A Test Battery Approach for the Ecotoxicological Evaluation
of Estuarine Sediments
M. DAVOREN,1,* S. NI´SHU´ILLEABHA´IN,1J. O’ HALLORAN,2M.G.J. HARTL,2D. SHEEHAN,2
N.M. O’BRIEN,2F.N.A.M. VAN PELT,2AND C. MOTHERSILL3
1Radiation and Environmental Science Centre, FOCAS Institute, Dublin Institute of Technology,
Kevin Street, Dublin 8, Ireland
2Environmental Research Institute, University College, Cork, Ireland
3McMaster University, Hamilton, Ontario, Canada
Accepted 9 February 2005/Published online 7 September 2005
Abstract. The purpose of this study was to evaluate the overall sensitivity and applicability of a number of
bioassays representing multiple trophic levels, for the preliminary ecotoxicological screening (Tier I) of
estuarine sediments. Chemical analyses were conducted on sediments from all sampling sites to assist in
interpreting results. As sediment is an inherently complex, heterogeneous geological matrix, the toxicity
associated with different exposure routes (solid, porewater and elutriate phases) was also assessed. A
stimulatory response was detected following exposure of some sediment phases to both the Microtox?and
algal bioassays. Of the bioassays and endpoints employed in this study, the algal test was the most
responsive to both elutriates and porewaters. Salinity controls, which corresponded to the salinity of the
neat porewater samples, were found to have significant effects on the growth of the algae. To our
knowledge, this is the first report of the inclusion of a salinity control in algal toxicity tests, the results of
which emphasise the importance of incorporating appropriate controls in experimental design. While
differential responses were observed, the site characterised as the most polluted on the basis of chemical
analysis was consistently ranked the most toxic with all test species and all test phases. In terms of
identifying appropriate Tier I screening tests for sediments, this study demonstrated both the Microtox?
and algal bioassays to be more sensitive than the bacterial enzyme assays and the invertebrate lethality
assay employing Artemia salina. The findings of this study highlight that salinity effects and geophysical
properties need to be taken into account when interpreting the results of the bioassays.
Keywords: sediment; elutriate; porewater; Microtox?; Skeletonema costatum; hormesis
Compliance with the EU Water Framework
Directive (WFD) (2000/60/EC) requires that all
member states implement comprehensive moni-
toring programmes for transitional and coastal
waters by 2006, with the ultimate objective of
achieving good quality water status by the year
2015. The quality of aquatic sediment is critical to
pollutants show a strong affinity to suspended
particulate matter (Harris and Cleary, 1987; Rag-
narsdottir, 2000) and can ultimately be sequestered
*To whom correspondence should be addressed:
Tel.: +353-1-402 7974; Fax: +353-1-402 7904;
E-mail: firstname.lastname@example.org (M.Davoren)
Ecotoxicology, 14, 741–755, 2005
? 2005 Springer Science+Business Media, Inc. Printed in The U.S.A.
from the water column and incorporated into the
underlying sediment. Contaminants accumulated
in the sediment have the potential to cause adverse
effects to indigenous biota and aquatic organisms
should they become bioavailable, or remobilised
following chemical (e.g. pH, salinity fluctuations),
physical (e.g. dredging) or biological (e.g. biotur-
bation) processes. The monitoring of contaminants
of any water quality management plan.
At present, assessment and monitoring of
sediment quality in Ireland is predominantly
reliant on chemical analyses (Nendza, 2002).
Quantifying contaminant concentrations alone is
often insufficient to derive accurate conclusions
on the potential toxicity of contaminated envi-
ronmental samples, as it is virtually impossible to
identify and measure the concentration of all
potential toxicants. In addition, the magnitude of
contamination does not necessarily reflect a
similar scale of ecotoxicological effects, as chemical
analyses cannot directly assess the combined effects
toxicants to organisms. An ideal monitoring strat-
egy should, therefore, incorporate chemical char-
acterisation and ecotoxicological analysis without
undue emphasis on any single test result (Cleveland
et al., 1997; Pedersen et al., 1998; Ghirardini et al.,
1999; Lee et al., 1999).
Sediment is an inherently complex, heteroge-
neous geological matrix, with a number of routes
by which biota may be exposed to sediment-asso-
ciated contaminants. Undoubtedly, simulation of
in situ exposure of aquatic organisms to contami-
nated sediments is most realistic using whole
sediment samples (Ankley et al., 1991). Many
organisms, however, cannot be employed to test
the toxicity of the solid phase directly and aqueous
extracts are, therefore, often used to circumvent
this problem. Contaminants in porewater repre-
sent the water-soluble, bioavailable fraction and as
a result may be a major route of exposure to
infaunal species (Carr et al., 1989; Carr, 1998;
Nipper et al., 2002). Comparisons of porewater
and whole sediment toxicity tests have also indi-
cated that porewater is similarly, if not more,
sensitive than whole sediment tests (Carr and
Chapman, 1992; Winger et al., 1993). The use
of elutriate extracts provides information on
the leaching capability of sediment-associated
contaminants, and as such may be identified as an
important route of toxicant exposure following the
disposal and relocation of dredged material and
sediments. Toxicity tests employing elutriates may
therefore yield important data on the potential
adverse effects towater
following disturbance of the underlying sediment
(Cheung et al., 1997; Wong et al., 1999). A com-
toxicity consequently requires the consideration of
multiple exposure phases. In addition, the use of a
battery of test species, from various phyla and
representing multiple trophic levels, has been
advocated by a number of researchers (Dutka
et al., 1989; Giesy and Hoke, 1989; Matthiessen
et al., 1998; Nendza, 2002), as testing of single or
few organisms may not detect contaminants with a
specific mode of action.
