Article

Integrating the fish embryo toxicity test as triad element for sediment toxicity assessment based on the Water Framework Directive approach

Helmholtz Centre for Environmental Research, UFZ Department Bioanalytical Ecotoxicology Permoserstr. 15 04318 Leipzig Germany
Journal of Soils and Sediments (Impact Factor: 2.14). 04/2010; 10(3):389-399. DOI: 10.1007/s11368-009-0170-1
ABSTRACT
Purpose
The objective of this study was to complement analyses according to the European Union Water Framework Directive (WFD) with a sediment toxicity analysis as part of an integrated river assessment. To this end, Hessian water courses were analyzed using the sediment quality triad concept according to Chapman with chemical analyses, in situ effect evaluations, and ecotoxicological assessments. For the ecotoxicological assessment (fish embryo toxicity test with Danio rerio), a new evaluation scheme was developed, the fish teratogenicity index (FTI), that allows for a classification of sediments into ecological quality classes compliant to the WFD.
Materials and methods
Sediment and macrozoobenthos samples were taken from tributaries of the rivers Fulda and Lahn. Sediments were characterized regarding particle size, carbon, heavy metals, and polyaromatic hydrocarbon content. Macroinvertebrate samples were taken via multi-habitat sampling. The fish embryo toxicity test with D. rerio was conducted as a contact assay on the basis of DIN 38415-6.
Results and discussion
The integrated assessment indicated a significant influence of heavy metals and carbon content on macroinvertebrate communities. The bioaccessibility of sediment pollutants were clearly demonstrated by the FTI, which showed a wide range of adverse effects. A significant linear relationship between metals and the FTI was detected. However, there was no statistically significant evidence that macroinvertebrate communities were affected by the hydromorphological quality elements at the sampling sites.
Conclusions
The new scheme for the assessment of fish embryo toxicity test was successfully applied. The results suggest that sediment compounds impact macroinvertebrate communities and early development of fish. It demonstrates that the quality of sediments should be evaluated on a routine basis as part of an integrated river assessment.

Full-text

Available from: Jörg Oehlmann
SEDIMENTS, SEC 2 SEDIMENT RISK MANAGEMENT AND COMMUNICATION RESEARCH ARTICLE
Integrating the fish embryo toxicity test as triad element
for sediment toxicity assessment based on the Water
Framework Directive approach
Mariana Bartzke & Vera Delov &
Petra Stahlschmidt-Allner & Bernhard Allner &
Jörg Oehlmann
Received: 24 July 2009 /Accepted: 10 December 2009 /Published online: 21 January 2010
#
Springer-Verlag 2010
Abstract
Purpose The objective of this study was to complement
analyses according to the European Union Water Frame-
work Directive (WFD) with a sediment toxicity analysis as
part of an integrated river assessment. To this end, Hessian
water courses were analyzed using the sediment quality
triad concept according to Chapman with chemical analy-
ses, in situ effect evaluations, and ecotox icological assess-
ments. For the ecotoxicological assessment (fish embryo
toxicity test with Danio rerio), a new evaluation schem e
was developed, the fish teratogenicity index (FTI), that
allows for a classification of sediments into ecological
quality classes compliant to the WFD.
Materials and methods Sediment and macrozoobenthos
samples were taken from tributaries of the rivers Fulda and
Lahn. Sediments were characterize d regarding particle size,
carbon, heavy metals, and polyaromatic hydrocarbon content.
Macroinvertebrate samples were taken via multi-habitat
sampling. The fish embryo toxicity test with D. rerio was
conducted as a contact assay on the basis of DIN 38415-6.
Results and discussion The integrated assessment indicated
a significant influence of heavy metals and carbon content
on macroinvertebrate communities. The bioaccessibility of
sediment pollutants were clearly demonstrated by the FTI,
which showed a wide range of adverse effects. A
significant linear relationship between metals and the FTI
was detected. However, there was no stat istically significant
evidence that macroinvertebrate communities were affected
by the hydromorphological quality elements at the sam-
pling sites.
Conclusions The new scheme for the assessment of fish
embryo toxicity test was succe ssfully applied. The results
suggest that s ediment compounds impact macroinverte-
brate commu ni ties an d ear ly de velo pm ent o f fish. It
demonstrates that the quality of sediments should be
evaluated on a routine basis as part of an integrated river
assessment.
Keywords Bioassay
.
Danio rerio
.
Fish teratogenicity
index
.
Macroinvertebrates
.
Metals
.
Sediment toxicity
Responsible editor: Marc Babut
Mariana Bartzke and Vera Delov contributed equally to this article.
Electronic supplementary material The online version of this article
(doi:10.1007/s11368-009-0170-1) contains supplementary material,
which is available to authorized users.
