Article

pubs.acs.org/est

Limited Waterborne Acute Toxicity of Native Polycyclic Aromatic

Compounds from Coals of Different Types Compared to Their Total
Hazard Potential
Wiebke Meyer,*,† Thomas-Benjamin Seiler,‡ Mathias Reininghaus,‡ Jan Schwarzbauer,§
Wilhelm Püttmann,∥ Henner Hollert,‡ and Christine Achten*,†
†
University of Münster, Institute of Geology and Palaeontology - Applied Geology, Corrensstrasse 24, 48149 Münster, Germany
‡
RWTH Aachen University, Institute for Environmental Research, Department of Ecosystem Analysis, Worringerweg 1, 52074
Aachen, Germany
§
RWTH Aachen University, Institute of Geology and Geochemistry of Petroleum and Coal, Lochnerstrasse 4-20, 52056 Aachen,
Germany
∥
J.W.-Goethe University Frankfurt am Main, Institute for Atmospheric and Environmental Sciences, Department of Analytical
Environmental Chemistry, Altenhöferallee 1, 60438 Frankfurt/Main, Germany
*
S Supporting Information

ABSTRACT: Coals contain native polycyclic aromatic

compounds (PACs), which include polycyclic aromatic
hydrocarbons (PAHs), and heterocyclic aromatic compounds
(NSO-PACs) in considerably varying amounts up to
2500 mg/kg. Whereas PAC bioavailability and toxicity from
coals are generally considered to be low, few studies have
considered potential variations arising from the composition of
different coal types including native PAC content. In the
present study, fine particles of different coal types exhibiting
variable properties were systematically investigated regarding
their PAC bioavailability. PAH content reached up to 79 mg/
kg EPA-PAH and 865 mg/kg total PAH. Determination of the
toxic potential of extracted PACs in bioassays showed
inhibition of Caenorhabditis elegans reproduction (up to
94%) and increased mortality of Danio rerio embryos (up to 100%) after exposure to extracts from lignite, sub-bituminous,
and bituminous coals. Anthracite extracts showed no effects. Contact assays using whole coal samples revealed no toxicity to D.
rerio embryos in any of the coal samples, suggesting low bioavailability of PACs. In contrast, C. elegans reproduction was inhibited
by direct coal contact; however, the observed toxicity probably resulted from other coal effects. The results suggest that despite
the high toxic potential of PACs present, their bioavailability from different coal types is very limited and independent of coal
properties and native PAH content.

■ INTRODUCTION
Coals contain native polycyclic aromatic compounds (PACs),
which include polycyclic aromatic hydrocarbons (PAHs), and
heterocyclic aromatic compounds (NSO-PACs) that are
generated during diagenetic formation of coal from plant and
microbial/fungal biomass due to prolonged exposure to
elevated temperatures and pressures in the subsurface.1 From
an organic geochemical perspective, the composition of coals
and of present PACs can vary significantly, comparing coals
from different coal basins worldwide. PAH contents of
numerous coal samples have been determined1−7 and reach
up to 165 mg/kg EPA-PAH and about 2500 mg/kg total PAH.
Higher PAH contents were detected in bituminous coals
compared to lower mature coals such as lignite/sub-bituminous
coals and higher mature coals such as anthracite. The PAH
content and composition is assumed to depend on biological
precursor material, geological settings, and maturity.1−3 In
contrast to PAHs and their alkylated derivatives, reports on
NSO-PACs in coals are limited.8−13 Since coal has been used as
an energy source for decades and transported worldwide,
released unburnt coal particles 14−19 and associated
PACs13,20−24 have contaminated soils and sediments and
pose a potential health risk for organisms. Yet, actual risk is
assumed to be generally low. From available literature, it is
concluded that PAH bioavailability from coals is generally very
Received: April 13, 2013
Revised: September 4, 2013
Accepted: September 11, 2013

© XXXX American Chemical Society A dx.doi.org/10.1021/es401609n | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
limited.25−29 However, the general conclusion about PAC
bioavailability from unburnt coal from the available literature
does not sufficiently take into account existing highly variable
coal heterogeneity, although this may have an impact on
bioavailability due to the variable sorption capacity.30 The
majority of previous studies investigating PAH bioavailability
from coals are restricted to only one sample of coal, coal-rich
sediment, or coal dust.31−37 If more samples were studied, coals
were of the same type38,39 or originated from the same
basin40−43 (with the exception of Stahl et al.44,45). In some
studies, the sample was not further specified.32,35,46 In others,
native PAC contents were neither determined nor related to
the obtained results.32,34,44−46 Moreover, the deduction of
generally valid conclusions derived from field studies40,47,48 may
be questioned because the conclusions are limited to the
unique samples and conditions at the distinct sites. To our
knowledge, a comparative study on PAC bioavailability from
different coal types characterized by their PAC contents and of
varying maturity/origin under the same experimental con-
ditions has not been performed so far. This is of special interest
with regard to an expected increase of coal loading and
transportation worldwide,49 which may pose a future risk for
soils and sediments due to accidental spills of coals or dumping
of coal containing wastes.