Here we employ a 1st tier multi-trophic, multi-
exposure phase assessment approach in order to
characterise Irish estuarine sediment samples.
Bacteria play a vital role as decomposers in
aquatic systems and hence there is justification
for their inclusion in a test battery for the
assessment of sediment toxicity. Three bacterial
bioassays were employed in this study, which
allowed the toxicological assessment of both
whole sediment (Microtox?Solid Phase Test
(SPT), Toxi-ChromPad?) and sediment pore-
water and elutriates (Microtox?, MetPAD?). The
marine diatom Skeletonema costatum was chosen
to assess the acute toxicity of sediment porewater
and elutriates due to its widespread distribution,
sensitivity, and availability as an axenic culture
(McKinney et al., 1997; Weideborg et al., 1997;
Sverdrup et al., 2002). Sediment contamination
has previously been investigated following expo-
sure of S. costatum to both sediment porewater
(Pederson et al., 1998) and elutriates (Cheung
et al., 1997). Finally, in this study, the standar-
dised crustacean bioassay with Artemia salina
was selected as an additional biological tool
species for screening sediment porewater and
elutriates. The objectives of the present study
were therefore, (1) to assess the overall sensitivity
and applicability of each test species and end-
points employed in the test battery and (2) to
sediment exposure phase.
742Davoren et al.
Materials and methods
Site selection and sediment sampling
Three sampling locations around the Irish coast
(Figure 1) were selected on the basis of previously
published data that demonstrated them to be
representative of a range of contaminant burdens
(Marine Institute, 1999; Byrne and O’ Halloran,
2000; Kilemade et al., 2004). Ballymacoda Estu-
ary, Co. Cork (07?55¢ W, 51?52¢ N) was chosen as
a relatively uncontaminated site while the Douglas
Estuary, Co. Cork (08?23¢ W, 51?52¢ N) and the
East Wall, Liffey Estuary, Dublin (06?07¢ W,
53?20¢ N) have moderate and high contaminant
For practical purposes sediment sampling was
based on a once-off representative, ‘‘snapshot’’
survey of surficial, inter-tidal sediment from each
of the three sites. At each sampling site the top
1–2 cm of sediment (the aerobic layer) was taken
from eight random sections in approximately a
300 cm2area, these samples were then pooled and
homogenised thoroughly and stored at 4 ?C in a
polyethylene bag and treated as one sample. The
mixing of the samples helped to minimize site
intra-variability, and ensured that the sediment
collected was truly representative of each site. All
ecotoxicity tests were carried out within 2 weeks of
the sampling episode as recommended by the
American Society for Testing and Materials
Physical and chemical characterisation of sediments
Physical and chemical characterisation of the
Ballymacoda and Douglas sediments were con-
ducted by Kilemade et al. (2004). Characterisation
of sediments from the East Wall site was per-
formed as part of this study. The percentage dry
weight of the sediments was determined after the
sub-samples were oven-dried at 105 ?C to a con-
stant weight. Total organic carbon (TOC) was
determined by ALcontrol laboratories (Chester,
UK) using the combustion method, which con-
formed to ISO 17025. The accuracy of this method
was ensured using a certified reference material
(CRM) (Alpha Resources Inc. CSN standard
AR873). The accuracy was further assessed by the
inclusion of a test material (obtained from the
QUASIMEME project office), which was found to
be within acceptable limits.
Sediments were wet sieved using the gravimetric
method (Loring and Rantala, 1992), to obtain the
fine (<63 lm) sediment fraction on which all
chemical analyses were conducted. Heavy metal
analyses (Pb, Cd, Cu, Zn) were conducted by the
Marine Institute (Abbotstown, Dublin, Ireland).
Lead, cadmium, and copper concentrations were
Absorption Spectrometry with Zeeman back-
ground correction (Varian SpectrAA 220Z). Zinc
atomic absorption spectroscopy (Varian SpectrAA
20 Plus). The East Wall sediment was sent to
Research, the Netherlands
STERLAB) for the analyses of polycyclic aromatic
hydrocarbons (PAHs), polychlorinated biphenyls
(PCBs), polybrominated diphenylethers (PBDEs),
brominated flame retardants (BFRs), organo-
chlorine pesticides (OCPs). Organotin (OT) anal-
yses were conducted by TNO-MEP (Apeldoorn,
the Netherlands). Table 1 summarizes the meth-
odology employed for the extraction, separation,
detection and quantification of each class of
organic contaminant. A comprehensive analytical
quality assurance program, involving the routine
testing of quality control samples such as blanks,
CRMs), underpinned all chemical testing.
