Hewitt and Marvin 2005

Mutation Research 589 (2005) 208232
www.elsevier.com/locate/reviewsmr
Community address: www.elsevier.com/locate/mutres
Review
Analytical methods in environmental effects-directed

investigations of effluents
L. Mark Hewitt a,*, Chris H. Marvin b
a
Aquatic Ecosystem Protection Research Branch, National Water Research Institute, Environment Canada,
867 Lakeshore Road, Burlington, Ont., Canada L7R 4A6
b
Aquatic Ecosystem Management Research Branch, National Water Research Institute,
Environment Canada, 867 Lakeshore Road, Burlington, Ont., Canada L7R 4A6
Received 2 September 2004; received in revised form 31 December 2004; accepted 10 February 2005
Available online 21 March 2005
Abstract
Effluent discharges are released into aquatic environments as complex mixtures for which there is commonly either no
knowledge of the toxic components or a lack of understanding of how known toxicants interact with other effluent components.
Effects-directed investigations consist of chemical extraction and iterative fractionation steps directed by a biological endpoint
that is designed to permit the identification or characterization of the chemical classes or compounds in a complex mixture
responsible for the observed biological activity. Our review of the literature on effects-directed analyses of effluents for nonmutagenic as well as mutagenic endpoints showed that common extraction and concentration methods have been used. Since the
mid-1980s, the methods have evolved from the use of XAD resins to C18 solid-phase extraction (SPE). Blue cotton, blue rayon,
and blue chitin have been used specifically for investigations of mutagenic activity where polycyclic compounds were involved
or suspected. After isolation, subsequent fractionations have been accomplished using SPE or a high-pressure liquid
chromatography (HPLC) system commonly fitted with a C18 reverse-phase column. Substances in active fractions are
characterized by gas chromatography/mass spectrometry (GCMS) and/or other spectrometric techniques for identification.
LCMS methods have been developed for difficult-to-analyze polar substances identified from effects-directed studies, but the
potential for LCMS to identify unknown polar compounds has yet to be fully realized. Salmonella-based assays (some
miniaturized) have been coupled with fractionation methods for most studies aimed at identifying mutagenic fractions and
chemical classes in mixtures. Effects-directed investigations of mutagens have focused mostly on drinking water and sewage,
whereas extensive investigations of non-mutagenic effects have also included runoff, pesticides, and pulp mill effluents. The
success of effects-directed investigations should be based on a realistic initial objective of each project. Identification of
chemical classes associated with the measured biological endpoint is frequently achievable; however, confirmation of individual
compounds is much more difficult and not always a necessary goal of effects-directed chemical analysis.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Effects-directed; Fractionation; Endpoint; Mutagen; Endocrine disruptor; Effluent
* Corresponding author. Tel.: +1 905 319 6924; fax: +1 905 336 6430.
E-mail address: mark.hewitt@ec.gc.ca (L.M. Hewitt).
1383-5742/$ see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrrev.2005.02.001
L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232
209
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effluent and drinking water mutagenic investigations . . . . .
Non-mutagenic effluent evaluations . . . . . . . . . . . . . . . . .
3.1. Pulp mill effluents . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Sewage effluents . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Other effluents . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Runoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Endpoint considerations in bioassay-directed investigations.
Conclusions and future directions . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Several million kilograms of genotoxic and other
biologically active substances are released into the
environment each year [1]. Most substances are
released as components of complex mixtures, such as
liquid effluents, airborne emissions and solid wastes.
There are also additional unknown toxicants released
in these mixtures and those produced through biotic
and abiotic processes. Because of the complexity of
the emissions, standard target-chemical analyses are
limited in their ability to generate adequate information on the toxic potential on a chemical-specific basis.
Non-target analysis of complex mixtures allows the
detection of a broader range of compounds; however
the results are often difficult to interpret since
toxicological data for compounds detected are not
available, especially as they exist in the matrix of
a given effluent. While bioassay assessments of
industrial wastes provide a means to evaluate and
compare effluents without detailed knowledge of their
chemical compositions [2] they are limited in their
predictive capacity of effluent effects. The coupling of
bioassay assessments with chemical analysis yields
more information than either of these assessments
individually. This is evident in studies where
combined chemical and biological evaluations of
complex environmental mixtures show measured
levels of priority pollutants are a poor indicator of
toxicity [3].
Effects-directed analysis allows a biological endpoint to direct chemical manipulations of a mixture to
separate active components from inactive ones
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209
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(Fig. 1). This approach allows analytical efforts to