PAH bioavailability from coal/coal-rich sediment or coal dust
samples has been studied using different approaches: (i)
Investigation of PAC sorption and/or desorption in water,
desorption by supercritical fluid extraction (SFE), or desorption
in digestive juice liquids; (ii) microbiological degradation of
PACs; and (iii) bioaccumulation or toxicity of coal-derived
PACs.
The investigation of PAC concentrations in coal leachates
revealed up to 48 μg/L for an individual compound
(phenanthrene).44 From sorption studies in water or using
SFE, it was concluded that coal acts as a very strong sorbent for
PAHs and other hydrophobic contaminants.36,37,50−55 In the
digestive tract fluid of the deposit feeding polychaete Arenicola
marina, only traces of phenanthrene and pyrene were
desorbed.33
No microbial degradation of PAHs was observed in coal-rich
soil samples, suggesting lacking bioavailability.56
Bioaccumulation studies carried out with fishes31,42 and
oysters32 revealed no significant PAH bioaccumulation
(compared to controls) from individual coal samples containing
up to 1000 mg/kg total PAH.32 Investigations of the toxicity of
coals, coal leachates, and coal dust-containing water samples
were heterogeneously designed and showed ambiguous results:
Some researchers detected mutagenic/clastogenic effects,45
cytotoxicity to human cell lines,39 and induction of cytochrome
P450 activity in fishes,46 while others found no mutagenicity
and only slight cytotoxic effects34 and no cytochrome 450
activity (Carlson et al., reported in Ahrens and Morrisey29). No
response of PAH-sensitive bioreporter bacteria was detected
after exposure to coal samples.40 While Chapman et al.47
concluded low bioavailability of PAHs at an investigated coal
contaminated site, another field study48 observed a correlation
between toxicity to mussels and metal as well as PAH content.
From the various studies, it may be assumed that PACs from
coals are often not available. However, this assumption is based
on results from very different and mostly case-specific studies
on often unspecified single coals which were frequently not
characterized for their PAC content. The aim of the present
study was to investigate whether the assumption of non-
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availability of PAHs from coals can be verified for a set of coals
with varying properties for exemplary organisms which are
exposed via different pathways to coal-bound PACs. For this
purpose, fine particles of coals of different origin, maturity,
geological age, depositional environment, and native PAH
content were compared in terms of their toxicity to two test
organisms, using standardized exposure conditions. The coals
of the sample set, including a lignite, sub- to low volatile
bituminous coals with elevated PAH contents, and an
anthracite sample (high maturity coal) were investigated in a
comparative study under the same conditions.
In a first tier, potential toxicities of the coals were determined
by assays with embryos of the zebrafish Danio rerio and the
nematode Caenorhabditis elegans using the organic extracts of
the coal samples (dissolved PACs). Extract tests are able to
yield the total hazard potential of compounds bound to solid
samples57 due to their full bioavailability (worst-case exposure
scenario). Second, contact assays (whole-sample toxicity tests)
with the same organisms employing the original whole fine coal
particles (PACs in solid phase) were used to study the effects of
bioavailable PACs in coals, which are released under natural
conditions. This approach provides the possibility to compare
the potential toxicity of coal-associated PACs to the effects of
the bioavailable fraction of different coals whereby information
on the actual risk posed by heterogeneous coals are obtained.
■ EXPERIMENTAL SECTION
Coal samples. Eight coal samples with preferentially
increased PAH contents and of varying origin, geological age,
coal type, and depositional environment from coal basins
worldwide were chosen (Table S1, Supporting Information).
Samples of bituminous coal were chosen from Poland (POL-
B), Great Britain (GB-B), and Germany (GE-B). Two different
bituminous coal samples from South Africa (SA-B1, SA-B2)
were selected due to their elevated EPA-PAH contents. As
representatives for low rank coals, a sub-bituminous coal from
Indonesia (IND-SB) and a lignite from Germany (GE-L) were
selected, whereas high rank coal was represented by an
anthracite from Germany (GE-A). The respective coal basins
are characterized by either paralic (both limnic and marine
influence) or only limnic depositional environments. Hence,
the chosen coal samples represent the main different stages of
coal maturity, main different depositional environments, and a
range of different origins, as these properties are relevant for the
PAH content and composition of coals.1,2 All samples were
“run-of-mine” samples (raw coal material before any kind of
processing). For the simulation of a worst-case-scenario
(maximum uptake) and for results independent of the grain
size distribution of each sample, all samples were ground to a
grain size of <200 μm (defined by the used rotor mill ZM 200,
Retsch) before use. The resulting fine coal particles represent a
major dispersed form of coal in the environment.15 Samples
were used for the study according to Figure 1.