Porewater extracts were prepared by centrifuging
25 ml sub-samples of sediment at 1200·g for
30 min at 4 ?C. The supernatant was collected as
porewater, and conductivity, salinity and pH
measured using the appropriate electrodes with a
Sension?multimeter (Hach, Loveland, CO, USA)
following filtration through a 0.2 lm filter (Nal-
gene, NY, USA). Porewater was stored in primed
glass bottles with minimal headspace at 4 ?C in the
dark prior to toxicity testing, which was conducted
within 24 h of preparation.
Elutriate extracts were prepared with modification
according to the Standard Elutriate Test (Keely
Ecotoxicological Evaluation of Estuarine Sediments743
Figure 1. Location of the sites sampled in this study.
744 Davoren et al.
and Engler, 1974; USEPA, 1977). Seawater (Sig-
ma, Ireland) was added to 10 g sub-samples of
sediment in a 1:4 (w/v) ratio, based on the sedi-
ment dry weight. The slurry was shaken at 240
rpm for 1 h and then centrifuged at 1200·g for
30 min at 4 ?C. The supernatant was collected as
elutriate and conductivity, salinity and pH were
measured following filtration through a 0.2 lm
filter. The elutriate was stored in primed glass
bottles with minimal headspace at 4 ?C in the dark
prior to toxicity testing, which was conducted
within 24 h of preparation.
Bioassays and exposure conditions
Lyophilised Vibrio fischeri bacteria (NRRL B-
11177) and all reagents used in the Microtox?as-
say were obtained from SDI Europe, Hampshire,
UK. Sediment elutriates and porewater were tested
using the 90% basic test for aqueous extract pro-
tocol (Azur Environmental Ltd., 1998) with the
slight modification of adjusting the salinity of the
Microtox?diluent, using the osmotic adjusting
solution (OAS), to that of the aqueous test sample.
This bioassay has also been adapted to a solid
phase test where toxicity to organisms in direct
contact with sediments can be assessed. Whole
sediment exposures were performed according to
the SPT protocol (Azur Environmental Ltd., 1998).
A basic test (Azur Environmental Ltd., 1998) using
phenol as a reference chemical was conducted to
ensure validity of all the tests. The bioluminescent
responses were measured using a Microtox?Model
500 analyser (SDI Europe, Hampshire, UK). The
SPT EC50data is expressed as concentration (mg/l)
of whole sediment corrected for moisture content,
which caused a 50% reduction in bacterial biolu-
minescence following a 20 min incubation period.
The Toxi-ChromoPad?bacterial bioassay (Kwan,
1995) is based on inhibition of the enzyme
b-galactosidase, in a mutant strain of Escherichia
coli. The test was purchased from Environmental
Canada) and performed on whole sediment
according to manufacturer’s instructions. Results
were determined by recording the lowest sample
concentration that gave complete b-galactosidase
synthesis inhibition, i.e. no colour development,
and are expressed as the sediments’ Effective
Concentration 100 (EC100) value expressed as a
The MetPAD?bioassay, which is also based on the
inhibition of the enzyme b-galactosidase in E. coli,
was obtained from the Department of Environ-
(Gainesville, FL, USA). This bioassay was specifi-
cally developed for the determination of heavy
metal contamination in aqueous samples (Bitton
et al., 1992). The test was performed on sediment
elutriates extracted using moderately hard water as
specified by the manufacturer’s instructions. Tox-
icity scores were assigned according to colour
intensity, ranging from 0+ for 100% sample
toxicity to 5+ for non-toxic samples. Spot colour
(moderately hard water) and a positive control
(copper sulphate). A reduction in colour intensity
in comparison to the negative control indicated
heavy metal toxicity in the sample.
Table 1. An overview of the analytical methodologies employed for the detection and quantification of the organic contami-
nants in the East Wall sediment
Compounds Extraction procedureSeparation procedure – chromatograph columns Detection
CP-Sil 8, DB-5
CP-Sil 8, CP Sil 19
CP-Sil 8, CP Sil 19
SGE 25QCE BPX5
aHigh pressure liquid chromatography.
bGas chromatography – mass spectrometry.
cGas chromatography – electron capture detection.