be focused on the compounds of greatest relevance,
which are not necessarily known. It conversely allows
confirmation of suspected mutagens or toxicants and
elimination of those compounds not associated with
the effect of concern. The concept of effects-directed
investigations is not new and has been applied since
the late 1970s to identify acutely toxic substances in
industrial effluents, as reviewed in Schuetzle and
Lewtas [4]. The early approaches have evolved into
the general toxicity identification evaluation (TIE)
protocols of the early 1990s [57] that were mainly
driven by US legislation [8].
The present review examines the analytical
approaches used to identify mutagenic compounds
as well as other biologically active substances in
effluents from the perspective of (i) the techniques used
to tackle various effluent matrices, (ii) the evolution of
these techniques with technological developments
and scientific questions, and (iii) the success level
attained. Mutagenic-directed investigations of a variety
of effluents are discussed separately from nonmutagenic research on point-source effluents from pulp
and paper mills, sewage plants, chemical manufacturing
and non-point source sources such as runoff.
2. Effluent and drinking water mutagenic

investigations
A wide range of industrial effluents have been
associated with mutagenic effects, including those
from organic chemical manufacturers, metal refining
210
Fig. 1. General analytical approach for conducting effects-directed investigations of effluents. Adapted from [12,127].
operations, dye manufacturers, petroleum refineries

and pulp and paper mills [9]. It has been reported that
while waste treatment reduces overall toxicity, it does
not always reduce the genotoxicity, and in some cases
can increase it [1]. Effects-directed investigations of
effluents have utilized a variety of approaches to
provide information on the sources and identities of
mutagenic substances entering the environment. The
type of approach used varies with the effluent matrix
being examined and the mutagenic endpoint used to
drive chemical fractionations, but, as will be shown,
the general approaches are similar to non-mutagenic
investigations. In addition to effluent investigations, a
great deal of effects-directed work for mutagenic
activity has focused on drinking water. While the
focus of this review concerns effluent investigations,
the same analytical approaches developed for drinking
water have been applied to effluents and are therefore
included.
Although a large number of genotoxicity assays

have been developed, only a small number have been
used in the evaluations of complex industrial
discharges. Nearly 60% of studies have employed
the Ames Salmonella mutagenicity assay, 22% used
other gene assays, 10% used chromosomal assays, 7%
used DNA damage assays, and 2.5% were in vivo
animal tests [2]. The coupling of the Ames Salmonella
assay to effects-directed investigations of mixtures
enhances the application of this assay, and it is
particularly suited to these investigations as it is easy
to use, cost-effective, and can provide rapid, reliable
results. Salmonella testing is also frequently conducted with the addition of metabolic activation (S9).
S9 is the supernatant resulting from centrifugation of
rat liver homogenate at a centrifugal force of
9000 g. It is used to simulate eukaryotic activation
of compounds in bacterial genotoxic tests and is
normally harvested from the liver of rats in which the
de novo synthesis of biotransformation enzymes is

induced by intraperitoneal injection of a PCB mixture.
In the case of effects-directed mutagenic investigations, care must be exercised when applying S9 and
interpreting data. Decreases in mutagenic activity
have been associated with S9 addition [9], but these
may be artifacts from sorption of genotoxic substances
to membranes and proteins in the S9 mix [10].
Therefore, this assay should only be used to determine
the presence of metabolically activated genotoxicants,
owing to the possibility of false negatives.
Application of mutagenicity tests directly to whole
effluents commonly results in negative or ambiguous
results [11]. To conduct effects-directed investigations
it is therefore usually necessary to concentrate the
active substances (Fig. 1). Pre-concentration has been
the method of choice in the literature [12] and the
method of choice depends on several factors, such as
the volatility of the active substances of interest, the
degree of concentration required and the biological
test system used. Studies prior to the 1980s frequently
employed the use of solvent extraction (sequentially,
with increasing polarity) to isolate bioactive substances from aqueous matrices (reviewed in [4]). This
technique was sufficient when higher concentrations
of the active substances were present. Adsorption
methods employing XAD resins gained increasing
popularity starting in the 1980s because of their
effectiveness at concentrating substances present in
trace quantities, their convenience, decreased solvent
use, and decreased costs. XAD resins are non-ionic
styrene divinylbenzene (SDB)-based polymeric adsorbents that are highly porous structures whose internal
surfaces can adsorb and then desorb a wide variety of
different species depending on the environment in
which they are used. For example, in polar solvents
such as water, polymeric adsorbents exhibit non-polar
or hydrophobic behavior and so can adsorb organic
species that are sparingly soluble. This hydrophobicity
is most pronounced with the styrenic adsorbents.
XAD-2 and XAD-4 are SDB based, are non-polar and
are therefore more popular in the isolation of organic
contaminants from aqueous matrices, while XAD-7,
being based on a polymethacrylate matrix and of
intermediate polarity, can absorb compounds such as
phenols from water.
There are challenges to the pre-concentration step
in any effects-directed investigation. Modifications of
211
substances can also occur during extraction and the

