Chemosphere 86 (2012) 105–110

Contents lists available at SciVerse ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Short Communication

New methodological improvements in the MicrotoxÒ solid phase assay

Karen F. Burga Pérez a,⇑, Rayna Charlatchka b, Leila Sahli c, Jean-François Férard a
a
LIEBE (Laboratoire des Interactions Ecotoxicologie, Biodiversité, Ecosystèmes), Université Paul Verlaine-Metz, UMR CNRS 7146, Campus Bridoux, Batiment IBISE,
Rue Delestraint, 57070 Metz, France
b
EDF – Electricité de France, R&D, Laboratoire National d’hydrologie et Environnement, 7 Chatou, France
c
LBE – Laboratoire de Biologie et Environnement, Campus Chaab-Ersas, Biopôle, Université Mentouri, Route Ain El Bey, 25000, Constantine 8401, Algeria

a r t i c l e i n f o a b s t r a c t

Article history: The classic MicrotoxÒ solid phase assay (MSPA) based on the inhibition of light production of the marine
Received 13 June 2011 bacteria recently renamed Aliivibrio fischeri suffers from various bias and interferences, mainly due to
Received in revised form 19 August 2011 physico-chemical characteristics of the tested solid phase. To precisely assess ecotoxicity of sediments,
Accepted 22 August 2011
we have developed an alternative method, named MicrotoxÒ leachate phase assay (MLPA), in order to
Available online 29 September 2011
measure the action of dissolved pollutants in the aqueous phase. Two hypotheses were formulated to
explain the observed difference between MSPA and MLPA results: a real ecotoxicity of the solid phase
Keywords:
or the fixation of bacteria to fine particles and/or organic matter. To estimate the latter, flow cytometry
Sediment
Adsorption
analyses were performed with two fluorochromes (known for their ability to stain bacterial DNA), allow-
Bacteria ing correction of MSPA measurements and generation of new (corrected) IC50. Comparison of results of
Ecotoxicity MLPA with the new IC50 MSPA allows differentiating real ecotoxic and fixation effect in classic MSPA
Bioluminescence especially for samples with high amount of fines and/or organic matter.
Flow cytometry Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction
The MicrotoxÒ solid phase assay (MSPA) initially developed by
Brouwer et al. (1990) is one of the most popular tests for solid
phase assessments (Van Beelen, 2003). The MicrotoxÒ test was
performed initially with liquid samples, as it is based on the inhi-
bition of light production of the marine bacterium recently re-
named Aliivibrio fischeri (Urbanczyk et al., 2007). The MSPA has
been recommended as a screening tool to test solid media like
soils, sludges and sediments (Burton et al., 1996; Ringwood et al.,
1999; Doherty, 2001; Doe et al., 2005). It has been reported that
the test responses showed Pearson correlation with responses of
the seed germination-root elongation in Cucumis sativus (Burton
et al., 1996). Dellamatrice et al. (2006) found also a relationship be-
tween MSPA and Luminotox solid phase assay results.
It has been demonstrated that the test is easy, rapid and repro-
ducible (Stronkhorst et al., 2004). The test protocol has been thor-
oughly detailed by Doe et al. (2005). Briefly, bacteria are exposed to
serial dilutions of sediments, and after 20 min of contact, dispos-
able filters are inserted into the test tubes in order to separate
the liquid phase containing the bacteria from the sediment. Light
output is then measured after 5, 10, 15 or 30 min with the bacteria
contained in the liquid phase.
⇑ Corresponding author. Tel.: +33 3 87 37 84 28/6 59 32 64 49; fax: +33 3 87 37
85 12.
E-mail address: karenburga@yahoo.com (K.F. Burga Pérez).
It has been reported that test sensitivity can be affected by some
chemicals often present in sediments such as ammonium (Ankley
et al., 1990), elemental sulphur (Pardos et al., 1999) or hydrogen
sulphide (Jacobs et al., 1992). As reported by Doe et al. (2005), inter-
ferences can also occur by optical factors linked to the filtrate, due
to colour (light absorption) and/or turbidity (light scatter) coming
from the fines particles not removed by filtration. Also, Campisi
et al. (2005) have reported that there were a poor correlation
(r2 = 0.13) between organic matter (OM) and initial light loss mea-
sured in the modified Basic Solid Phase Test that they proposed. A
better linear correlation (r2 = 0.41, p < 0.05) was reached between
initial light loss and the calculated amount of OM in the cuvette.
But, with the traditional MSPA, Guerra et al. (2007) did not find cor-
relation between responses in MSPA and organic matter. Volpi Ghi-
rardini et al. (2009) have also listed as confounding factors pH
variations linked to sample dilutions. Another important confound-
ing factor is related to the fixation of bacteria to fine-grained sedi-
ments during the exposure time, leading to a significant loss of
bacteria in the recovered liquid phase. Different authors have
pointed out IC50 value overestimations. For example Benton et al.
(1995) found that apparent artificial sediment toxicity was related
to particle size, especially fine particles (i.e. clay + silt). Quintino
et al. (1995) also showed a negative relationship between IC50 val-
ues and percentage of fines content. Ringwood et al. (1997) tested
the hypothesis that bacteria are fixed by clay particles and they
showed that light output decreased in a predictable manner as clay
content increases and proposed a log–log relationship between