Ecotoxicological Evaluation of Estuarine Sediments745
Algal toxicity test
Axenic cultures of S. costatum (CCAP 1077/3)
originally obtained from the Culture Collection of
Algae and Protozoa (CCAP) (Argyll, Scotland),
were employed. Guillard’s medium for diatoms
(Guillard and Ryther, 1962) was purchased as a
concentrated nutrient stock (CCAP, Scotland) and
diluted using seawater (Sigma, Ireland) to prepare
Algal Growth Medium (AGM). The algae were
pre-cultured for 3 days in AGM before testing. A
modified test protocol based on the British
Standard procedure (BS EN ISO 10253, 1998) was
employed. Briefly, nutrient stock solution was
added to the neat porewater and elutriate samples
(100% sample) to ensure nutrients were not limit-
ing. The pH of the samples was adjusted to
8.0±0.2 when necessary. Neat samples were seri-
ally diluted using AGM to obtain five test
concentrations (20, 40, 60, 80 and 100%) and each
concentration was tested in triplicate. Six controls
were incorporated for each test containing AGM
and algal inoculum only. A salinity control (based
on 100% porewater sample salinity) was included
in each test in addition to AGM controls. The
initial algal density in each flask was 2.5·103cells
per ml in a final test volume of 20 ml. The test was
conducted for 72 h exposure period at 20±1 ?C
with continuous shaking at 100 rpm and illumi-
nationof 10,000 lux.
measured using a Neubauer Improved (Bright-
Line) chamber (Brand, Germany). The average
specific growth rate (l) and percentage inhibition
of average specific growth rate (% Ir) relative to
controls was calculated. Test validity criteria
required algal growth in control flasks to increase
by more than a factor of 16 within the 72 h test
period and pH not to have varied by more than
±1.0 unit during the test. Potassium dichromate
(K2Cr2O7) was employed as a reference chemical
to ensure the validity of the test method.
The cell density was
Brine shrimp larvae assay
Porewater and elutriate extracts were further as-
sayed for toxicity using the brine shrimp A. salina
according to Vanhaecke and Persoone (1981).
A. salina cysts were obtained from Galway
Aquatic Ltd. (Galway, Ireland) and hatched in
seawater (Sigma, Ireland) to produce instar larvae.
Using instar II–III larvae, an acute toxicity test
was performed for 24 h at 25 ?C in the dark. Tests
were conducted using a 24-well plate (Nunc,
Denmark) with one control and five test dilutions
(20, 40, 60, 80 and 100%) with three replicates per
concentration. Lethality for each sample dilution
was recorded and the percentage mortality (LC50)
compared to the control was determined. Potas-
sium dichromate was employed as a reference
chemical and tested in tandem with test samples to
ensure the validity of the test method.
Experiments were performed in triplicate in at
least two independent experiments. Coefficient of
variation (CV) for the controls of each test was
calculated to ascertain reproducibility. Data are
expressed as the arithmetic mean±standard error
of the mean (SEM). The acute toxicity data for the
Microtox?assays was analysed using the Micro-
toxOmni?software (SDI Europe, Hampshire,
UK). Toxicity data was fitted to a sigmoidal curve
and a four parameter logistic model was used to
calculate EC10and EC50values. This analysis was
performed using Xlfit3?(ID Business Solutions,
UK) a curve fitting add-in for Microsoft?Excel.
Statistical analyses for the algal tests were carried
out using the Kruskal–Wallis and Mann–Whitney
U tests for non-parametric data. These data anal-
yses were performed using MINITAB?release 12
(MINITAB Inc. PA, USA). Statistical significance
was accepted at p £ 0.05.
The results of the physicochemical characterisa-
tion of each sediment are presented in Table 2.
Total organic carbon concentrations were rela-
tively uniform between sites (2.2–3.6%). The per-
centage of sediment fine particles (<63 lm) was
highest at Ballymacoda and lowest at the Douglas
site. Salinity of the Ballymacoda porewater (39&)
was found to exceed that of normal seawater
Comprehensive chemical analysis was con-
ducted on sediments to assist in the interpretation
of any observed ecotoxicity (Table 3). The highest
concentrations of each contaminant class were
746 Davoren et al.
measured at the East Wall site. Based on the
results of the chemical analyses the sites were
ranked in order of most contaminated as East
The mean EC50 value for the positive phenol
control (seven analyses) was 19.2±0.7 mg/l. The
sensitivity of the Microtox?bioassay towards
whole sediment, porewater and elutriate samples is
summarised in Table 4.
Exposure to Ballymacoda porewater resulted in
a maximal reduction in bioluminescence of 13.2%.
Reduction in bioluminescence was not found to
differ significantly over time. Douglas porewater
elicited a maximal reduction in bacterial light
output of 21% following a 30-min exposure.
Biostimulation, however, was noted at an earlier
time point. The East Wall site was found to elicit
the greatest toxicity with porewater concentrations
ranging from 5.9–8.9% and 49–54% resulting in a
10 and 50% reduction in bacterial light output,
respectively over the time points investigated.
following exposure to elutriate extracts from both
the Ballymacoda and the Douglas sediment sam-
ples. This stimulatory response ranged from a 3 to
25% increase in light output when compared to
Table 2. Physicochemical properties of the three sediment-sampling sites
% Dry weight
Total organic carbon
Sediment fraction<63 lm
aS=whole sediment; P=porewater; E=elutriate extract.