issue of solvent carry-over to the bioassay and its
effects must be accounted for. Further, only a small
proportion of the effluents organic material may be
retained by extraction and the efficiency of the
extraction technique on recovery of the biological
activity needs to be assessed. Gauthier et al. [11]
reported extraction efficiencies of tannery effluents
before and after XAD-4 resin extractions at pH 7 and
2, and also gravimetrically measured the dissolved
organic carbon (DOC) recovered from each extraction, but more often than not this information is not
reported. This is also the case with evaluating activity
following extraction or fractionations steps. For
example, Cerna et al. [13] examined genotoxicity
using the Ames test on water collected from the Labe
River in the Czech Republic in a study that involved
effluent and river water extractions using macroporous
polystyrene gel Separon SE resin columns. Acetone
column extracts were then subdivided into five
fractions based on polarity, acidity, and volatility,
and screened by gas chromatography/mass spectrometry (GCMS), but only 25% of the activity was
recovered after fractionation.
While it is possible that interactions between
individual compounds or matrix components may be
related to the total effect observed in the whole
sample, this could easily be addressed in studies by
recombining fractions and testing the difference from
the original. This is infrequently done and it is thought
that interactive effects (synergism, antagonism) play
a role in the cumulative response. While this may
occur, it has been rarely shown to be the case and
it is much more likely than non-additivity in
recombined fractions is due to losses during extraction and fractionation. For example, alterations
during solvent extraction as well as acid/base
partitioning can induce chemical changes in the
compounds of interest, resulting in apparent losses.
Nevertheless, most studies have adopted successful
extraction approaches to investigate mutagenic
substances, as well as non-mutagenic substances
(see below). Advantages of the extraction approach
include (i) the method of extraction provides
immediate information on the types of chemicals
involved in the effect being studied, (ii) the preservation of the active substances from microbial
degradation once they are contained in a solvent and
212
removed from the effluent matrix, and (iii) concentration of the active substances facilitates the tracking
of the biological response by resolving it from
background and additional mutagens (or other
compounds of interest) that may be present.
One of the most commonly employed methods of
extracting and concentrating mutagens from sewage
effluents and drinking water has been XAD resins.
One of the first studies to utilize XAD resins in these
types of applications was by Kool et al. [14]. It was
found that a combination of XAD-4/8 was as effective
in adsorbing mutagens from surface waters as XAD-2.
Dimethylsulfoxide (DMSO) was found to be as
efficient as acetone in eluting mutagens from XAD
resins and provides adequate delivery of compounds
to the Ames test. In surface waters, the majority of
mutagens were found to be adsorbed at neutral pH.
Optimal recoveries of mutagens in drinking water with
XAD resins have been found using flow rates at 24
bed volumes/min [15]. While convenient and nonresponsive in mutagenicity assays, the use of DMSO
as an elution solvent should be used with caution
since, because of its high boiling point, further
manipulation of extracts or solvent exchange is not
possible.
In a later study, Filipic and Toman [16] used XAD2 resins to extract dissolved mutagenic substances
sequentially at neutral and acidic pH from influents
and effluents from a municipal sewage plant processing both industrial and domestic wastes. The XAD
resins were extracted sequentially with acetone and
dichloromethane (DCM) and the extracts were tested
with and without S9 activation using the Ames test.
Ono et al. [17] used simple extractions of filtered
samples with C18 solid-phase extraction (SPE) to
detect error-prone DNA repair induced by chemicals
in Japanese sewage and nightsoil. Following SPE, the
authors used semi-preparative reverse phase-high
pressure liquid chromatography (RP-HPLC) fractionation and fraction collection every 10 mL. While the
endpoint biased this study for aromatic amines,
ozonation treatment was found to be a treatment
option that removed the activity. Takigami et al. [18]
also used XAD-2 resin under neutral pH at the ratio of
18 L to 50 mL sorbent and a miniaturized Bacillus
subtilis assay to examine genotoxicity in extracts of
Japanese sewage, river water and tap waters. Resins
were eluted sequentially with ethanol followed by
ethyl ether, but no mention was provided as to whether