M. Bartzke (*)
:
V. Delov
:
J. Oehlmann
Department Aquatic Ecotoxicology,
Goethe University Frankfurt am Main,
Siesmayerstr. 70,
60054 Frankfurt, Germany
e-mail: mariana.bartzke@ufz.de
M. Bartzke
:
V. Delov
:
P. Stahlschmidt-Allner
:
B. Allner
Gobio GmbH,
Scheidertalstr. 69a,
65326 Aarbergen/Kettenbach, Germany
Present Address:
M. Bartzke
Department Bioanalytical Ecotoxicology,
Helmholtz Centre for Environmental Research, UFZ,
Permoserstr. 15,
04318 Leipzig, Germany
Present Address:
V. Delov
Ecotoxicology, Fraunhofer Institute for
Molecular Biology and Applied Ecology IME,
Forckenbeckstrasse 6,
52074 Aachen, Germany
e-mail: vera.delov@rwth-aachen.de
J Soils Sediments (2010) 10:389399
DOI 10.1007/s11368-009-0170-1
Page 1
1 Introduction
The aim of the European Water Framework Directive
(WFD) is a good ecological and chemical status for all
European surface waters by 2015. Furthermore, the WFD
regulates the use of standard methods to monitor the quality
of water courses Europe-wide (European Union 2000).
According to recent investigations commissioned by the
Hessian State Agency for the Environment and Geology
(Völker and Borchardt 2007), hydromorphological distur-
bance of water courses is the main cause for not meeting
the t arget of a good ecological s tatus. However, the
sediment quality and ecotoxicological effects of sediment-
borne contaminants are currently not considered for an
ecological assessment of water courses according to the
WFD. A range of e nvi ronmental pollutants is removed
from the water phase by adsorption to suspended
particulat e matt er an d solid s (Rie ß et al. 1997). H ence,
sediments can se rve as a sink an d source f or contaminan ts
and may affect aquatic organisms. The sediment toxicity
due to adsorbed substances is mostly underestimated
(Hollert et al. 2003; Ulrich et al. 2002). Therefore, this
study aimed at analyzing the impact of sediments on
biological communities by means of an integrated river
assessmen t. Twenty-three Hessian br ooks and ri vers of
low mountain ranges in the catchments of the rivers Fulda
and Lahn were surveyed. The investigation was based on
the sediment quality triad accor di ng to Chap man (19 9 0)to
detect chemical pollutants, in situ effects, and ecotoxico-
logical impacts.
With regard to chemical pollutants, the metal and
polyaromatic hydrocarbon (PAH) contents of sediment s
were analyzed. Metals can be introduced into water courses
by sewage treatment plant effluents, fertilizers, or landfill
leachate. PAHs are mainly produced by coalification. When
taken up by organisms, they can be subject to a metabolic
activation with genotoxic epoxides appearing (Slaga et al.
2004). In situ effects were analyzed by an assessment of
macroinvertebrate communities. Invertebrates live on the
sediment and are the main food source for higher trophic
levels. Hence, any reduction or change of the benthos
coenosis may have far-reaching consequences for the
ecosystem (Chapman 1990).
To detect ecotoxicological impacts, the fish embryo
toxicity test with Danio rerio was applied, and a new
assessment system for the evaluation of the results was
developed. The evaluation scheme reveals not only the
number of effected embryos as purposed by recent studies
(Braunbeck et al. 2005; Hollert et al. 2003; Nagel 2002) but
also weights the effects according to thei r intensity. Hereby,
the test results are arranged into ecological quality classes.
This allows for a comparison with test results of river
assessment systems as defined in the EU WFD. For a
comprehensive interpretation of the results, hyd romorpho-
logical quality elements at the sampling sites and of the
catchment areas were analyzed as well as the organic
content and particle size of sediments.
2 Materials and methods
2.1 Sampling sites
Sediment samples were taken from 12 siliceous brooks of
the river Fulda catchment and from 11 tributaries of the
river Lahn catchment (Table 1). Both areas are situated in
Hessian low mountain ranges. The brooks of the Fulda
catchment are typified as small fine substrate dominated
siliceous highland rivers (type 5.1), according to the
categories of Pottgiesser and Sommerhäuser (2004 ), and
the rivers of the Lahn catchment as mid-sized fine to
coarse substrate dominated siliceous highland rivers (type
9) and large highland rivers (type 9.2).
The reference sediment sample was taken from the brook
Elbrighäuserbach. This is typified as small coarse substrate
dominated siliceous highland river
and serves as a
reference for the EU WFD macroinvertebrate monitoring
for stream type 5 (HMULV 2007a).
2.2 Classification of the hydrom orphological quality
Data of the hydromorphological quality were taken from
the GESIS information system (HMULV 2007c). The
assessment is based on a habitat-specific classification of
riverbeds, riverbanks, and surrounding areas. The quality
classes 1 (not modified) to 7 (completely modified) are
defined though comparison to pristine water courses with
no or negligible anthropogenic impact as references.