Extraction and Fractionation. A total of 30.00 g of
ground coal samples was extracted by accelerated solvent
extraction (ASE 350, Dionex). Extraction was performed at
120 °C and 110 bar in two cycles (7 min static time each) with
dichloromethane (DCM, Rotisolv GC-Ultra grade, Carl Roth).
For the removal of elemental sulfur, activated copper shots
(Alfa Aesar) were added to the extracts overnight. Thereafter, a
SARA (saturates, aromatics, resins, asphaltenes) extract
fractionation was performed: After the removal of asphal-
tenes,58 extracts were separated into three fractions (F1−F3) by
Environmental Science & Technology
Figure 1. Flow diagram describing sample processing procedure.
open column flash-chromatography based on EPA method
3611B59 (for details on asphaltene removal, fractionation, and
recovery of PAHs, see the Supporting Information). The
fractions were subsequently reduced to 3 mL each, and 1 mL
was used for chemical analysis. Prior to analysis, an internal
standard (16 deuterated EPA-PAH, Chiron) was added for
quantification. The remaining 2 mL of the F2 and F3 fractions
was used for extract testing with D. rerio and C. elegans. For this,
extracts were reduced to dryness in a gentle nitrogen stream
and reconstituted in dimethyl sulfoxide (DMSO, analytical
grade, Fluka). Resulting extract concentrations were 10 g of
extracted coal equivalent per milliliter of DMSO. Extracts were
kept at −20 °C in darkness.
PAH Analysis. Gas chromatography−mass spectrometry
was performed using a GCMS-QP-2010Plus system (Shimad-
zu) with a 60 m Optima 5 MS Accent capillary column
(Macherey-Nagel). Quantification was carried out using an
external calibration where the amount of target compounds was
corrected for analytical deviations via the response factor ratios
of target compound peak areas and surrogate standard peak
areas. The following 40 target PAH compounds were analyzed:
naphthalene, 2-methylnaphthalene, acenaphthylene, acenaph-
thene, fluorene, 1-methylfluorene, phenanthrene, anthracene, 9-
methylphenanthrene, 9-methylanthracene, fluoranthene, pyr-
ene, 9,10-dimethylanthracene, 1-methylpyrene, 7H-benzo[c]-
fluorene, benzo[ghi]fluoranthene, benzo[c]phenanthrene,
cyclopenta[cd]pyrene, benz[a]anthracene, chrysene, 6-
methylbenz[a]anthracene, 5-methylchrysene, benzo[b]-
fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene,
7,12-dimethylbenz[a]anthracene, benzo[e]pyrene, benzo[a]-
pyrene, perylene, 3-methylcholanthrene, 6-methylbenzo[a]-
pyrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene, benzo-
[ghi]perylene, anthanthrene, dibenzo[a,e]fluoranthene,
dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,i]pyrene,
and dibenzo[a,h]pyrene (for details concerning applied GC-
MS parameter and detection limits, see the Supporting
Information, Tables S3 and S4). Additionally, a simultaneous
full scan was performed to obtain the content of total PAH. For
this, common C1−C4 alkylated PAHs (naphthalenes, phenan-
threnes/anthracenes, fluorenes, pyrenes/fluoranthenes, chrys-
enes/benzanthracenes, and benzopyrenes/benzofluoranthenes)
were identified by their mass spectra. The peak area of the
intensive fragment ion and a related internal standard
compound (e.g., for alkylated naphthalenes, naphthalene-d8
was used) was used to calculate response factors. For
determination of concentration, a substitute calibration curve
of a replacement standard compound (the corresponding C1 or
C0 alkylated PAH available in the external standard) was used.
Thus, the measurement of alkylated PAH was merely
semiquantitative.
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Selection of Test Organisms. Two test organisms were
chosen because they are exposed to contaminants via two
different pathways: (1) the liquid phase and (2) the liquid and
particulate phase.