recovery of genotoxic substances was in any way
quantitative.
Quantitative recovery of mutagenic activity has
been reported for drinking water evaluations using
XAD-2 and XAD-8 resins with varying water pH [19].
The mmutagenicity of pH 2 drinking water concentrates were sevenfold higher than those of the pH 8
extracts, suggesting that acidic compounds accounted
for the majority of the mutagenicity. The presence of
residual chlorine did not affect mutagenicity. Comparisons of the mutagenic activity for the pH 2 versus
pH 8 extracts prepared by lyophilization further
indicated that the acidic mutagens were chlorine
disinfection products [19], which proved that earlier
results associating the formation of mutagens with
residual chlorine and XAD-4 resin [20] were minor
contributors. The indication that mutagenic compounds were present in drinking waters led to the
general acceptance of chlorinated humic material to
cause the formation of 3-chloro-4-(dichloromethyl)-5hydroxy-2L(5H)-furanone, or (MX), initially discovered in drinking water by Hemming et al. [21]. The
identification of MX in drinking water was preceded
by the effects-directed effort, which led to its initial
discovery in spent bleaching liquors as the major
mutagen in pulp mill effluents [22] (see below). MX
was found to occur after chlorination of drinking water
and natural surface waters containing humic material.
In the drinking water study, MX was recovered by
acidifying the samples to pH 2 and passing them
through a column containing a 1:1 mixture of XAD-4
and XAD-8 [21]. After its discovery in drinking water,
a quantitative effects-directed analysis of drinking
waters was conducted by Kronberg et al. [23] where
mutagenic compounds in XAD extracts of chlorinated
humic water were separated in two stages of HPLC
fractionation. XAD extracts were fractionated first by
a preparative C18 column and mutagenic fractions
were then sub-fractionated on a C6 analytical column.
GCMS analyses of mutagenic fractions identified
MX and its geometric isomer, (E)-2-chloro-3(dichloromethyl)-4-oxobutenoic acid (EMX). Both
compounds were detected in extracts of chlorinated
drinking waters, with MX accounting for 2050% of
the total mutagenic activity and EMX accounting for
2% of the activity. Subsequent studies have shown
MX to be the most potent mutagen present in drinking
waters, accounting for the largest proportion of

mutagenic activity. In an analysis of the mutation
spectra of drinking waters treated under pilot-plant
conditions of chlorination, cloramination and ozonation, DeMarini et al. [24] found high frameshift
frequencies in TA98 associated with MX and MXlike compounds. The authors suggested that halogenated aromatics, such as halogenated polycyclic
aromatic hydrocarbons account for much of the
mutagenic activity and specificity of the non-volatile
organics in drinking water.
The discovery that chlorination of humic material
could produce mutagenic substances led to efforts
directed to the mutagenicity of natural waters and
sewage effluents. In the late 1990s, an extensive series
of studies on the mutagenicity of sewage effluents in
Japan covered all aspects of effects-directed investigations, including extraction of mutagens from
effluents, their isolation from inactive components,
proposed structural identifications [25], confirmation
with custom-synthesized authentic standards [26], and
measurements in surface waters of additional analogous chemicals [2729]. In the initial effects-directed
work, Nukaya et al. [25] used blue cotton and blue
rayon to isolate mutagens from sewage effluents in
Japan. These materials consist of rayon or cotton
covalently bound to the blue pigment copper
phthalocyanine trisulfonate (CPT), which selectively
binds multi-cyclic planar compounds. Five mutagens
were recovered using methanol/ammonia water (50:1)
and residues subjected to RP-HPLC were eluted with
an acetonitrile and phosphate buffer mobile phase.
Specific compounds were eventually isolated in two
more levels of subfractionation using RP columns.
Structural work was conducted on a bulk-scale workup
of river water just below sewage treatment plants with
preparative HPLC. Using X-ray crystallography, UV
vis spectrometry, 1H-NMR, and high-resolution mass
spectrometry, the compound with the highest mutagenic activity (21% of the total) against Salmonella
typhimurium was identified as 2-[2-(acetylamino)4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5amino-7-bromo-4-chloro-2H-benzotriazole, or PBTA1. Employing the blue rayon passive sampling
procedure, a total of eight PBTA isomers were
subsequently discovered [2729]; these compounds
account for approximately one-third of the total
effluent mutagenicity [30], and are thought to be
213
produced from azo dyes during effluent treatment