According to the Hessian Ministry of Agriculture and
Forests (2000), the quality target for surface waters in
Hesse is class 3 (moderately modified). In order to detect a
potential additional anthropogenic influence, the hydro-
morphological quality in the catchment area was assessed
also. Therefore, the whole catchment area was partitioned
in 100-m sections. River sections with a class worse than
class 3 were determined as disturbed. The percentage of
river sections with hydromorphological disturbances was
calculated. Thes e data were received from HMULV
(2007b).
2.3 Invertebrate sampling
The invertebrate sampling and assessment was performed
according to Meier et al. (2006b). Brooks were sampled in
April 2007 and rivers in July 20 07. Based on the
proportional representation of the bottom substrates at the
390 J Soils Sediments (2010) 10:389399
Page 2
sampling site, a qualitative multi-habitat composite sample
within a 100-m stream reach was taken for all water
courses. Twenty subsamples per samp ling site were taken
by kicksampling with a Surber sampler (25×25 cm, 500-µm
mesh net) and preserved in 96% ethanol.
In the laboratory, a subsample of one sixth of the
main sample was taken which included at least 350
macroinvertebrates. Sorted specimens were counted and
separated into taxonomic groups and preserved in
separate vials. The level of taxonomic identification
refers to the operational taxa list for German rivers
(AQEM 2006). For calculating the ecological status of
rivers based on the benthic invertebrate community, the
software Perlodes was used, a component of the European
assessment system Asterics (version 3.1.1, 2008). The
output of t he assessment system is a multim etric index
based on the autecology of the observed taxa. The index
was developed to assess the impact of hydromorpholog-
ical degradation ( Lorenz et al. 2004). It combines sev er al
individual biotic matrices and integrates multiple attrib-
utes of stream communities. Thus, it is useful to evaluate
the ecological status of running waters and i s able t o
detect changes in biota along a gradient of different
stressors (Ofenböck et al. 2004). For the evaluation of
the ecological status, the index is cl assified into quality
classes (class 1, goodclass 5, bad) according to the EU
WFD (European Union 2000), which indicate the anthro-
pogenic burden of the ecosystem.
2.4 Sediment sampling
Twenty sediment samples were taken along the same
100-m stret ch at each sampling site, which was also
investigated during the macroinvertebrate assessment.
The sediment s amples were collected in a bucket using
a small stainless steel shovel and were homogenized. The
samples w ere th en fr oz en at 20°C. Hence, a modifica-
tion of sediment pollutants was minimized, and a
potential impact of the natural sediment infauna was
excluded.
2.5 Chemical analysis
The freeze-dried sediments were used to determine heavy
metals and PAHs. The heavy metal analyses was conducted
with ICP-MS and ICP-OES according to DIN 38406 E29
(1996). Thereby, the sediment contents of Cd, Co, Cr, Cu,
Fe, Mn, Mo, Ni, Pb, and Zn were determined. Based on the
Table 1 Sampling sites of tributaries of the rivers Fulda and Lahn in Germany
Name of sampling site Abbreviation y-Coordinate x-Coordinate Hydromorphological quality class
Untere Haune H1 5634576 3550935 4
Haune/Hünefeld H2 5610747 3559578 4
Steinbach/Haunetal H3 5620600 3551221 3
Eubach F1 5659224 3542875 6
Wichte F2 5658519 3542066 5
Pfieffe F3 5664200 3539522 5
Kehrenbach F4 5666213 3539784 5
Grenff G1 5638085 3519295 6
Buchbach G2 5635640 3525506 4
Oberer Grenzebach G3 5642738 3521237 4
Wälze-Bach S1 5659542 3509911 4
Riedwiesengraben S2 5663213 3526259 5
Lahn/Biedenkopf La1 5643395 3464987 6
Lahn/Cölbe La2 5635520 3485400 4
Lahn/Gießen La3 5608475 3475825 5
Lahn/Gießen La4 5604014 3474500 6
Untere Dill Di 5605200 3462560 5
Lahn/Solms La5 5601450 3459600 5
Lahn/Biskirchen La6 5599299 3451560 4
Untere Weil We 5591819 3449824 4
Lahn/Weilburg La7 5593504 3446798 6
Lahn/Limburg La8 5584737 3431836 6
Lahn/Limburg La9 5583700 3429950 6
J Soils Sediments (2010) 10:389399 391
Page 3
contents of Cd, Cr, Cu, Ni, Pb, and Zn, the sediments were
arranged in quality classes IIV according to Wachs (1991);
Table 2). The final quality class was determined by the
metal with the worst class.
The PAHs (16 priority pollutants of the US-EPA) were
analyzed with high-performance liquid chromatography
according to DIN 38414 S21 (1996). Pursuant to Rieß et
al. (1997), the PAH assessment of sediments was based on
the benzo(a)pyrene and fluoranthene content. The quality
target for fluoranthene was 250µg/kg and for benzo(a)
pyrene, 180µg/kg. These quality targets were used to
calculate q uality classes according to LAWA (1998);
Table 3), with the worse class for both substances
determining the quality class of the analyzed sediment for
PAHs.