The fish embryo toxicity test with the zebra fish Danio rerio is
commonly used for testing of chemicals, water samples,
sediment extracts,60−62 and whole sediments.63−67 The
embryonic membrane (chorion) shields the embryo from
direct contact, which results in exposure only to dissolved
contaminants. PAHs are known to cause severe developmental
effects to D. rerio embryos, mainly malformations and
cardiovascular dysfunctions.68−74
The soil nematode Caenorhabditis elegans is used for
ecotoxicological testing of liquid samples,75 sediments,67,76,77
and soils.78,79 During feeding by pharynx pumping, the
bacterivorous nematode unselectively ingests smaller particles
and is thus exposed to particle-bound contaminants that can
become solubilized and bioavailable by digestive fluids in the
gut.80 Thus, the nematode is exposed to both contaminants
dissolved in pore water and to particle-bound contaminants. It
has been shown that C. elegans survival, growth, and
reproduction is affected by PAHs.79,81−83
Fish Embryo Toxicity Test with Danio rerio. Zebrafish
(Danio rerio, Hamilton-Buchanan 1922) maintenance and
breeding were carried out according to DIN EN ISO 1508884
and Hollert et al.59 For extract testing in liquid medium, F2 and
F3 fractions of coals (in DMSO) were used. Each test vessel
(20 mL glass crystallizing dishes) contained 4995 μL of artificial
water according to OECD 20385 and 5 μL of coal extract (only
the highest extract concentration was tested in the fish embryo
toxicity test). Two test vessels for each coal extract, four vessels
containing reconstituted water as negative controls, two test
vessels containing 3.7 mg/L of 3,4- dichloroaniline (3,4-DCA,
Sigma−Aldrich) as positive controls, and two vessels containing
5 μL of DMSO as solvent controls were prepared.
For the contact assay, coal-water slurries contained 60%
particulate coal (<200 μm) and 40% artificial water according
to OECD 203.85 To each test vessel, 3.0 g of moist coal sample
was transferred, and 5 mL of reconstituted water was added on
top. Four test vessels were prepared for each coal sample; eight
vessels containing reconstituted water and four vessels
containing 3.0 g of pure quartz sand (F36, Quarzwerke
Frechen) were included as negative controls. Four vessels
containing reconstituted water with 3.7 mg/L of 3,4-DCA and
two vessels containing quartz sand spiked with 3.7 mg/L of 3,4-
DCA were included as positive controls. Upon checking the pH
of the overlying water in the test vessels, a value of 3.4 was
found in the sample SA-B1. Calcium carbonate (Sigma-Aldrich)
was added until pH 6.8 was reached (approximately 2% (w/w)
per test vessel).
Fertilized fish eggs of at least four-cell stage and not older
than 128-cell stage were chosen using a stereomicroscope
(Nikon), and five eggs were transferred to each of the prepared
test vessels. They were sealed with laboratory film (Parafilm M,
Brand) and incubated in darkness at 26 ± 1 °C for 48 h while
gently rotating (30 rpm). For test evaluation, fish embryos were
examined with an inverted microscope at 100-fold magnifica-
tion (Nikon). Embryos which were coagulated, lacked a
heartbeat, or whose tail was not detached were considered
dead. Mortality was calculated as percentage of dead embryos.
Three independent assays were carried out. The third
independent assay was omitted, if no significant effect was
observed in the former two.
Environmental Science & Technology
Nematode Bioassay with Caenorhabditis elegans. C.
elegans (var. Bristol, strain N2) maintenance and breeding were
carried out according to ISO 10872.86 For extract testing in a
liquid medium, the fractions F2 and F3 were used. To obtain a
dose−response relationship and EC50 values, a prior serial
dilution of coal extracts in DMSO was performed resulting in
10 g/L, 5 g/L, 2.5 g/L, 1.25 g/L, and 0.625 g/L extracted coal
equivalent per milliliter of DMSO. Thereafter, test vessels
(10 mL glass snap cap vials) containing 499.5 μL of M9
medium84 and 0.5 μL of prediluted coal extract in DMSO were
prepared (three replicate test vessels per concentration).
Negative controls containing M9 medium, solvent controls
containing 0.5 μL DMSO, and positive controls containing 5
mg/L benzylcetyldimethylammonium chloride monohydrate
(BAC-C16) were included. The employed BAC-C16 concen-
tration was derived from the EC50 for reproduction inhibition,
which was estimated before in a preliminary test.
For the contact assay, coal-water slurries contained 60%
ground coal (<200 μm) and 40% M9-medium.86 A total of
0.50 g of coal-water slurry was transferred to 10 mL glass snap-
cap vials. Adjustment of the pH was not necessary due to the
buffering characteristics of the M9-medium (pH was controlled
at the beginning and end of the assay). A natural sediment with
low level anthropogenic contamination from a Rhine back
water (Altrip, Germany)75 was used as a negative control
sample. Additionally, liquid negative controls and positive
controls as described above were included. For each treatment,
three replicate test vessels were prepared.