0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2011.08.042
106 K.F. Burga Pérez et al. / Chemosphere 86 (2012) 105–110
IC50 and clay (+silt) content. All these authors used artificial sedi-
ments, and this paper thus seeks to review the relationship be-
tween MSPA IC50 and fines (clay + silt) by quantifying the
percentage of bacteria not recovered in the liquid phase for each
concentration of sediment tested using flow cytometric techniques.
To discriminate between leachate and bulk effects, we have also
developed an alternative to the MSPA, that is a MicrotoxÒ leachate
phase assay (MLPA) carried out exactly in the same conditions as
the MSPA in terms of volume, processing time (20 min of contact
of bacteria with sediments), except for the time of introduction of
bacteria into the sediment solution, which are added after filtration
of the sediment dilution and not before like in the MSPA (for details
see Section 2).
2. Materials and methods
2.1. Natural sediments
Sediments were collected from different sites in France. They
were selected on the basis of their putative ecotoxicity and their
different quantities of fines. Dry weights were determined on three
replicates after 3 d at 60 °C. The grain-size distribution was mea-
sured on wet sediment with a Coulter LS-100, which can measure
in a range of measurement between 0.4 and 900 lm (Loizeau et al.,
1994).
2.2. MicrotoxÒ assays
(a) The MicrotoxÒ solid phase assay (MSPA) was performed
according to Doe et al. (2005) with slight modifications
(see below). All materials and reagents were purchased from
R-Biopharm (Lyon, France). In this test, luminescent bacteria
were regenerated with 1 mL of reconstitution solution and
placed in the reagent well of the MicrotoxÒ analyzer for at
least 1 h. During this time, sediment dilutions were prepared
in triplicates. Seven grams of wet sediment were taken from
the sample and diluted with 35 mL 2% sodium chloride
(NaCl), the solution was mixed on a magnetic stirrer for
10 min. From this solution, 1:2 dilutions were prepared in
borosilicate tubes in which 1.5 mL of 2% NaCl was previously
placed. Bacteria (approx. 1  106 cell mL1 per assay) were
exposed to control (1.5 mL of 2% NaCl) and each dilution of
sediment for 20 min, and, at end of exposure, a column filter
of polyethylene was pushed in and 0.5 mL of supernatant
containing exposed bacteria was pipetted and transferred
to a new borosilicate tube. According to the supplier, the fil-
ter has a diameter of 6 mm and a thickness of 4 mm, and the
pore size ranges from 15 to 45 lm. Finally, light output was
measured after 5 min with a MicrotoxÒ Analyzer 500.
(b) The MicrotoxÒ leachate phase assay (MLPA) is a MSPA mod-
ified assay, where the only difference concerns the fact that
A. fischeri is exposed after filtration of sediment dilutions,
and not before (Fig. 1). Afterwards, it becomes a lixiviate
MicrotoxÒ assay conducted in the same conditions as the
MSPA assay. This new assay allows to differentiate the
action of contaminants that passed through the column filter
(i.e. those which are dissolved in the aqueous phase and also
those attached to fines that are recovered in the filtrate)
from those that do not pass through the filter. The same
kinds of supplies of MSPA (tubes, filters and reagents) were
used in this test. The control was prepared by 2% NaCl fil-
tered with the MSPA column filter. Bacteria were exposed
to each filtrate during 20 min after filtration of the sediment
dilutions, then 0.5 mL of filtrate which contains exposed
bacteria were pipetted and light output was measured after
5 min (with a MicrotoxÒ Analyzer 500). It is important to
note that for each MSPA and MLPA IC50 determinations,
the same concentrations of sediment were tested, using
the same regenerated bacterial reagent. So, results of both
tests are directly comparable.
2.3. Flow cytometry analysis
DNA of bacteria was stained with Syto 13 (Invitrogen S7575)
and Sybr-Green I (Invitrogen S7563) dyes. Both were used to quan-
tify the percentage of bacteria retained by the sediment and the fil-
ter. For each determination, we carried out two different assays (in