[25].
In related works, a similar technique unique to
mutagenicity investigations has been developed in a
column-based preparation using chitin (poly-N-acetylglucosamine) powder bearing covalently linked CPT
residues [31]. The chief disadvantage of rayon or cotton
supports lies in their limited ability to passively batchtreat water samples, whereas the chitin based solid
phase preparations allow for construction of SPE
columns in Sep-pak cartridges. Constructed cartridges
can then be handled under laboratory conditions, with
smaller sample volumes, and provide quantitative data.
All sorbents bearing CPT have been found to be highly
selective for polycyclic planar structures that can
function as mutagens and thus offer a unique method of
extracting them from aqueous matrices, food and
human excreta [32]. CPTchitin columns have been
used to investigate mutagens in river water contaminated with sewage by elution with methanolammonia
and testing with S. typhimurium TA98 activated with S9
[31]. In one of the few cases of evaluating extraction
efficiency of biological activity, the authors confirmed
the chitin residue contained no residual activity. Further
experiments also showed no effect of water volume,
sample pH, methanol fortification up to 50% (v/v) and
extraction flow rate on recoveries of known mutagens.
A follow-up study examined the extraction efficiencies
of CPTchitin columns, hanging blue-rayon and XAD2 columns for mutagens in two Japanese rivers known
to contain mutagens [33]. The results showed that the
CPTchitin column was more efficient than XAD-2,
and interestingly, that the blue-rayon technique of
hanging in directly in the river was the most sensitive
and convenient. A more recent extensive survey of six
rivers in north-eastern North America using the
hanging blue rayon technique showed that Salmonella
strains YG1041 and YG1024 were much more
sensitive than TA98 with S9 mix and that rivers
flowing through major North American cities contain
frameshift-type, aromatic amine-like mutagenic activity [29].
The CPTchitin technique has since been investigated further and has found numerous applications in
the studies of environmental mutagens, particularly
those that are polyaromatic hydrocarbon (PAH)based. PAH mutagens have been evaluated in detail
and their elution conditions from modified blue chitin
214
columns optimized for the study of river waters [34].

Methanolammonia, followed by dichloromethane,
were found to provide adequate recoveries of 22 PAHs
with three to six rings and NO2PAHs with four to five
rings from river or lake waters. Blue chitin, blue rayon
and blue cotton have been used to recover heterocyclic
amines from various environmental matrices, including river waters [35], with widespread applications to
cooked meats matrices [36].
Kummrow et al. [37] found that by using a
combination of mutagenicity tests and selective
extraction methodologies, the classes of mutagenic
organic contaminants found in surface waters might be
elucidated and linked to their source. In this study,
mutagenicity comparisons of blue rayon and XAD-4
resin extracts of river water below an azo dyeprocessing plant discharge and a reservoir contaminated with untreated sewage were performed. Only
samples collected below the azo dye plant showed
mutagenic activity with the blue rayon extraction,
suggesting the presence of polycyclic compounds in
those samples. In order to better characterize the
classes of mutagens present, Kummrow et al. [37]
recommend using YG strains of Salmonella which are
more sensitive to aryl amines, if they are suspected to
be present. A similar recommendation was recently
made by Umbuzeiro et al. [38] in comparing XAD-4
and blue rayon extracts of river and drinking water in
Brazil. Elevated mutagenicity with YG-strains suggested that nitro-aromatics and/or aromatic amines
were causing the mutagenicity and this was supported
by positive responses from blue rayon.
Application of the blue chitin technique directly to
an effects-directed investigation of municipal sewage
effluents and river waters in Japan was recently
conducted by Nagai et al. [39]. Mutagenicity was
determined by the Ames assay, and strain TA98 was
used to estimate the quantitative contribution rate of
mutagenicity estimated from PAHs. Through the use
of blue chitin columns it was found that in several
surface waters and effluents that the contribution of the
total mutagenic activity from routinely measured
PAHs ranged from 1 to 64%, demonstrating the
contributions of other, non-planar heterocyclics in
mutagenic activity [39]. It is this remaining fraction of
unknown non-PAH type mutagens that presents the
next challenge in mutagenic-directed investigations of
surface waters and effluents.
Beginning in the early 1980s and continuing into