For C. elegans feeding during both the contact assay and
extract test, 0.5 mL of food medium containing Escherichia coli
(OP 50) with the required optical density according to ISO
1087286 was added to each test vessel. After the introduction of
10 first-stage juvenile nematodes to each test vessel, sealed
vessels were incubated for 96 h at 20 ± 0.5 °C in darkness. The
assay was stopped by heating the test vessels for 15 min at
80 °C. To improve visibility of the nematodes, 1 mL of a
solution of bengal rose (Fluka) was added before heating.
Nematodes were extracted from coal or sediment with a
colloidal silica suspension (Ludox 50, Sigma-Aldrich) according
to ISO 10872.86 Tests were evaluated using a stereomicroscope.
Adult nematodes and offspring were counted followed by a
calculation of inhibition of nematode reproduction (offspring
per adult hermaphrodite) relative to control reproduction from
the negative control sediment for the contact assay and from
solvent control for the extract test. Reproduction as the end
point of the tests was chosen because reproduction is more
sensitive to PAH exposure than growth.81 Three independent
assays were carried out (each with three replicate treatments).
Again, the third independent assay was omitted, if no significant
effect was observed in the former two.
Statistical Analysis. Statistical data evaluation was carried
out using GraphPad Prism 5.01, IBM SPSS Statistics 21.0, and
ToxRat Professional 2.10. To investigate whether the effects
from coal samples or extracts are significantly different from
controls or each other, an analysis of variance (ANOVA)
followed by Dunnett’s or Tukey’s posthoc test was used. If data
were not normally distributed, a Kruskal−Wallis test with
Dunn’s posthoc test was performed. In the case of lacking
homogeneity of variance, a Dunnett-T3 test was performed
after ANOVA. EC50 values for nematode reproduction were
calculated from the dose−response relationship using a Weibull
regression.
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RESULTS AND DISCUSSION
PAH Analysis of Coal Samples. Fractionation of coal
extracts and PAH analysis showed that very high contents of
PAH were present in F2 fractions, and only traces of individual
PAHs occurred in some F1 and F3 fractions. These were added
to the content of F2 fractions. EPA-PAH, Σ40 PAH, and total
PAH contents of coal samples are shown in Figure 2 (for
Figure 2. Content of EPA-PAH, Σ40 PAH, and total PAHa [mg/kg]
in coal samples (sum of all fractions). aTotal PAHs include C1−C4
alkylated PAH, which were determined semiquantitatively.
content of individual PAH compounds, see Table S5,
Supporting Information). As expected, bituminous coals
showed the highest PAH contents: In our sample set, SA-B1
and SA-B2 had the highest contents of EPA-PAH (about
80 mg/kg each) and Σ40 PAH (about 145 mg/kg each), while
GB-B, POL-B, and GE-B contained slightly smaller contents
(about 50−60 mg/kg EPA-PAH and 100−130 mg/kg
Σ40 PAH). The total PAH content was highest in GE-B
(>800 mg/kg) followed by GB-B, POL-B, SA-B1, and SA-B2
(about 400−600 mg/kg). Lower PAH contents were detected
with lower rank: in the sub-bituminous coal IND-SB (6 mg/kg
EPA-PAH, 13 mg/kg Σ40 PAH, and 88 mg/kg total PAH) and
the lignite GE-L (lowest rank, 3.5 mg/kg EPA-PAH, 7 mg/kg
Σ40 PAH, and 17 mg/kg total PAH). The high mature GE-A
sample contained only traces of <1 mg/kg total PAH. The
EPA-PAH content was less than 55% of the Σ40 PAH content
and less than 21% of the total PAH content in all samples
except for the anthracite (80% and 53%, respectively),
indicating that exclusive analysis of the 16 EPA-PAH may
underestimate the overall PAH content of coal samples.
Elevated EPA-PAH and total PAH contents in bituminous
coals are in line with former findings (up to 165 mg/kg EPA-
PAH and 2500 mg/kg total PAH)1,3 as are the lower PAH
concentrations obtained for lignite, sub-bituminous coal, and
anthracite (previous studies found up to 6 mg/kg EPA-PAH
and 14 mg/kg total PAH in lignite/sub-bituminous coals and
up to 4.6 mg/kg EPA-PAH in anthracites1,3).