Table 3. Chemical characterisation of the fine (<63 lm) sediment fraction for the three sampling sites
Metals (mg kg)1dry weight)
Organic contaminants (lg kg)1dry weight)
Organotins (lg kg)1dry weight)
aAnalysis conducted by Kilemade et al. (2004).
bS=Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, Benzo(a)anthracene, Chrysene, Benzo(e)pyrene,
Benzo(b)fluoranthene, Benzo(k)fluoranthene, Benzo(a)pyrene, Dibenzo(a, h)anthracene, Benzo(g, h, i)perylene, Indeno(1, 2, 3-cd)
cS=Congeners #28, 52, 101, 118, 138, 153, 180.
dS=a-, b-, c-HCH, HCB, QCB, o,p¢-DDD, -DDE, -DDT, p,p¢-DDD, -DDE, -DDT
eS=Hexabromocyclododecane, Polybrominated diphenylether #28, 47, 66, 71, 75, 77, 85, 99, 100, 119, 138, 154, 183, 190, 209.
Ecotoxicological Evaluation of Estuarine Sediments747
controls. Exposure to the East Wall elutriate
resulted in a maximal reduction in biolumines-
cence of 19.7% after 30-min exposure.
Solid phase exposure to each of the sediments
elicited a significant reduction in bioluminescence
(p £ 0.05)when compared
maximum test concentration exposed to the
bacteria was 197,400 mg/l of sediment. Based on
these results the East Wall sediment was again
Toxicity (EC100) was ranked according to the per-
required to produce an EC100effect: (a) very toxic,
if the sample concentration was
moderately toxic, if the sample concentration was
>12.5% and £ 50%; and (c), non-toxic, if the
sample concentration was >50%. The Toxi-Chro-
moPad?ranked the sampling sites in the following
of sample whichwas
£ 12.5%; (b),
order of toxicity, East Wall>Douglas>Ballyma-
coda with EC100values of 6.25, 50 and >50%,
The Ballymacoda and Douglas elutriates did not
inhibit enzyme synthesis when compared to the
controls and were thus assigned a toxicity score
of 5+ (non-toxic). There was notable inhibition
of b-galactosidase synthesis following exposure to
the East wall sample in comparison to the neg-
ative control. As inhibition of colour develop-
ment was not as pronounced as in the positive
control (0+), this sample was assigned a toxicity
score of 2+.
The mean EC50value for the reference chemical
potassium dichromate was 1.6±0.08 mg/l. Algal
growth was completely inhibited by all concen-
trations of the Ballymacoda porewater tested,
Table 4. Sensitivity of the Microtox?bioassay towards whole sediment, porewater and elutriate samples
Incubation time (min)Maximum % reduction in bioluminescenceb
aP=porewater; E=elutriate extract; SPT=whole sediment.
bMaximum percentage reduction in bioluminescence following exposure when compared to control.
cConcentration (%) of test sample that caused a 10% (EC10) or 50% (EC50) reduction in bioluminescence. Data represents mean
eConcentration (mg/l) of sediment sample that caused a 50% (EC50) reduction in bioluminescence. These values are corrected for the
moisture content of each sediment. Data represents mean value±SEM
748Davoren et al.
while exposure to the Ballymacoda elutriates yiel-
ded a significant (p £ 0.05) stimulatory response
from S. costatum (Figure 2A). A dose response
curve was fitted for this data and Ballymacoda
elutriate concentrations of 22.3 and 80.2% were
estimated to cause a 10 and 50% increase in
controls. There was no significant difference
determined between the AGM control and the
Ballymacoda salinity control.
Douglas porewater concentrations from 20 to
80% were found to cause a significant stimulation
(p £ 0.05) of growth when compared to controls
(Figure 2B). An inhibitory effect was recorded at
the top concentration of 100% porewater but this
was not found to be statistically significant. It
should be noted, however, that a significant
in comparison to
(p £ 0.05) difference did exist between the Douglas
salinity and AGM controls. A dose response curve
was fitted for this data and a concentration of
50.7% Douglas porewater was estimated to cause
a 10% stimulation of algal growth in comparison
to AGM controls. While a significant (p £ 0.05)
exposure of S. costatum to 40 and 60% Douglas
elutriate, this response was not dose-dependent as
a significant difference was not found between the
other exposure concentrations and the AGM
control. Toxicity values (e.g. EC10) could not
therefore be extrapolated from the Douglas elu-
Significant inhibition (p £ 0.05) of algal growth
following exposure to the East Wall sediment was
Figure 2. Effect of salinity control (n), porewater (h) and elutriate (n) exposure from (a) Ballymacoda, (b) Douglas and (c) East
Wall site to Skeletonema costatum. Stimulatory effects are plotted above the x-axis while inhibition is plotted below. Data is ex-
pressed as a percentage of unexposed controls±SEM of three replicates for each exposure concentration. *denotes significant dif-
ference from the control (p £ 0.05). CV for the controls ranged from 7.2–14.1%.