2.6 Fish embryo toxicity test
To investigate the toxicity of native sediment samples
on vertebrates, the embryo toxicity test with the zebra-
fish (D. rerio, HamiltonBuchanan 1922) was performed
as a contact assay based on DIN 38415-6 (2001); cf.
below).
2.6.1 Maintenance and egg production of zebrafish
Sexually mature zebrafish (D. rerio), aged 6 to 12 months,
were kept in groups of up to 30 specimens in 30-L tanks
under a 16:8 h light:dark ph otoperiod. Fish were fed once
daily with a commercial diet (TetraMin flakes) and once
daily with Ar temia salina nauplii.
To collect freshly fertilized fish eggs, glass dishes
covered with a stainless steel mesh with a mesh size of
1.25 mm were placed on the bottom of the breeding tanks
30 min before photoperiod started in the morning. Artificial
green plastic plant imitates on the steel mesh were used as
spawning stimulants. Spawning and fertilization took place
within 30 min after the light had been turned on in the
morning (Hollert et al. 2003; Nagel 2002). Afterwards,
glass dishes were removed, and eggs were transferred into
petri dishes with reconstituted water.
2.6.2 Preparing the sediments
Sediment samples were prepared 12 h before starting the
test. For this purpose, each well of a 24-well microtiter
plate was filled up with 250 mg defrosted native
sediment, covered with 1,750µL reconstituted water.
The microti ter plates were sealed with adhesive film
and kept at 4°C. The plates were heated to 26°C directly
before starting the test.
2.6.3 Exposure
Not later than 30 min post fertilization, viable eggs were
individually selected using an inverse microscope and
subsequently transferred into the sediment-containing
cavities of the 24-well plates. Ten replicates per sediment
sample were used for each run. 3,4-dichloraniline solution
was applied as positive control at a final test concentration
of 3.7 mg/L. Reconstituted water (standard water according
to DIN 38415-6) and the native reference sediment from the
Elbrighäuserbach served as negative control. All sediment
samples were tested three times (n=30).
The microtiter plates were incubated at 26°C for 48 h.
After 48 h, the eggs were transferred into separate petri
dishes, filled with reconstituted water, and incubated at
26°C for further 96 h. Embryos were inspected after 24, 48,
72, and 144 h using an inverse microscope. The following
endpoints were noted during the inspections: egg coagula-
tion, lack of somite development, no tail detachment, lack
Quality
class
Degree of burden
I No or very low
burden
III Low burden
II Moderate burden
IIIII Critical burden
III Enhanced burden
IIIIV Very great burden
IV Excessive burden
Table 2 Characterization of the
quality classes according to
Wachs (1991)
Quality class Degree of burden Calculation
I Unpolluted Geogenic background value or 0
III Very low burden Until half of the quality target value
II Moderate burden Until the quality target value
IIIII Significant burden Until double of the quality target value
III Enhanced burden Until fourfold of the quality target value
IIIIV Great burden Until eightfold of the quality target value
IV Very great burden Higher than eightfold of the quality target value
Table 3 Characterization of the
quality classes according to
LAWA 1998)
392 J Soils Sediments (2010) 10:389399
Page 4
of heartbeat and blood circulation, gross morphological
malformations, edema, growth retardation, and decreased or
missing pigmentation. A test was considered as valid if
more than 90% of the control embryos survived without
abnormalities.
2.6.4 Toxicity evaluation
For the interpretation of results, the test parameters were
classified in lethal, sublethal, and other effects (Table 4).
For this purpose, each embryo was assigned only the worst
singular effect. The arrangement of parameters is based on
the observations which have been made during this study.
To classify the parameters into ecological quality classes
analog to the EU WFD, a fish teratogenicity index (FTI)
was developed. Here, the different effects were weighted
according to the effect intensity as follows:
FTI ¼
E
other
1 þ E
subl:
2 þ E
lethal
3
N
ð1Þ
FTI Fish teratogenicity index
E
other
Number of embryos with other effects
E
subl.
Number of embryos with sublethal effects
E
lethal
Number of embryos with lethal effects
N Number of inserted eggs
The quality class limits were determined according to
Table 5. (For the development of the FTI, see section 3.4.1).
2.7 Statistical analysis
Statistical analyses were performed using GraphPad Prism®,
Version 4.03 (GraphPad Software, San Diego, CA, USA). The
Chi-squared test was used to evaluate significant differences
between the reference and the samples with respect to lethal,
sublethal, and other effects of the fish embryo toxicity test.
In order to compare the results of the fish toxicity test
and of the macroinvertebrate assessment with abiotic
factors, data were analyzed with linear regression after
confirming a normal distribution.