Strongly carcinogenic dibenzopyrenes87 were most concen-
trated in the bituminous coals (Table S5, Supporting
Information). The measured concentrations ranged between
0.26 and 1.80 mg/kg for dibenzo[a,l]pyrene, between 0.44 and
1.90 mg/kg for dibenzo[a,e]pyrene, between 0.64 and
2.06 mg/kg for dibenzo[a,i]pyrene, and between 0.74 and
1.67 mg/kg for dibenzo[a,h]pyrene. The highest content of the
Environmental Science & Technology
Figure 3. Mortality of D. rerio embryos (a) after 48 h exposure to fractions F2 and F3 of coal extracts in a liquid medium, mean ± SD, n = 3 (n:
independent assays, each with 10 embryos), extract concentration: 10 mg coal equivalent per mL medium, and (b) after 48 h exposure to whole coal
samples (100% coal as substrate) in contact assays, mean ± SD, n = 2 (n: independent assays, each with 20 embryos).
sum of the four isomers (7.52 mg/kg) was detected in the
sample SA-B1. No dibenzopyrenes were detected in the
anthracite or lignite, and only a low content was detected in
the sub-bituminous coal (0.05 mg/kg of dibenzo[a,l]pyrene).
PAH patterns of the bituminous coals exhibit characteristic
petrogenic bell-shaped distributions of C0−C4 alkylated
naphthalenes, phenanthrenes/anthracenes, pyrenes/fluoran-
thenes, and chrysenes/benzanthracenes (Figure S1, Supporting
Information). Although the anthracite sample (GE-A) con-
tained only traces of PAH, these compounds reveal a typical
slope-shape distribution of alkylated phenanthrenes/anthra-
cenes and pyrenes/fluoranthenes (indicating pyrogenic origin).
This distribution was also observed in anthracites in a former
study.2 The PAH pattern of the sub-bituminous coal (IND-SB)
is dominated by naphthalenes and their alkylated derivatives,
which is in accordance with former findings1 whereby C4
alkylated naphthalenes show the highest contents. Except for
the notable dominance of C4 alkylated phenanthrenes/
anthracenes, the lowest rank coal of the sample set, the lignite
GE-L, exhibited no characteristic PAH pattern. In contrast to
most other PAHs, acenaphthylene, 9,10-dimethylanthracene,
7,12-dimethylbenz[a]anthracene, and cyclopenta[cd]pyrene
showed the highest contents in lignite, sub-bituminous coal,
and anthracite but not bituminous coals. Among the
bituminous coals of the sample set, limnic coals tended to
higher contents of higher molecular PAHs compared to paralic
coals.
Fish Embryo Toxicity Test with Danio rerio. The results
from the fish embryo toxicity test show a clear difference
between extract testing and contact assay (Figure 3). The coal
extracts induced severe effects in D. rerio embryos (Figure 3a).
The PAH-containing F2 fractions of the bituminous coals and
GE-L led to 100% mortality in fish embryos, whereas 56.7 ±
41.6% mortality was observed in IND-SB. Mortality was highly
increased in coal extracts compared to the negative control and
solvent control, where mortality was only 3.3 ± 1.4% and 3.3 ±
5.8%, respectively. In contrast, the exposure of fish embryos to
ground coal samples as a substrate did not lead to any lethal or
teratogenic effects (Figure 3b). The coal samples GE-L, SA-B1,
SA-B2, and POL-B produced no mortality at all. The results of
the contact assays containing whole coal samples compared to
the extract tests clearly suggest that despite a very high toxic
potential of the compounds in all coals (except for anthracite),
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no bioavailability leading to acute mortality or teratogenicity in
D. rerio embryos is concluded. The findings of the extract tests
are in accordance with the expectations derived from the PAH
analysis of these samples: In the present study, EPA-PAH,
Σ40 PAH, and total PAH concentrations in the treatments
where mortality was observed ranged from 35 to 789, 71 to
1479, and 170 to 8647 μg/L (nominal). Closer inspection of
the fish embryos further revealed the lower toxic potential of
the F2 extract of IND-SB. While the exposure of embryos to
the F2 fractions of the bituminous coals and GE-L caused
coagulation of nearly all embryos, even the embryos exposed to
the F2 of IND-SB which were considered dead had generally
further progressed in their development. Here, the recorded
lethal end point was mostly a lacking heartbeat instead of
coagulation of embryos. Pericardial edema and bradycardia, as
observed here in surviving embryos in the sample IND-SB, are
a previously reported consequence of PAH exposure.71
The finding of high zebra fish mortality for the lignite F2
extract was contrary to expectations, given the lower PAH
content of the coal extract, but could be due to the presence of
a few specific, highly toxic PAHs. Notably, the full scan of the
lignite sample GE-L showed highly intensive peaks of m/z 274
and m/z 324. The content of these compounds was >400 mg/
kg (semiquantitative estimation). Compounds of m/z 274 and
324 were observed before in coals13,88,89 and would be worth
studying in detail, particularly for their toxic potential. The F2
fraction of the anthracite sample (GE-A) led only to 6.7 ± 5.8%
mortality (no significant difference from controls, Dunn’s
multiple comparison p > 0.05), which is in line with the
negligible PAH content (<1 mg/kg total PAH) of this sample.