Ecotoxicological Evaluation of Estuarine Sediments 749
60–100%. The maximum observed effect concen-
tration (MOEC) was recorded at 80% porewater
exposure. Stimulation was observed at the lower
concentrations but was found only to be signifi-
cantly different from the AGM control at the 20%
porewater exposure. A significant (p £ 0.05) dif-
ference was again recorded between the East Wall
salinity and AGM controls. A porewater concen-
tration of 59% was estimated to inhibit algal
growth by 50% when compared to AGM controls.
Algal growth was significantly inhibited following
exposure to all concentrations of the East Wall
elutriate. An EC50could not be derived for the
East Wall elutriate, as the dose response was not
Brine shrimp bioassay
The A. salina bioassay was performed in triplicate
and yielded a 24-h LC50value of 22.7±2.7 mg/l
for the reference chemical potassium dichromate.
A toxic effect was not observed following exposure
to porewater or elutriate samples from any of the
It is well recognised that comprehensive sediment
hazard assessment necessitates the use of an inte-
grated approach, which considers contaminant
concentrations in tandem with biological effects
(Ahlf et al., 2002; Nendza, 2002; Nipper et al.,
2002). There is no universally sensitive test species
that can reliably predict the potential hazards
associated with contaminated sediments. A multi-
trophic battery of tests, which incorporates a
number of different test species, is therefore
advocated to reduce uncertainty in sediment
Bombardier and Bermingham, 1999; Chapman
et al., 2002). In addition, a test strategy, which
includes the assessment of multiple exposure
phases, to evaluate the potential toxicity exerted
by both dissolved and bound contaminants,
affords a more thorough appraisal of potential
Tests with V. fischeri using phenol as a reference
chemical resulted in a value, which was within the
recommended EC50value, range of 13 to 26 mg/l
(Azur Environmental Ltd., 1998). A slight toxic
and Hoke, 1989;
response was observed following exposure of
bacteria to porewaters extracted from the Bal-
lymacoda and Douglas sites. The reduction in light
levels was found to be time-dependent following
exposure to Douglas porewater. This time-depen-
dent decrease in light levels is a typical response of
V. fischeri following exposure to heavy metal
compounds (Azur Environmental Ltd., 1998), and
thus the use of multi-time point measurements,
especially when evaluating the toxicity of complex
mixtures is recommended. Porewater extracted
from the East Wall sample was the most toxic
aqueous phase tested using the Microtox?bioas-
say. The reduction in luminescent responses de-
tected for this sample, were relatively constant
over the incubation time points examined, which is
suggestive of toxicity elicited by organic com-
pounds (Azur Environmental Ltd., 1998).
Exposure of V. fischeri to elutriate extracts from
the East Wall site resulted in a reduction in
bioluminescence, while stimulation of light output
was recorded following exposure to both the Bal-
lymacoda and Douglas elutriate samples. Based on
these results the East Wall site can be ranked as the
most toxic, using the Microtox?assay.
It is noteworthy that seawater (salinity 35&)
was employed as a diluent in this study. Salinity
(ionic strength) is a known determinant of parti-
tioning coefficients for pollutants in estuarine
environments (Chapman and Wang, 2001). Previ-
ous research using the Microtox?assay, has
diluents of varying ionic strength resulted in
marked differences in the toxic response detected
(Ankley et al., 1989; Dombroski et al., 1996; Cook
et al., 2000; Onorati and Mecozzi, 2004). Dom-
broski et al. (1996) found that elutriates extracted
with distilled deionised water exerted greater toxic
responses than those extracted with a 20& NaCl
solution (equivalent to the salinity of Microtox?
diluent). Conversely, Onarati and Mecozzi (2004)
demonstrated that elutriates prepared with the
Microtox?solid phase diluent (salinity 35&) re-
sulted in a greater reduction in bacterial light
output than those prepared with the Microtox?
aqueous diluent (salinity 20&).
Bacterial responses (inhibitory or stimulatory)
following toxicant exposure are expressed as a
percentage of control values. The biostimulatory
responses measured following exposure to the
750Davoren et al.
Ballymacoda and Douglas aqueous extracts, in
this study, were therefore not attributable to os-
motic stress, as the salinity of the controls were
adjusted to the salinity of the test samples using
OAS. A biostimulatory or ‘hormetic’ response is a
phenomenon known to cause a stimulatory
response over that of controls in the presence of
sub-inhibitory levels of toxicants (Stebbing, 1982,
Calabrese and Baldwin, 2003; Calabrese, 2004).