the mid-1990s, mutagenic studies of pulp mill
effluents have employed extraction techniques based
solely on XAD resins. Unlike studies with drinking
water and other surface waters, CPT-based solid
phases have not been employed during investigations
of mutagenic compounds in pulp mill effluents. This is
likely due to matrix effects, in particular the large
amount of lignin material present in pulp mill effluents
that would affect the adsorption of planar polycyclic
mutagens. Early pulp mill studies used in vitro
Salmonella assays to indicate mutagenic activity, and
later studies investigated fish-specific mutagenic
responses. Most of the pulp mill-derived mutagens
are derived from polar compounds produced from
individual waste streams, with corresponding weak
evidence of final effluents containing mutagenic
substances. Efforts were first directed towards
chlorination stage effluents of mill bleach plants that
provided the strongest activity and led to the
association of chloroacetones with mutagenic activity
[40]. Kinae et al. [41] detected genotoxins in livers of
wild fish collected from areas receiving pulp mill
wastes, indicating the potential for exposure and
bioaccumulation. Holmbom et al. [22] used a
combination of ethyl acetate and XAD-4 resins to
quantitatively recover 7090% of the mutagenic
activity from chlorination bleachery effluents. The
majority of the recovered activity was removed by
partitioning with aqueous NaHCO3. Preparative thinlayer chromatography (TLC) was used as a first step in
isolation, followed by C8 RP-HPLC, further preparative TLC, C18 HPLC, and a final TLC step that
allowed the isolation of MX. As this compound was
not amenable to GCMS analyses, methylated,
acetylated and trimethylsilyl derivates were synthesized and analyzed to facilitate structural interpretation.
XAD resin extractions have been used in attempts
to isolate compounds from pulp mill effluents that are
mutagenic in fish-based assays [22,42] but the results
indicate weak activity, and that fish are not affected by
mutagens in final treated effluents. Rao et al. [43] was
able to elute weakly acidic and polar mutagens from
final effluent using an XAD-8 column eluted with
NaOH or methanol. The authors also employed
diethylaminoethyl (DEAE) cellulose to adsorb high
molecular weight lignin interferences from final
effluents, which was also effective in studies of aryl

hydrocarbon receptor agonists (see Section 3.1 and
[44]). Metcalfe et al. [42] found evidence of directacting mutagens in extracts of XAD-7 (methanol) and
XAD-4 (diethyl ether) using a S. typhimurium
fluctuation assay, but not with trout eggs or sac fry
exposed in vivo; further characterizations were not
undertaken.
A spectrum of other municipal and industrial
effluents has been studied for mutagenic activity, and
these studies have employed different analytical
approaches to isolate genotoxic substances, fractionate
them and, in some cases, characterize unknown
mutagens. White et al. [9] conducted a screening study
using the SOS Chromotest to evaluate DCM extracts of
effluents from a cross-section of industries. Samples
were fractionated according to whether substances
were dissolved or particulate-bound. Acid/base partitioning was used to further differentiate those
substances that were water-soluble. S9 metabolic
activation was found to almost exclusively decrease
genotoxic potency. The highest loadings, expressed in
benzo(a)pyrene equivalents, were from sewage plants,
pulp and paper mills and metal refining processes.
Higher loadings were usually associated with effluent
particulate material, and generation of genotoxic
sorption partition coefficients (Kd-genotox), were later
found to generally agree with octanolwater partition
coefficient (Kow) values of known genotoxic substances
[45]. Given that particulate materials originating from
effluents contained higher genotoxic potential, White
et al. [46] further investigated their bioaccumulation
potential in the St. Lawrence and Saguenay River
systems in Canada. Genotoxic concentrations in fish
and invertebrates indicated that metabolism in higher
vertebrates plays a role in lower body burdens.
3. Non-mutagenic effluent evaluations