The concentration ranges where mortality was observed
correspond to former studies. Carls et al.68 observed
embryotoxic effects (cardiac dysfunctions/malformations) in
zebrafish after exposure to mechanically dispersed oil at an
estimated dissolved PAH concentration of 350 μg/L (sum of
44 individual 2−5 ring PAHs). Incardona et al.72 investigated
the mode of action of different individual PAH compounds and
reported various malformations and cardiovascular dysfunctions
at individual PAH concentrations of >10 000 μg/L. They
deduce from this72 and former studies69−71 that the mode of
action is distinct for different PAHs and conclude that toxic
effects from complex PAH mixtures are induced via different
pathways. Notably, in the fish embryo toxicity test, the more
Environmental Science & Technology
Figure 4. Inhibition of C. elegans reproduction (a) after 96 h exposure to fractions F2 and F3 of coal extracts in liquid medium, mean ± SD, F2: n =
3, F3: n = 2 (n: independent assays, each with three replicates), 5 mg of coal equivalent per mL medium. *Significant difference of reproduction
(juveniles per adult) from solvent control and (b) after 96 h exposure to whole coal samples (100% coal as substrate) in contact assays, mean ± SD,
n = 3 (n: independent assays, each with three replicates).
polar F3 fraction of all coal extracts caused 100% mortality
except for with the anthracite. The zebrafish embryo is also
susceptible to the more polar compounds of the coal extract,
which indicates that this fraction of coal-derived compounds,
which can contain NSO-PACs, can have severe impacts on
organisms. The toxicity of NSO-PACs was observed before,
where it was reported that 4-azapyrene induced severe
malformations in D. rerio embryos after 120 h of exposure at
a concentration of 120 μg/L.73 Additionally, Peddinghaus et
al.90 noted nominal EC50 values for mortality after 48 h of
660−17 000 μg/L for individual NSO-PAC compounds. It may
be of general interest to identify the compounds present in the
F3 fractions of coal extracts because they possibly also occur in
other environmental matrices.
Nematode Bioassay with Caenorhabditis elegans. In
the extract test with C. elegans, the PAH containing F2 fraction
of extracts from the bituminous coals showed inhibition of
reproduction from 69 ± 16% to 94 ± 7% (Figure 4a), which
corresponds with the high PAH contents detected in these
fractions of coal extracts. The F2 fractions of GE-L and IND-SB
showed comparatively low inhibition (10 ± 8% and 12 ± 9%,
respectively, no significant difference to control reproduction,
Dunnett’s multiple comparison, p > 0.05), and the anthracite
extract was characterized by a complete lack of any effect.
These findings are supported by the PAH contents of the coals,
which revealed much smaller contents in these samples.
EC50 values for inhibition of C. elegans reproduction by PAHs
were calculated using the nominal PAH content. EC50 values of
the F2 fractions of the bituminous coals ranged from 45 to
84 μg/L for EPA-PAH, 101 to 165 μg/L for Σ40 PAH, and 432
to 781 μg/L for total PAH. The EC50 values obtained from the
different coals of the sample set are on the same order of
magnitude; slight differences may result from the different PAH
compositions in coal extracts.
The observed toxicity values for C. elegans are also within the
range of reported literature data. However, it has to be noted
that there are no data available concerning the effects on
reproduction after exposure to complex PAH mixtures like coal
or sediment extracts. Researchers mainly used single PAH
compounds81,82 or mixtures containing up to seven PAHs.83
Menzel et al.82 reported an EC50 of 899 μg/L for C. elegans
reproduction after 96 h of exposure to fluoranthene in aquatic
F dx.doi.org/10.1021/es401609n | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
media. Sese et al.81 investigated the inhibition of reproduction
after 72 h of exposure to individual PAHs (acenaphthene,
phenanthrene, anthracene fluoranthene, pyrene, and benzo[a]-
pyrene) and determined EC50 values ranging from 59 to
26 862 μg/L. Liuzzi et al.83 observed reduced survival after 72 h
of exposure to a mixture of 4−6-ring PAHs (benz[a]-
anthracene, benzo[b+j]fluoranthene, benzo[k]fluoranthene,
benzo[a]pyrene, indeno[1,2,3-cd]pyrene, benzo[ghi]perylene,
and dibenz[a,h]anthracene (0.7 μg/L each) at 4.9 μg/L.