triplicates): one with sediment and bacteria and another with sed-
iment but without bacteria to take into account the endogenous
bacterial DNA quantity. We also carried out one control (also in
triplicate) without filtration. For each assay or control, 1.5 mL of
samples were stained at a final concentration of 5 lM Syto 13
and 10 lL per mL of sample from a 102 dilution of the Sybr-Green
I commercial solution in distilled water. They were incubated with
experimental media for 15 min at room temperature in the dark
(Marie et al., 1997; Troussellier et al., 1993; Gasol and Del Giorgio,
2000).
Samples were analysed using a FacsCalibur flow cytometer
(Becton Dickinson) equipped with an air cooled argon laser
(488 nm, 15 mW). Stained bacterial cells were discriminated and
enumerated according to their right angle light scatter (SSC, related
to cell size) and green fluorescence (FL1) emission measured at
530 ± 30 nm. Both parameters were collected at logarithmic scale.
Controls were first analysed to eliminate the R1 zone where bacte-
ria were detected in order to exclude background noise (Fig. 2A).
Calculation of the percentages of bacteria retained by each sedi-
ment concentration and the filter were made by subtraction from
the sediment and bacterial assay count (Fig. 2B), the bacterial
count generated by the endogenous bacteria (Fig. 2C).
2.4. Data analysis
Five minute IC50s and their 95% confidence intervals were calcu-
lated with the REGTOX EV7.0 package (http://www.normale
sup.org/~vindimian/fr_index.html) and expressed in g of dry wei
ght sediment per litre (g L1). Differences in sensitivity between
both tests were judged according to overlapping of 95% confidence
intervals. Statistically significant differences between two fluoro-
chromes were tested with the Student t-test.
3. Results and discussion
3.1. MicrotoxÒ solid phase assay (MSPA) and MicrotoxÒ leachate phase
assay (MLPA)
These companion tests are representative of direct and indirect
tests, respectively (Pandard et al., 2006). Nevertheless, the first
(MSPA) is not a true direct test because bacteria are in contact with
a solid phase (i.e. sediment particles) but also with a liquid phase
(i.e. saline medium that is an inherent component of the MicrotoxÒ
test). Thus in MSPA, bacteria are impacted by contaminants either
fixed on particles or dissolved in the saline medium. The second
(MLPA) is a leachate test where bacteria are mainly exposed to
contaminants dissolved in the saline medium. Nevertheless bacte-
ria could also be exposed to fines that could have passed through
the filter.
Results of the seven sediments tested with both assays are pre-
sented in Fig. 3. They were ranked according to their IC50 values for
the MSPA (series 1) and the MLPA (series 2). There is a monotonous
relationship between these two sets of data (p < 0.01, Spearman’s
 K.F. Burga Pérez et al. / Chemosphere 86 (2012) 105–110
Conventional solid phase assay
Sediment + NaCl (2%)
Addition of bacteria
20 minutes of contact
Filtration
Light output measurement
after 5 minutes
Fig. 1. MicrotoxÒ solid phase assay and MicrotoxÒ leachate phase assay protocols.
rank correlation coefficient test). The IC50s of series 1 are all signif-
icantly lower (p < 0.05) than those of series 2 (Fig. 3). This result was
expected because MSPA and MLPA could be regarded as solid + li-
quid assay and liquid assay (at a glance), respectively.
To explain the differences between MSPA and MLPA IC50 val-
ues, two hypotheses were formulated:
(a) The differences are linked to a real ecotoxicity of the solid
phase: this means that decrease of luminescence comes
from the direct contact of bacteria with contaminants fixed
on sediment particles.
(b) The differences are linked to fixation of bacteria to fines par-
ticles and/or organic matter and it can be explained by a
quantification of no-recovered bacteria in the filtrates (see
below).
3.2. Flow cytometric analyses
In a first experiment, we quantified the percentage of bacte-
rial loss after filtration with fluorochromes Syto 13 and Sybr-
Green I on two sediments (Fig. 4A and B). Both fluorochromes