3 Results
3.1 Status of hydromorphological quality
The status of hydromorphological quality at the sample
sites is shown in Table 1. Except site H3, none of the
investigated sample sites reaches the quality target class 3.
Hence, these sampled water courses show deficits in their
hydromorphological quality.
The anthropogeni c impact is also reflected in Fig. 1 with
data on the hydromorphological quality of the entire
catchment area around the sampling sites. At 11 of the 23
sites, more than 50% of the river sections in the catchment
area show hydromorphological disturbances, i.e., worse
than class 3. The most impacted sites are La8 and La9 with
86% hydromorphological disturbance.
3.2 Sediment analysis
Figure 2 shows the results of the heavy metal analysis
with quality classes III (low burden) to I II (high burden)
according to Wachs (1991). The mean sum concentration
of total heavy metals in the analyzed samples is 26.9 g/kg.
Lower concentrations were detected in the brook sedi-
ments of t he river Fulda catchment (samples H1-S2)
compared to the sedim ents from the river Lahn tributaries
(La1-La9), except F1 (40.9 g/kg). The highest concentra-
Observation after 48h Observation after 72h Observation after 144h
Lethal effects Coagulation Coagulation/death Death
Edema Edema Edema
Gross malformation Gross malformation Gross malformation
Absence of somits Limited blood circulation
Non-detachment of tail Limited heartbeat
No hatch
Sublethal effects Absence of somits Limited blood circulation Limited pigmentation
Non-detachment of tail Limited heartbeat Growth retardation
Other effects Limited blood circulation Limited pigmentation
Limited heartbeat Growth retardation
Limited pigmentation
Growth retardation
Table 4 Assignment of end-
points in the fish embryo toxic-
ity test to lethal, sublethal,
and other effects
J Soils Sediments (2010) 10:389399 393
Page 5
tion of 54.8 g/kg was detected in sample La3. The highest
classified contamination level was found in sediments G1,
La1, Di, and La5 with class III (high contamination).
With respect to the PAH content, only sample We was
classified as class IIIII (significant burden). For sample
We, a fluoranthene concentration of 0.260 mg/kg and a
benzo(a)pyrene concentration of 0.164 mg/kg was detected.
All other samples showed a lower PAH contamination.
Hence, only sample We exceeded the target value according
to Rieß et al. (1997).
3.3 Macroinvertebrate communities
The analysis of invertebrate communities resulted in a wide
range of multimetric indices (Fig. 3). The multimetric index
serves as the overall measure for the degradation of the
macrozoobenthos communities. The ecological q uality
classes from II (good) to V (bad) were detected, whereby
only four of the 24 samples (H2, H3, F2, and G2) reached
the EU target of a good ecological status. Ten of 23 samples
were even assigned to the ecological status class V (bad).
3.4 Fish embryo toxicity test
3.4.1 Development of the fish teratogenicity index
For the evaluation of the fish embryo toxicity test, a rank
sum system was used to grade different effects depending
on their intensity as lethal, sublethal, and other effects (cf.
Table 4). Using this scheme, all observed effects at the
different inspection intervals (48, 72, and 144 h) were
assessed. During the investigation, we could perceive that
some effects were reversible, i.e., they receded to the next
inspection interval while others persisted or aggravated.
Based on these observations, the detected effects were
classified as follows. If the mean incidence of an observed
effect increased to the next inspectio n, the effect was
evaluated as lethal. If the mean incidence of an observed
effect decreased between 0% and 5%, the effect was
classified as sublethal. If the mean incidence of an observed
effect decre ased by more than 5%, the effect was
categorized as other effect. For example, altogether, 655
embryos in the exposed D. rerio eggs exhibited no effects
after 72 h, while 12 embryos showed edema; after 144 h, 21
more embryos showed edemas or died. Thus, the amount of
embryos with edema increased by 3.21% and was classified
as lethal for the 72-h inspection interval. In contrast, the
mean incidence of embryos with growth retardation and
limited pigmentation recovered by 14% and 17% at the
48- an d 72-h inspection, respectively. Therefore, these
effects were classified as other effects. After 144 h, no
observed effect was classified as other effect, because
every observed effect at this point in time was considered as
a serious limitation for survival.
The different effects were weighted and combined in the
formula of the FTI (cf. Eq. 1). For the FTI, quality classes
according to the WFD were defined thus allowing a
comparison with other assessment systems. Pursuant to
WFD, class III is defined as a significant increase of
damaging effects in comparison to the refere nce (European
Union 2000). To test for differences between sample and
reference regarding lethal, sublethal, and other effects of the
FTI, a Chi-squared test with three grouping variables was
used. These results were utilized to develop the limits of
ecological quality classes shown in Table 5.
3.4.2 Results of the fish embr yo toxicity test
For an integrated analysis of the data of the fish embryo
toxicity test, the results of the 144-h observation were used.