In contrast to the fish embryo tests, the F3 fraction of all coal
extracts led to no significant inhibition of C. elegans
reproduction. Inhibition reached up to 8 ± 15% (Figure 4a)
and was independent of coal type. For the F3 fraction, after two
independent assays showed no significant deviation of
reproduction from the solvent control resulting from all coal
extractscalculated with replicates within the independent
assaysno third independent assay was performed. The F3
fractions of all coal extracts led to no adverse effects in C.
elegans reproduction. Thus, C. elegans reproduction seems not
to be inhibited by the more polar, possibly NSO-PAC
containing fraction of coal extracts at the tested concentrations.
It was concluded before that C. elegans is less sensitive to N-
heterocyclic compounds in aquatic toxicity testing (LC50
between 8100 and 131 300 μg/L after 48 h of exposure to
quinoline, acridine, phenazine, or 1,10-phenanthroline).91
Using whole coal samples as a substrate for C. elegans in the
contact assay, reproduction was highly reduced by all
investigated coal samples (Figure 4b) compared to the negative
controls in M9 and Altrip sediment (reproduction inhibition
was calculated relative to Altrip control). Regarding bituminous
coal samples and GE-A, inhibition of reproduction nearly
reached 100% (94 ± 8% to 97 ± 2%). The inhibition of
reproduction by GE-L and IND-SB was slightly lower (83 ±
5% in GE-1 and 80 ± 15% in IND-SB). According to statistical
evaluation, this slightly lower inhibition was not significantly
different from the other coal samples (Tukey’s multiple
comparisons, p > 0.05). However, the lower PAH content of
the two low mature coals compared to bituminous coals
(Figure 2 and Table S5) may lead to the conclusion that the
lower reproduction inhibition was a result of lower PAH
content. Yet, this assumption is not supported by the results of
the anthracite sample (GE-A). This coal has by far the lowest
Environmental Science & Technology
PAH content (<1 mg/kg) and led to an inhibition of
reproduction similar to that resulting from the high PAH-
containing bituminous coals. The lacking inhibition resulting
from fraction F2 of GE-L, IND-SB, and GE-A in the extract test
further supports that the PAHs are not responsible for the
inhibition in the contact assay. The extract test results show
that the low PAH contents of GE-A, GE-L, and IND-SB, even if
fully available, did not inhibit reproduction as severely as
displayed in the contact assay. The strong inhibitory effect of
whole coal samples on C. elegans reproduction might, thus, be
related to toxic properties of coal other than PACs
(summarized by Ahrens and Morrisey).29
The results of this study suggest that despite a partly very
high toxic potential of PACs in the different investigated coals,
there was no bioavailability of PACs leading to acute mortality/
teratogenicity in D. rerio embryos. PACs were not desorbed
from the coal particles into the water to an extent that induces
significant adverse effects, which corresponds to former findings
of a strong sorption capacity of coals.36,37,50−55 This indicates
that PACs from fine coal particles are poorly available to
organisms which are exposed solely to dissolved contaminants.
With regard to this exposure pathway, we have shown that the
low apparent availability of PACs is independent of native PAH
content, origin, maturity, geological age, or depositional
environment of the coals. In contrast, from the C. elegans
test, where the nematode is additionally exposed to particle-
bound contaminants via the digestive pathway, no conclusion
about PAC availability could be drawn because other effects
resulting from particles or inorganic substances dominated.
However, a severe inhibition of nematode reproduction in the
presence of fine coal particles was observed. To further
investigate if there is any bioavailability of PAC from coals via
the digestive pathway, a bioaccumulation test with the deposit-
feeding oligochaete Lumbriculus variegatus is currently being
performed.
This study further revealed that the more polar fractions of
coal extracts (which likely contain NSO-PAC) are toxic for
zebrafish embryos. This is in accordance with other studies
which recognized NSO-PAC as an environmental risk.90,92−94
Nevertheless, their bioavailability from unburnt coal particles
has received less attention. Finally, the contents of the polar F3
fractions and the PAH with m/z 274 and m/z 324 which were
present in the lignite warrant further study regarding their
identity, toxicity, and bioavailability.
■
ASSOCIATED CONTENT
*
S Supporting Information
Description of asphaltene removal and extract fractionation,
Tables S1−S5, and Figure S1. This information is available free
of charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Authors
*Phone: +49 251 8336170. Fax: +49 251 8333933. E-mail:
achten@uni-muenster.de
*Phone: +49 251 8336172. Fax: +49 251 8333933. E-mail:
wiebke.meyer@uni-muenster.de
Notes
The authors declare no competing financial interest.
G dx.doi.org/10.1021/es401609n | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
■
Article
ACKNOWLEDGMENTS
We kindly acknowledge Dr. Sebastian Höss, ECOSSA, for
providing C. elegans and E. coli strains and Dr. Ing. P. Goerke-
Mallet, RAG Anthrazit Ibbenbüren GmbH, for providing coal
samples. Resources for carrying out this project came from the
University of Münster.
■
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