showed an increase of bacterial loss as a function of sediment
concentrations surrounding the IC50s. The bacterial loss was
estimated to be between 26.1% and 89.8% according to the tested
sediment concentration. Comparison of the percentages of loss
evaluated with both fluorochromes for each sediment concentra-
tion showed significant differences in four cases on eight tested
sediment concentrations. In the four other cases, the percentages
of bacterial loss evaluated with Syber-Green I were slightly
greater than those evaluated with Syto 13. Since either fluoro-
chrome can be used for quantification of bacterial loss, we then
decided to use Sybr Green I to test five other natural sediments
and to use it for correction of bioluminescence measurements.
3.3. Correction of IC50 values with Sybr-Green I measurements
There is a log–log significant relationship between the percent-
ages of bacterial loss and the percentages of fines (r2 = 0.82, p-
value = 2  1013) present in the different sediment concentration
(Fig. 5).
This relationship therefore allows us to use the regression
parameters to calculate adjusted bacterial loss for each percentage
of fines present in the tubes, so as to adjust bioluminescence mea-
surements according to the following formula:
107
Leachate phase assay
Sediment + NaCl (2%)
Filtration
Addition of bacteria
20 minutes of contact
Light output measurement
after 5 minutes
Corrected bioluminescence ¼ Measured bioluminescence ð1
þ bacterial loss in%ÞÞ
Table 1 shows the new (corrected) IC50s recalculated from cor-
rected bioluminescence. For five sediments (PG2, LCB2, BB2, CMC2,
ETAB) the new MSPA IC50s were not different from their respective
MLPA IC50s. It can be then concluded that the first (non-adjusted)
IC50s were overestimated due to bacterial fixation onto fines. For
two sediments (CMA2 and LAV) new (adjusted) IC50s were differ-
ent from their respective MLPA IC50s and it can be concluded that
there is a real toxic effect due to contact of bacteria with sediment
particles.
This study supports the results obtained by Benton et al. (1995),
Ringwood et al. (1997) and Quintino et al. (1995) who demon-
strated that bacterial loss is directly related to the percentage of
fines (i.e. clay + silt) in sediments. Bacteria often tend to adhere
to insoluble particles, usually via specific components located on
the bacterial surface (Costerton et al., 1985) or via elevated produc-
tion of extracellular polymeric substances (Ivanova et al., 2008).
For example, Bacillus subtilis may be adsorbed by humic sub-
stances (Maurice et al., 2004).
Here also we checked that bacteria remaining fixed by sediment
particles were not recovered after filtration causing a decrease in
the bioluminescence signal and then an overestimation of IC50s.
In our study the IC50 was significantly related with grain size
of sediments and a different relationship was found from that
reported by Ringwood et al. (1997) in their pilot study (Fig. 6).
This difference could be due to the fact that Ringwood et al.
(1997) used sediments with a wide range of fines percentage
(0.2–82.7), thereby establishing a better directed linear regression.
The difference between both linear regressions in Fig. 6 could be
due to different physicochemical characteristics of both sample
series (i.e. types of clay or silt, clay/silt ratio, etc.). It is also interest-
ing to note that our flow cytometric correction method includes
different kinds of confounding factors (clay, silt, organic matter,
etc.). Comparing the percentage of bacterial loss calculated from
the results of bacterial concentration found by Ringwood et al.
(1997) in their clay mixtures, it was found that in our study the
percentage of bacterial loss was up to 94% of bacterial loss (for
75% of silt + clay) whereas Ringwood et al. (1997) have found up
to 87% of bacterial loss (for 100% of one kind of clay, i.e. kaolin).
These differences could also be due to the fact that we have tested
natural sediments, and consequently the interaction between bac-
teria and different kinds of fines and organic matter as for example
108 K.F. Burga Pérez et al. / Chemosphere 86 (2012) 105–110