Fig. 1 Percentage of river sec-
tions with hydromorphological
disturbance (worse than quality
class 3) in the catchment
area, received from HMULV
(2007b)
Ecological quality
class
FTI
I <0.1
II 0.10.25
III 0.250.4
IV 0.40.6
V 0.6
Table 5 Classification of the
fish teratogenicity index (FTI)
in ecological quality
classes
394 J Soils Sediments (2010) 10:389399
Page 6
This permits a detection of effects appearing only after a longer
time. Furthermore, a recovery from sublethal and other effects
found at earlier inspections during the test can be detected.
The analysis of the fish embryo toxicity test showed a
wide range of toxic effect intensities (Fig. 4). Class I (high)
was determined for only one sediment: G1 with a FTI value
of 0 showed no effects in this bioassay. Sixteen of 23
samples showed a quality class III or higher and did not
achieve a good ecological status. Seven samples attained
FTI values beyond 0.6, indicating a bad status (quality
class V). The worst effects were detected in sample La 7
with an FTI of 1.24.
4 Discussion
4.1 Condition of the macroinver tebrate community
According to the Hessian Ministry for the Environment
(Völker and Borchardt 2007), hydromorphological distur-
bance at the sampling sites is the main cause for the bad
condition of the macroinvertebrate communities. However,
our own analyses did not provide evidence for a relationship
between the multimetric index and the hydromorphological
quality at the sampling sites (Table 6). In contrast, the
multimetric indices are significantly correlated to the organic
carbon content (ignition loss determined according DIN
38414 S3 1985) and heavy metal concentrations in sedi-
ments (particularly Fe, Zn, Cr, Co, Ni, Cu, Mo, and Pb, data
not shown) as well as to the hydromorphological quality in
the catchment area at the 5% level.
Heavy metals are potentially harmful to aquatic organ-
isms. They can pass ion channels and pores and can be
reabsorbed from the digestive tract and thus are particularly
ingested by detritus feeders and sediment-dwelling organ-
isms (Wachs 1991). Species numbers of macroinvertebrates
in the field are reduced by toxic metals (Bass et al. 2008;
Iwasaki et al. 2009). A bioaccumulation of Cd, Ni, Ag, and
Zn in invertebrates has been shown by Lee et al. (2000),
revealing the relation between metals and macroinverte-
brates. Macroinvertebrate communities are even suggested
as tool for detecting metal toxicity (Bass et al. 2008;
Fig. 2 Sum of heavy metal
concentration of Cd, Co, Cr,
Cu, Fe, Mn, Mo, Ni, Pb, and Zn
in grams per kilogram. Shading
of the columns indicates the
ecological quality classes
according to Wachs (1991)
Fig. 3 The multimetric index of
macroinvertebrate communities.
Left ordinate: scale of the index;
right ordinate: resulting
ecological quality classes
J Soils Sediments (2010) 10:389399 395
Page 7
Winner et al. 1980). Aside from metals, organic pollutants
play an important role for macrozoobenthos communities
because of their influence on food and sediments. The
influence of organic pollution has been shown by Ofenböck
et al. (2004). In addition, the organic carbon content
correlates significantly with the heavy metal concent ration
at the 5% level (r
2
=0.284), a relation which was already
proved in former studies (Coquery and Welbourn 1995;
Lin and Chen 1998; Hart 1982). In sewage treatment plants,
metals adsorb to activated sludge flakes. Solved metals bind
to extracellular polymers of bacteria and the bacterial cell
cytoplasm. Thereby, metals may flush with treated sewage
into recipient water courses. Under anaerobic conditions,
which can be found in deeper sediment layers, the hydro-
carbons are hydrolyzed, and metals are released out of the
sediment (Chapman 1986).
Next to metals and organic content, macroinver te-
brates are related to the hydromorphological quality in
the catchment area. According to the Hessian State
Agency for the Environment and Geology, the catchment
regions of the investigated water courses are mostly used
agriculturally: 53% of the river Fulda catchment
(HMLUV 2007a) and 48% of the river Lahn catchment
(HMLUV 2004) are agricultural areas, which implies a
huge impact on sediments by soil erosion. In addition, the
use of fertilizers causes an exposure to organic pollutants
and metals due to surface runoff (Moolenaar 1999;
Meier et al. 2006a). Consequently, t he hydromorpholog-
ical quality in the catchment area correlates significantly
with the organic carbon content (0.1% level, r
2
=0.439)
and heavy metal concentration of sediments (5% level,
r
2
=0.250).
Deduced from these data and from these relationships,
an influence of sediment compounds on macroinvertebrate
communities is likely. Wherefore, the hydromorphological
disturbance of water courses cannot be the only reason for
the impaired macroinvertebrate coenosis.
