments.
Pilňok-sediment
Pilňok-extrakt
% of embryos per female
compared to control
160
**
120
80
40
0
1
2
3
4
5
week of exposure
6
7
8
Fig. 2. Number of embryos per female P. antipodarum examined after 2, 5 and 8 weeks of
exposure to Pilňok sediment or its sediment extract. Data expressed in % to control values,
mean and standard deviation obtained from 30 females examined in weeks 2 and 5, and from
26-30 females examined in week 8. Significance of t-test at p<0.05 marked with star.
Interestingly, the in vitro assay with AhR-responsive cells has shown unusually high AhRligand activity of crude Pilňok sediment extract (TCDD-EQ = 123.4 ng/g dry sediment
weight) that was substantially decreased in samples treated with sulfuric acid (TCDD-EQ =
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X
13.5 ng/g dry sediment weight). These observations imply significant contribution of
polyaromatic hydrocarbons (PAHs) that were removed by treatment with sulfuric acid. The
chemical analyses confirmed high concentrations of PAHs (data not shown) that might reflect
the natural character of the coal. It is well known, that AhR activity and AhR-related effects
(dioxin-like toxicity) are mostly caused by PCDDs/Fs, PCBs as well as other (poly)aromatic
compounds (Hilscherová et al., 2000). Our chemical analyses revealed relatively low
concentrations of PCBs, and hypothetically, also PCDD/Fs (polychlorinated dioxins and
furans - concentrations were not analysed) might be responsible for the AhR-mediated effects
remaining in acid-treated sediment extracts. These results demonstrate that coal is a rich
source of organic compounds that might modulate endocrine balance and cause the effects
related to antiestrogenicity (Hilscherová et al., 2000). Although, the estrogenicity of
environmental samples has been studied extensively, to the best of our knowledge, this is the
first report that focuses on the ER-mediated effects of waste coal. In spite of apparent
reproductive effects in invertebrate in vivo assays (see above), the MVLN cellular bioassay
didn’t show any estrogen receptor-mediated activity in either crude or acid-treated sediment
extract (Tab. 1).
Cell line
H4IIE-luc
MVLN
aryl hydrocarbon
estrogen
TCDD-EQ
E2-EQ
(ng/g dry weight)
(ng/g dry weight)
crude extract
123.4
n.d.
acid-treated extract
13.5
n.d.
transfected receptor
signalling
activity unit
Tab. 1. The results obtained from two in vitro cellular bioassays for AhR- and ER-mediated
effects. The activities of the sediment extracts were compared to the effects of reference
TCDD and 17β-estradiol (E2) and expressed as mass equivalents.
Conclusion
In the present study, the contamination of the sediment by powdered waste coal caused
serious effects in two invertebrate model organisms, the amphipod Gammarus fossarum and
the prosobranchian snail Potamopryrgus antipodarum. In both studied model invertebrate
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X
species, modulations of reproductive parameters were observed. Moreover, high dioxin-like
activity of sediment extract indicates that waste coal might elicit endocrine disruptive effects
in vertebrate system such as anti-estrogenicity. Although AhR wasn’t proven in invertebrates
yet, these animals have shown a wide range of responses to dioxine-like active compounds
(Verslycke et al., 2004). Our laboratory experiments indicate the presence of endocrine
disruptive compounds in studied sediments and they provide further insights into the frequent
occurrence of intersexual specimens in unusually rich population of the narrow-clawed
crayfish Pontastacus leptodactylus (Decapoda, Crustacea) that was previously observed at the
Pilňok locality.
References
Duft, M., Schulte-Oehlmann, U., Weltje, L., Tillmann, M. & Oehlmann, J. (2003): Stimulated
embryo production as a parameter of estrogenic exposure via sediments in the
freshwater mudsnail Potamopyrgus antipodarum. Aquatic Toxicology 64: 437-449.
Ďuriš, Z. & Horká, I. (2001): K výskytu raka bahenního Pontastacus (=Astacus)
leptodactylus Eschscholtz na Karvinsku. Časopis Slezského Muzea 50 (A): 78-84.
Hilscherová, K., Machala, M., Kannan, K., Blankenship, A. L. & Giesy, J. P. (2000): Cell
bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER)
mediated activity in environmental samples. Environmental Science and Pollution
Research 7: 159-171.
Jobling, S., Casey, D., Rodgers-Gray, T., Oehlmann, J., Schulte-Oehlmann, U., Pawlowski,
S., Baunbeck, T., Turner, A. P. & Tyler, C. R. (2003): Comparative responses of
molluscs and fish to environmental estrogens and an estrogenic effluent. Aquatic
Toxicology 65: 205-220.
Lindsay, R. H., Hill, J. B., Gaitan, E., Cooksey, R. C. & Jolley, R. L. (1992): Antithyroid
effects of coal-derived pollutants. Journal of Toxicology & Environmental Health 37:
467-81.
Mu, X. & LeBlanc, G. A. (2002): Developmental toxicity of testosterone in the crustacean
Daphnia magna involves anti-ecdysteroidal activity. General and Comparative
Endocrinology 129: 127-133.
Tillmann, M. (1999): Untersuchungen zum Bau des Genitaltraktes von Viviparus viviparus
sowie zum Fortpflanzungserfolg eines weiteren ovoviviparen Prosobranchiers
(Potamopyrgus antipodarum) unter Schwermetallstreß. Diploma thesis, International
Graduate School (IHI) Zittau.
Verslycke, T., De Wasch, K., De Brabander, H. F. & Janssen, C. R. (2002): Testosterone
metabolism in the estuarine mysid Neomysis integer (Crustacea; Mysidacea):
Identification of testosterone metabolites and endogenous vertebrate-type steroids.
General and Comparative Endocrinology 126: 190-199.
Verslycke, T. A., Fockedey, N., McKenney, C. L., Roast, S. D., Jones, M. B., Mees, J. &
Janssen, C. R. (2004): Mysid crustaceans as potential test organisms for the evaluation
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X
of environmental endocrine disruption: A review. Environmental Toxicology and
Chemistry 23: 1219-1234.
Withers, P., C. (1992). Comparative Animal Physiology. Harcourt Brace Jovanovich College
Publishers, Saunders College Publishing, Fort Worth, Philadelphia, San Diego, New
York, Orlando, Austin, San Antonio, Toronto, Montreal, London, Sydney, Tokyo.
Information about the author
Edita Mazurová
Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX)
Masaryk University
Kamenice 3
625 00 Brno
Czech Republic
emazurova@yahoo.com
Mazurová, E., L. Bláha, K. Hilscherová, Z. Ďuriš, I. Horká, J.P. Giesy, and R. Triebskorn. 2006.
Ecotoxicological evaluation of sediments contaminated by waste coal by in vivo and in vitro bioassays”.
Proceedings of Environmental Changes and Biological Assessment III. Scripta Facultatis Rerum Naturalium
Universitatis Ostraviensis, 163:274-278. ISBN 80-7368-201-X