Fig. 2. Flow cytometry dot plots FL1 vs. SSC showing from the left to the right: (A) Control test (Aliivibrio fischeri in the zone named R1), (B) bacteria with 1.25% of sediment
LCB2, and (C) 1.25% of sediment LCB2 where endogenous bacteria were found.
 K.F. Burga Pérez et al. / Chemosphere 86 (2012) 105–110 109

* 2.10

LOG (% bacterial loss +1)

(g dry weight sediment/L)

50 2.00

40 1.90

30 1.80
IC50

* 1.70 y = 0.4491x + 1.5367

20
* * * R2 = 0.8198
* 1.60
10 *
1.50
0
PG2 LCB2 BB2 CMA2 CMC2 ETAB LAV 1.40
Sediments 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40
LOG ( Silt + Clay +1)
Fig. 3. IC50s related to solid phase (white bars) and leachate phase (dark bars)
assays for all samples (indicates no overlapping of 95% confidence intervals). Fig. 5. Plot of percentages of bacterial loss in relation to percentages of silt + clay
for each tested sediment concentration.
100
90 (A) *
Bacteria loss (%)

80
70 2.00 Our study
60 y = -0.7927x + 1.9353
1.50 2
50 R = 0.5418
40

Log (IC50 + 1)
30 1.00
20
10 0.50
0
0.00 0.47 0.94 1.88 3.76
0.00 Ringwood et al. 1997
Sediment concentration (g dry weight /L) (Pilot study)
-0.50 y = -0.6775x + 1.2662
100 * * 2
R = 0.7593
90 (B) -1.00
Bacteria loss (%)

80 * 0.00 0.50 1.00 1.50 2.00 2.50

70
60 Log (Clay+ Silt %)+1
50
40 Fig. 6. Plot of IC50 values in MSPA (dark triangles) and the Ringwood et al. (1997)
30 study (dark squares) in relation to percentage of silt + clay for each sediment tested.
20
10
0
0 1.60 3.20 6.40 12.79
Sediment concentration (g dry weight /L)
(b) The second is a method of correction based on flow cytome-
Fig. 4. Percentages of bacterial loss for each sediment concentration tested with try analyses. As suggested by Ringwood et al. (1997), we also
Syto 13 (stripped bars) and Sybr-Green I (filled bars): (A) sediment CMA2 and (B) proposed to use a log–log plot to make corrections of MSPA
sediment PG2. Significant differences at p < 0.01, test t-student. IC50s.

Hence, a combination of both methods constitutes a useful tool

humic substances could be more complex (Fein et al., 1999; War- for differentiating between liquid and true solid effects. As a per-
ren and Haack, 2001). spective, it would be also very interesting to look at the viability
of fixed and not recovered bacteria when there is a true solid phase
4. Conclusions assay effect.

This study demonstrated the bias related to fixation of bacteria

on particles in natural sediments when performing the MSPA. Acknowledgements
Moreover, we proposed two improvements for this test:
Funding for this project was provided by the ANR-PRECODD
(a) The first is the development of MLPA to differentiate (DIESE). The authors would like to thank Christian Blaise for his
between leachate and bulk effects. feedback on this document.

Table 1
MSPA, MLPA and new MSPA IC50s (recalculated from loss of bacteria for each sediment).