4.2 Effects of the fish embryo toxicity test (144 h)
In line with the potential effects on macroinvetebrates, the
results of the fish embryo toxicity test indicated an impact
from the heavy metal content of the sediments. A linear
regression analysis proved a significant relationship be-
Abiotic parameter Multimetric index
Mean particle size r
2
0.039
Slope 139±151
Organic carbon content r
2
0.243*
Slope 72.6±27.9
Sum of PAHs r
2
0.060
Slope 0.3±0.3
Sum of heavy metals r
2
0.181*
Slope 458±162
Hydromorphological quality at sampling site r
2
0.106
Slope 1.4±0.9
Hydromorphological quality of catchment area r
2
0.267*
Slope 58.1±21.0
Table 6 Linear regression
between abiotic parameters and
the multimetric index
(macroinvertebrates)
*p<0.05
Fig. 4 The fish teratogenicity
index after 144 h. Left ordinate:
scale of the index; right ordi-
nate: resulting ecological
quality classes
396 J Soils Sediments (2010) 10:389399
Page 8
tween the FTI and the sum of heavy met als on the 1% level
(Table 7). For the same elements (except Pb), which
correlate significantly with the multimetric index, we found
a significant relationship with the FTI (for Ni on the 5%
level and for Fe, Zn, Cr, Co, Cu, and Mo on the 1% level).
Furthermore, the FTI correlates significantly with Mg on
the 5% level and with Al and Cd on the 1% level (data not
shown). Hence, for the detected effects in the fish
teratogenicity test, metals in the sediments may play an
important role. Metals can be taken up by D. rerio directly
through the respiratory organs (Bury and Grosell 2003;
Fracacio et al. 2003). The accumulation of heavy metals in
embryos of D. rerio has already been proven by Hallare et
al. (2005) and Dave and Xiu (1991). Toxic effects of heavy
metals have also been described, e.g., for cadmium, which
causes pericardial edema and hatch delay (Fraysse et al.
2006) and for copper, which induces neurotoxic effects
(Hernandez et al. 2006), liver damage (Paris-Palacios et al.
2000), and formation of reactive oxygen species (Sanrini et
al. 2009)inD. rerio. Beyond that, nickel leads to a
significant decrease of hatching success (Scheil and Köhler
2009). Therefore, an accessibility of heavy metals in the
fish embryo toxicity test is to assume. However, other
chemicals which are likely to be present in the tested
sediments but were not included in the analytical program
may also have contributed to the observed effects in the
bioassay.
A positive linear relationship was found between the
organic carbon content and the FTI (r
2
=0.476; p<0.01),
which is similar to the statistical results of the macro-
invertebrate analysis. The relation between the organic
carbon content and the FTI results seems to be plausible,
because a high organic content increases the biological
activity and thereby redox conditions and metal redox
conversions. Furthermore, the degradation of organic matter
influences the complexation of contaminants, which is
directly related with the bioaccessibility of contaminants
like metals (van Leeuwen and Vermeire 2007). However, a
direct toxic effect of organic carbon on fish embryos is
improbable. Oxygen, ammonium, nitrite, and nitrate con-
centrations were measured in the water phase during the
sediment test after an incubation period of 48 h. Maximum
ammonium concentrations were 0.49 mg/L at a pH of 7.9.
To analyze a potential impact of these ammonium concen-
trations, a separate 48-h fish e mbryo acute test w as
performed. The results gave no indication for any adverse
effects caused by ammonium up to a concentration of
1.6 mg/L at a pH of 8.0. The highest nitrite concentration
measured was 3.1 mg/L. Voslarova et al. (2006 ) report a
96-h LC
50
of 386±29.8 mg/L for adult D. rerio, more than
100-fold of the concentration in the tested water phase.
For all samples, an oxygen concentration of more than
7.9 mg/L was measured. This clearly exceeds the OECD
guide value of minimum 5 mg/L (OECD 1992). So, any
harm on the fish embryos by the lack of oxygen is unlikely
(Braunbeck et al. 2005). Furthermore, after 48-h sediment
exposure, the embryos were transferred into separate petri
dishes, filled with oxygen-rich reconstituted water. Hence,
there was enough time to recover from any possible oxygen
lacks until the observation time of 144 h.
4.3 Application of the fish teratogenicity index (FTI)
We developed a new scheme for the assessment of fish
embryo toxicity in the sediment contact test. This scheme
can easily be used by environmental managers for
classification of sediments in the WFD approach. The
consideration of sediment assessment in the WFD would be
viable to identify deficits of aquatic ecosystems. Hence,
these identified deficits could be reduced to recover a good
ecological status. This would be a substantial improvement
of the current WFD approach where mainly morphological
properties are remediated (Wharton and Gilvear 2006). For
European-wide monitoring, we suggest to review the FTI in
a comprehensive study covering a wider range of sediment
samples from different river types. For more detailed
information about the sediment toxicit y, we suggest using
not only one single biotest but also a battery of sediment
tests adapted to the WFD.