Hormetic responses have previously been reported
with Microtox?studies (Christofi et al., 2002). As
contaminant burdens in elutriate extracts are
generally expected to be less than those in pore-
water extracts, it is not surprising that a stimula-
tory response is observed following exposure to
elutriate extracts, while exposure to the corre-
sponding porewater resulted in a reduction of
bioluminescence (Table 4). In addition, slight
stimulation was observed at lower dilutions of
porewater, which further supports the hypothesis
that sub-inhibitory levels of toxicants caused the
stimulatory response. Toxicity values were not
derived where biostimulation was observed, as the
detection of stimulatory responses at lower doses
resulted in an atypical concentration response
curve, and thus did not permit accurate estimation
of toxicity (EC10and EC50).
The Microtox?SPT assay was also employed in
this study to provide information on solid-phase
associated contaminants, the toxicity of which
may not be addressed in aqueous phase testing but
can still exert adverse toxic effects on benthic-
dwelling organisms. Data obtained using the
SPT assay have previously
correlated with results of invertebrate toxicity
bioassays (Day et al., 1995; Doherty, 2001). This
multiphase approach permits a more holistic
appraisal of the potential toxicity of each of the
sediments to V. fischeri.
Direct sediment contact increases the test’s
sensitivity to potential toxicants, but also makes
this assay more susceptible to interferences e.g.
loss of bacteria adhering to the particles and fil-
tered from the test suspension, such that a signif-
icant bias can occur in the interpretation of the
results (Benton et al., 1995; Ringwood et al., 1997;
Stronkhorst et al., 2003). Ringwood et al. (1997)
reported that a high rate of false positive results
was obtained following exposure to estuarine
sediments that were characterised by high silt–clay
content. The significant toxicity observed follow-
ing exposure of V. fischeri to the Ballymacoda
sediment, may be explained by the adsorption of
the bacteria to the fine grained sediments, as this
sample contained the highest percentage silt–clay
fraction (<63 lm). Conversely, the Douglas sed-
iment, which consisted of the lowest percentage of
fine grains, was classified as non-toxic on the basis
of its high SPT EC50value. This does not concur
with the results obtained using Douglas aqueous
extracts or with the chemical characterisation data
for the Douglas sediment. These findings illus-
trates that grain size was a major confounding
factor in this study. In addition, it emphasises the
importance of identifying a suitable reference site
that can be employed for grain size normalisation
purposes. Given the diverse nature of natural
sediments, this, however, is very difficult to
Finally, the East Wall site was ranked the most
toxic based on SPT EC50 data, which was in
agreement with the results of the aqueous phase
tests for this sediment. While the East Wall sedi-
ment consisted of a lower silt-clay fraction, the
EC50 derived was approximately 50% less than
that derived for the Ballymacoda sediment. This
suggests that reduction
following exposure to the East Wall sediment was
a result of contaminant toxicity rather than loss of
bacteria through sediment adsorption.
Results from tests with S. costatum using
potassium dichromate as a reference chemical were
in good agreement with the mean value of 2.5 mg/l
cited by the British Standard procedure (BS EN
ISO 10253, 1998). Porewater extracted from the
Ballymacoda sediment caused complete inhibition
of algal growth at all test dilutions, while the
corresponding elutriate stimulated algal growth.
These results concur with the Microtox?data,
where the same trend was observed but to a lesser
extent. As previously discussed, salinity is a sig-
nificant factor in the sediment–water partitioning
of pollutants. It has been demonstrated that high
ionic strength enhances the extraction of NH4
from sediments (Laima, 1992). As the salinity
measured in Ballymacoda porewater was signifi-
cantly higher than in the other samples investi-
gated, it is possible that the toxicity observed was
due to this naturally occurring contaminant.
Previous research has demonstrated elevated levels
Ecotoxicological Evaluation of Estuarine Sediments 751
of ammonia-N are a potent factor in inhibiting
algal growth (Pun et al., 1995; Cheung et al., 1997;
Wong et al., 1999). The stimulation of algal
growth observed following exposure to the Bal-
lymacoda elutriate samples may be due to a hor-
metic response. As the reclaimed land adjacent to
the Ballymacoda site is subject to intensive agri-
cultural use, with cattle grazing and silage being
the most common activities, it is also possible that
this is a eutrophic response caused by elevated
levels of nutrients. Unfortunately, it was not pos-
sible to accurately measure the levels of ammonia-
N or perform nutrient analyses in this study.
A stimulatory response was also noted follow-
ing exposure to the Douglas elutriate samples.
These results were, however, inconclusive as the
response observed was neither dose-dependent or
significantly different with respect to the controls
at all concentrations tested. A dose-dependent
decrease in stimulation was evident with Douglas
porewater, which was indicative of a hormetic ef-
fect. While the toxic effect observed following
exposure to the top concentration of Douglas
porewater, was not statistically significant, it
should be noted that a stimulatory response was
recorded following exposure to the corresponding
salinity control. It is therefore, possible that the
toxic effect was ameliorated by the lower sample
salinity. To our knowledge, this is the first report
of the inclusion of a salinity control in algal tox-
icity tests. The results obtained in this study
highlight the importance of incorporating appro-
priate controls in experimental design.