Station MSPA MLPA New MSPA

IC 50% (g dry weight L1) Confidence intervals IC 50% (g dry weight L1) Confidence intervals IC 50% (g dry weight L1) Confidence intervals
95% 95% 95%
PG2 3.30 2.81 3.97 7.83 6.52 9.19 9.60 7.50 12.00
LCB2 5.00 4.27 5.76 8.02 7.31 8.83 6.70 5.29 8.26
BB2 25.55 19.33 31.89 52.30 47.67 56.59 48.74 37.00 71.50
CMA2 0.69 0.63 0.74 1.01 0.96 1.06 0.88 0.81 0.95
CMC2 2.78 2.62 2.96 4.02 3.80 4.27 3.70 3.50 4.00
ETAB 3.00 2.25 3.78 5.61 4.83 6.50 3.46 2.50 5.88
LAV 5.50 3.95 7.10 14.07 13.81 14.36 11.30 10.20 12.70
110 K.F. Burga Pérez et al. / Chemosphere 86 (2012) 105–110

References Jacobs, M.W., Delfino, J.J., Bitton, G., 1992. The toxicity of sulfur to Microtox from
acetonitrile extracts of contaminated sediments. Environ. Toxicol. Chem. 11,
1137–1143.
Loizeau, J.-L., Arbouille, D., Santiago, S., Vernet, J.P., 1994. Evaluation of a wide range
Ankley, G.T., Katko, A., Arthur, J.W., 1990. Identification of ammonia as a major
laser diffraction grain size analyser for use with sediments. Sedimentology 41,
sediment associated toxicant in the lower Fox River and Green Bay, Wisconsin.
353–361.
Environ. Toxicol. Chem. 9, 313–322.
Marie, D., Partensky, F., Jacquet, S., Vaulot, D., 1997. Enumeration and cell cycle
Benton, M.J., Malott, M.L., Knight, S.S., Cooper, C.M., Benson, W.H., 1995. Influence of
analysis of natural populations of marine picoplankton by flow cytometry using
sediment composition on apparent toxicity in a solid-phase test using
the nucleic acid stain SYBR Green I. Appl. Environ. Microbiol. 63, 186–193.
bioluminescent bacteria. Environ. Toxicol. Chem. 14, 411–414.
Maurice, P.A., Manecki, M., Fein, J.B., Schaefer, J., 2004. Fractionation of an aquatic
Brouwer, H., Murphy, T., McArdle, L., 1990. A sediment contact bioassay with
fulvic acid upon adsorption to the bacterium, Bacillus subtilis. Geomicrobiol. J.
Photobacterium phosphoreum. Environ. Toxicol. Chem. 9, 1353–1358.
21, 69–78.
Burton, G.A., Ingersoll, C.G., Burnett, L.C., Henry, M., Hinman, M.L., Klaine, S.J.,
Pandard, P., Devillers, J., Charissou, A.M., Poulsen, V., Jourdain, M.J., Férard, J.F.,
Landrum, P.F., Ross, P., Tuchman, M., 1996. A comparison of sediment toxicity
Grand, C., Bispo, A., 2006. Selecting a battery of bioassays for ecotoxicological
test methods at three great lakes areas of concern. J. Great Lakes Res. 22, 495–
characterization of wastes. Sci. Total Environ. 363, 114–125.
511.
Pardos, M., Benninghoff, C., Thomas, R.L., Khim-Heang, S., 1999. Confirmation of
Costerton, J.W., Marrie, T.J., Cheng, K.J., 1985. Phenomena of bacterial adhesion. In:
elemental sulfur toxicity in the microtoxt assay during organic extracts
Savage, D.C., Fletcher, M. (Eds.), Bacterial Adhesion, Mechanisms and
assessment of freshwater sediments. Environ. Toxicol. Chem. 18, 188–193.
Physiological Significance. Plenum Press, New-York, pp. 3–43.
Quintino, V., Picado, A.M., Rodrigues, A.M., Mendonça, E., Costa, M.H., Bordalo Costa,
Campisi, T., Abbondanzi, F., Casado-Martinez, C., Del Valls, T.A., Guerra, R., Iacondini,
M., Lindgaard-Jorgensen, P., Pearson, T.H., 1995. Sediment chemistry – infaunal
A., 2005. Effects of sediment turbidity and color on light output measurements