Several environmental investigations of cause and
effect have employed bioassay-directed fractionation
approaches to identify agents responsible for a variety
of non-mutagenic endpoints of interest. Historically,
these investigations were directed by acute toxicity to
various aquatic species (e.g. rainbow trout, Daphnia
magna). This has changed over the past two decades
since industrial wastes are for the most part regulated on
215
acute parameters and their causal factors have been

established (e.g. low dissolved oxygen (DO), ammonia,
heavy metals). Since global incidences of fish kills have
declined, concern has shifted to other endpoints
associated with chronic effects. One of the best
examples of this has been the focus on endocrine
disruptors associated with reproductive effects in
aquatic biota, wildlife, and the human population
[47]. The following sections of this review highlight
trends in effects-directed investigations of point source
discharges to the environment, in particular effluents,
but also other studies in emerging non-point source
stressors.
3.1. Pulp mill effluents
The effects of pulp mill effluents on aquatic
environments have been examined for over 40 years,
and effects-directed studies have been conducted since
the 1970s. During this period, environmental effects
have been observed, regulations have been implemented, and the industry has responded to these regulations
resulting in significant reductions in acute environmental effects. However, other environmental
responses have persisted, and have become the main
focus of cause and effect research concerning these
discharges since the early 1990s. Because of effluent
complexities and changes in effluent compositions over
the last two decades, this matrix has represented one of
the greatest analytical challenges to overcome in
identifying bioactive substances in complex mixtures.
Early studies on the acute toxicity of effluents from
pulping operations were largely successful. One of the
first effects-directed investigations concerning pulp
mill effluents was by Das et al. [48] who indirectly
implicated tetrachloro-o-benzoquinone and other
chlorodihydroxybenzenes in the acute toxicity of
kraft chlorination liquors to fish. Studies conducted
during the 1970s and 1980s continued to focus on kraft
mill process streams, particularly the chlorination and
extraction stages of bleaching and the chemicals
responsible for acute toxicity to salmonids [49,50].
These studies utilized XAD resins for extraction, acid
partitioning with aqueous base, and fractionation
using silica gel and/or preparative TLC. From these
investigations, resin acids, unsaturated fatty acids and
chlorinated phenolics were determined to be the major
sources of acute toxicity. Diterpene alcohols, pitch
216
dispersants, juvenile insect hormone analogues and

unidentified neutral compounds also contributed to
lesser degrees. This led to increased attention to these
compounds [51], and the discovery of dioxin and
furans led to regulations restricting their discharge in
whole (adsorbable organic halide) or in part (dioxins
and furans) [52]. As a result, the industry adopted
process changes and effluent treatment throughout the
1990s to reduce the loadings of these compounds to
the environment. While obvious benefits have been
accrued from these efforts, subtle effects on fish
reproduction, first noticed in the late 1980s, have
persisted to the present day (reviewed in [53]).
Despite the level of effort over the last decade, and
the various approaches used to identify the acutely
toxic compounds in pulp mill effluents, the compounds
responsible for the persistent reproductive changes in
fish have remained elusive. Historically, studies of
final effluents from pulping operations in general have
proven to be problematic. Difficulties encountered
include: (i) fractionation experiments conducted on
grab samples of effluent do not reflect temporal
fluctuations in active chemicals, (ii) toxicological
potencies of effluent samples are influenced by sample
handling and storage conditions [54], (iii) the large
amount of high molecular weight lignin material is
a significant interference when investigating low
molecular weight extractives [44,55], (iv) the complexity of low molecular weight effluent extractives
[51], and (v) uncertainties regarding the bioavailability
of identified bioactive components [56].
In addition to the obstacles confronting chemical
aspects of bioassay-directed investigations, there has
also been uncertainty surrounding which biological
endpoint to use in directing fractionations. This
uncertainty is chiefly derived from the complexity
of the responses, and lack of definition of a mechanism
associated with the observed effects. While individual
effluent constituents such as b-sitosterol [57], abietic
acid, pinosylvin, and betulin [58] have the potential to
affect fish reproduction when tested individually,
definitive cause and effect relationships have not been
established because of effluent complexity, differences in species response patterns (e.g. between
laboratory species and wild fish), and a lack of
information on the mechanisms of action [59,60].
Despite the difficulty in defining the mechanisms
surrounding the reproductive effects, research in the
area of bioassay-directed compound identification has