5 Conclusions
One of the objectives of this study was to determine the
influence of sediment compounds and hydromorphological
quality on the macroinvertebrate communities. Further-
more, to determine the bioaccessibility and toxicity of
sediment pollutants, a new sensitive assessment scheme for
the fish embryo toxicity test was developed and success-
fully applied. It allows an arrangement of the results into
quality classes and thus a comparison with other river
assessment data according to the WFD. Moreover, a
significant correlation between metal concentrations and
impacts on fish embryos and macroinvertebrates was
Table 7 Results of linear regression analysis between abiotic
sediment parameters and fish teratogenicity index (FTI; after 144 h)
Particle
size
Content of
organic carbon
Sum of
PAHs
Sum of heavy
metals
r
2
0.012 0.476** 0.033 0.321*
Slope 55.1±107 71.1±16.3 0.2±0.2 26,100±8,300
r
2
correlation coefficient
*p<0.01
**p<0.001
J Soils Sediments (2010) 10:389399 397
Page 9
detected, indicating a potential impact by this contaminant
class on the analyzed freshwater communities. In contrast,
the influence of habitat structure at the sampling site on
macrozoobenthos was found to be not statistically signifi-
cant. These results suggest that harmful effects on fish
embryos as well as on macroinvertebrate communities were
generated by sediment compounds. Finally, an important
role of sediment composition for the quality of the macro-
zoobenthos community has been proven. This demonstrates
that traditional river assessments may underestimate the
role of sediment quality. Therefore, bioassays should be
included in the river assessment according to the WFD. The
fish embryo toxicity test with D. rerio could be utilized to
detect the potential ecological hazard of sediment s to a
vertebrate organism. Fo r the evaluation of the fish embryo
toxicity test and for a comparison with other assessment
data, the FTI appears promising. However, before its
routine application, the FTI should be reviewed in a
comprehensive study covering a wider range of samples
from different river types.
Acknowledgements The authors are particularly grateful for the
support of sediment analysis by Heike Heidenreich and Gerlinde
Liepelt from the International Graduate School Zittau as well as for
collaborative support by Simone Galluba and Maren Heß from Goethe
University Frankfurt am Main and Dietmar Bernauer. Moreover, the
authors wish to thank Mirco Weil from ECT Oekotoxikologie GmbH
for support with ammonia toxicity testing.
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  • Source
    • "Sediments have been recognized as major sink for persistent toxic substances in the aquatic environment, but also as a potential source (Bartzke et al. 2010, Hollert et al. 2003); thus, contaminated sediments may affect aquatic organisms. However, the majority of existing bioassays for sediment toxicity testing do not provide sufficient information concerning "
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    • "Then, the results from ecological indices and ecotoxicological analyses were analysed through Pearson correlations. Similarly, Bartzke et al. (2010) developed an index based on fish embryo toxicity test, the fish teratogenicity index. Observed effects were weighted according to the effect intensity and mathematically combined in an index. "
    [Show abstract] [Hide abstract] ABSTRACT: The European Water Framework Directive (WFD) represents a transformation of the guidelines for water quality assessment and monitoring across all EU Member States. At present, it is widely accepted that the WFD requires holistic and multidisciplinary ecological approaches by integrating multiple lines of evidence. Within the scope of the WFD, the scientific community identified clear opportunities to take advantage of an ecotoxicological line of evidence. In this context, ecotoxicological tools, namely biomarkers and bioassays, were proposed to contribute to the integration of the chemical and biological indicators, and thus to provide an overall insight into the quality of a water body. More than one decade after the publication of the WFD, we reviewed the studies that have attempted to integrate ecotoxicological tools in the assessment of surface water bodies. For this purpose, we reviewed studies providing an ecological water status assessment through more conventional community based approaches, in which biomarkers and/or bioassays were also applied to complement the evaluation. Overall, from our review emerges that studies at community level appear suitable for assessing the ecological quality of water bodies, whereas the bioassays/biomarkers are especially useful as early warning systems and to investigate the causes of ecological impairment, allowing a better understanding of the cause–effect-relationships. In this sense, community level responses and biomarkers/bioassays seem to be clearly complementary, reinforcing the need of combining the approaches of different disciplines to achieve the best evaluation of ecosystem communities’ health.
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    • "The assessment of the bioavailable hazard potential of contaminated sediments is of major concern in environmental sciences. Chemicals with low and medium polarity are wellknown to accumulate in aquatic sediments at concentrations many times higher than those in the free water column, and thus, sediments are able to act as secondary sources for pollutants after remobilization (Ahlf et al. 2002; Bartzke et al. 2010; Brils 2008; Gerbersdorf et al. 2011; Hollert et al. 2002). Therefore, sediment toxicology plays an increasingly important role for, e.g., the recruitment of information required for the successful implementation of the European Water Framework Directive (EWFD 2000/60/EC). "
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