community structure in a southern european estuary related to solid-phase
for Microtox Basic Solid-Phase Test. Chemosphere 60, 9–15.
microtox toxicity testing. Neth. J. Aquat. Ecol. 29, 427–436.
Doe, K., Scroggins, R., Mcleay, D., Wohlgeschaffen, G., 2005. Solid-phase test for
Ringwood, A.H., Delorenzo, M.E., Ross, P.E., Holland, A.F., 1997. Interpretation of
sediment toxicity using the luminescent bacterium Vibrio fischeri. In: Blaise, C.,
Microtox™ solid-phase toxicity tests: the effects of sediment composition.
Férard, J.F. (Eds.), Small-scale Freshwater Toxicity Investigations, vol. 1.
Environ. Toxicol. Chem. 16, 1135–1140.
Springer, Dordrecht, pp. 107–136.
Stronkhorst, J., Ciarelli, S., Schipper, C.A., Postma, J.F., Dubbeldam, M., Vangheluwe,
Doherty, F.G., 2001. A review of the Microtox toxicity test system for assessing the
M., Brils, J.M., Hooftman, R., 2004. Inter-laboratory comparison of five marine
toxicity of sediments and soils. Water Qual. Res. J. Canada 36, 475–518.
bioassays for evaluating the toxicity of dredged material. Aquat. Ecosyst. Health
Dellamatrice, P.M., Monteiro, R.T.R., Blaise, C., Slabbert, J.L., Gagné, F., Alleau, S.,
Manage. 7, 147–159.
2006. Toxicity assessment of reference and natural freshwater sediments with
Troussellier, M., Courties, C., Vaquer, A., 1993. Recent applications of flow cytometry
the Luminotox assay. Environ. Toxicol. 21, 395–402.
in aquatic microbial ecology. Biol. Cell 78, 111–121.
Fein, J.B., Boily, J.F., Guçlu, K., Kaulbach, E., 1999. Experimental study of humic acid
Urbanczyk, H., Ast, J.C., Higgins, M.J., Carson, J., Dunlap, P.V., 2007. Reclassification of
adsorption onto bacteria and Al-oxide mineral surfaces. Chem. Geol. 162, 33–
Vibrio fischeri, Vibrio logei, Vibrio salmonicida and Vibrio wodanis as Aliivibrio
45.
fischeri gen. nov., comb. nov., Aliivibrio logei comb. nov., Aliivibrio salmonicida
Gasol, J.M., Del Giorgio, P.A., 2000. Using flow cytometry for counting natural
comb. nov. and Aliivibrio wodanis comb. nov. Int. J. Syst. Evol. Microbiol. 57,
planktonic bacteria and understanding the structure of planktonic bacterial
2823–2829.
communities. Sci. Mar. 64, 197–224.
Van Beelen, P., 2003. A review of the application of microbial toxicity test for
Guerra, R., Pasteris, A., Ponti, M., Fabbri, D., Bruzzi, L., 2007. Impact of dredging in a
deriving sediment quality guidelines. Chemosphere 53, 795–808.
shallow coastal lagoon: Microtox Basic Solid-Phase Test, trace metals and
Volpi Ghirardini, A., Girardini, M., Marchetto, D., Pantani, C., 2009. Microtox solid
Corophium bioassays. Environ. Int. 33, 469–473.
phase test: effect of diluents used in toxicity test. Ecotoxicol. Environ. Saf. 72,
Ivanova, E.P., Dineva, N.M., Mocanasu, R.C., Murphy, S., Wang, J., Riessen, G.,
851–861.
Crawford, R., 2008. Vibrio fischeri and Escherichia coli adhesion tendencies
Warren, L.A., Haack, E.A., 2001. Biogeochemical controls on metal behaviour in
towards photolithographically modified nanosmooth poly (tert-butyl
freshwater environments. Earth Sci. Rev. 54, 261–320.
methacrylate) polymer surfaces. Nanotechnol., Sci. Appl. 1, 33–44.

View publication stats