A typical hormetic response curve is apparent
for the East Wall porewater exposure with total
inhibition of algal growth recorded at the higher
test concentrations, and stimulation at the lower
test concentrations. An inhibitory response was
observed following exposure to all concentrations
of the East Wall elutriate. This was the only
elutriate sample to exert an algicidal effect. Of all
the bioassays and endpoints employed in this
study, the algal test was the most responsive to
both elutriates and porewaters. Cheung et al.
(1997) have previously demonstrated the suitabil-
ity of this diatom for testing sediment elutriates.
The demonstrated sensitivity to elutriate samples
endorses the inclusion of this bioassay in a test
battery for the toxicological assessment of dredged
Previous research has demonstrated the Toxi-
ChromoPad?solid phase test to be sensitive for
(Kwan and Dutka, 1995). Based on the EC50
results for this bioassay, Ballymacoda, Douglas
and the East Wall site were ranked as non-,
moderately- and very toxic, respectively. The
MetPAD?assay has been evaluated for toxicity
assessment of sediment elutriates from hazardous
waste sites in Florida and was found to be useful in
pinpointing heavy metal contaminated sediments
(Bitton et al., 1992). This bioassay ranked the East
Wall site as the most toxic, which again is in
agreement with the chemical analyses results for
this sediment. The Douglas sediment was unex-
pectedly ranked as non-toxic. It should be noted
however, that the MetPAD?assay is conducted
with elutriates extracted with moderately hard
water, and on the basis of the results presented for
this bioassay, heavy metals in the Douglas or
Ballymacoda samples were not bioavailable. To
permit a more accurate representation of in situ
conditions, there would be clear advantages to the
use of a solid phase biotest capable of assessing the
bioavailability of metals.
Persoone et al. (1989) previously reported an
LC50value for potassium dichromate of 22.2 mg/l
(25 ?C and 35&) with A. salina, which was in
excellent agreement with results established in this
study. Toxicity was not observed following expo-
sure of this test species to the aqueous phases
extracted from any of the three sediment samples
tested. These results concur with the findings of
Weideborg et al. (1997) who also demonstrated
A. salina to have too low a sensitivity to be con-
sidered as an appropriate bioassay organism for
While it was not possible to correlate the ob-
served ecotoxicological effects with a specific and/
or class of contaminants it is notable that the East
Wall site was consistently ranked the most toxic
with all test species and all test phases. This finding
concurs with the results of chemical characterisa-
tion conducted on this site, which demonstrated it
to be the most polluted based on the contaminants
analysed. The East Wall site is situated in the
largest container port on the East coast of Ireland,
and the surrounding area supports a large popu-
lation (2000+ persons km)2). As the Republic’s
principal port, Dublin port provides roll-on
752 Davoren et al.
roll-off facilities for a variety of different opera-
tions including liquid bulk (e.g. oil bitumen), and
dry bulk (e.g. concentrate coal and animal feed-
stuffs, fertiliser). In addition, the high population
density and the proximity of the site to a major
transport corridor are potential sources of PAHs,
BFRs and heavy metals. The results of both the
chemical characterisation and ecotoxicity tests,
especially those of the elutriate tests; indicate that
further hazard assessment of the East Wall site is
While comparatively high contaminant levels
were also observed in the Douglas site, ecotoxi-
colgical responses were not as pronounced as those
following exposure to the East Wall site. This
disparity would suggest that the contaminants in
the Douglas sediment were less bioavailable than
in the East Wall site. Previous research on the
Douglas site, has demonstrated this sediment to
elicit a genotoxic effect in Tapes semidecussatus
(Coughlan et al., 2002; Hartl et al., 2004), which
highlights that identification of sub-lethal specific
effects requires more detailed investigation. This
underlines that each test has its own specific sen-
sitivity and, thus, to reliably predict the potential
hazards associated with contaminated sediment
undue emphasis should not be placed on any single
In conclusion, the use of a multi-battery and
multi-phase approach is strongly recommended on
the basis of results from this study. In terms of
identifying appropriate Tier I screening tests for
Irish sediments, the use of both the Microtox?and
algal bioassays are recommended. The bioassay
results obtained in this study highlight the inherent
problems of conducting assays on estuarine sys-
tems. In particular, the effects of salinity and
geophysical properties need to be taken into
account in the interpretation of the results.
Finally, as hormesis is increasingly being recogni-
sed as a toxicological response, the detection of
biostimulation should not be overlooked, but
rather, indicates that further monitoring of the site
may be warranted.
We gratefully acknowledge Andy Fogarty and
the staff of Athlone Institute of Technology for
Microtox?and algal tests. Sincere gratitude to
Kathleen O’ Rourke for supplying the algal cul-
ture and Linda Tyrell of the Marine Institute,
Abbotstown, Dublin for conducting the metal
analysis on the sediment samples. This research
was funded by The Higher Education Authority
under the Program of Research in Third Level
Institutions (Cycle 2) as part of the Environ-
mental Research Institute, University College
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