progressed on mechanisms that have been derived
from effects assessments of wild fish populations,
namely induction of P450IAI enzymes (measured as
ethoxyresorufin-O-deethylase or EROD activity) and
impacts on levels of gonadal sex steroids. Hewitt et al.
[54] fractionated effluents before and after treatment,
and after a maintenance shutdown at a bleached kraft
mill in one of the first studies to address the role of
secondary treatment in affecting EROD activity.
Laboratory rainbow trout were exposed to treated
and untreated effluent, whole and filtered (<1 mm)
effluent, resuspended solids, and two fractions
of effluent generated by nanofiltration (>400 Da,
<400 Da). These analyses found correlations of
EROD activity with several chlorophenolics, including tetrachloroguaiacol. Subsequent exposures confirmed that tetrachloroguaiacol did not cause
induction, but Hewitt et al. [54] concluded that the
correlations might indicate the potential source of the
compounds is derived from lignin in the wood furnish.
Burnison et al. [44] attempted to directly isolate
chemicals inducing EROD activity in fish by
following an effects-directed approach on final
effluent from two bleached kraft mills located in
Ontario. Using centrifugation, tangential flow filtration, and C18 solid-phase extraction, effluents after
secondary treatment were investigated using a 4-day
rainbow trout in vivo bioassay. It was determined that
methanol extracts of particulates/colloidal material
and SPE fractions contained active substances. Work
focused on the particulate material and showed that
activity could be isolated using methanol extractions.
HPLC isolations determined that the active substances
were present in a relatively non-polar region of the
chromatographic separation, with a log Kow of 4.6
5.1. As a result of follow-up studies using rainbow
trout exposures and incubations with a rat hepatic
carcinoma cell line (H4IIE), which directed HPLC
fractionations of the methanol extract of the high
molecular weight material, a chlorinated ligninderived pterostilbene structure was postulated for an
unknown compound strongly associated with induction [55]. This was significant in that it showed a
natural product, modified in the bleach plant, was
eliciting the biological response.
In a comprehensive study, Martel et al. [61]
determined the source and identities of two substances
associated with induction present in the primarytreated effluent of a newsprint thermomechanical pulp
(TMP) mill. To determine the sources of activity
within the mill, rainbow trout were exposed static for
96 h to TMP condensate, deinking, paper machine
effluents, TMP whitewater, and various process
effluents sampled throughout the mill. Exposure
concentrations were based on the flow of these
process streams in relation to final effluent. Contaminated TMP steam condensates were identified as
the major process source of EROD-inducing substances. Using conventional liquid/liquid extraction,
silica gel fractionation and preparative thin-layer
chromatography procedures, an EROD-inducing fraction was isolated. The major constituents were
identified by gas chromatography/mass spectrometry
as juvabione, dehydrojuvabione, and manool, which
are naturally occurring extractives in balsam fir. After
extraction and isolation from balsam fir and TMP
condensates using the methodology developed, trout
exposed to juvabione and dehydrojuvabione exhibited
significant hepatic EROD induction.
Subsequent studies from the mid-1990s to the
present day have attempted to address the more
complex issue of reproductive effects in wild fish. This
approach of focusing on in vivo effects of biota in the
receiving environment and then working towards cause
and effect solutions represents a unique and highly
appropriate application of effects-directed studies.
Although ecologically relevant, reproductive dysfunction in wild fish has represented a much greater
challenge to address because the mechanisms involved
are not understood. The responses have included effects
on gonad size, depressed levels of circulating steroids
[62], perturbations in the sex steroid biosynthesis
pathway [63], and effects on gonadotropin production
and peripheral sex steroid metabolism [64], indicating
multiple mechanisms and chemicals are involved. In the
late 1990s, development of suitable bioassays, such as
fish-specific sex steroid receptor assays [65,66], life
cycle tests [67] and short-term in vivo tests for steroid
effects [68,69], has provided the opportunity to couple
mechanistically linked endpoints to chemical fractionations. This has led to the ability to formulate
questions regarding the characteristics of bioactive
substances, their relationship to production type, and
whether compounds associated with sex steroid
depressions are related to other reproductive impacts.
217
In the late 1990s, an approach similar to that used

by Kinae et al. [41] for mutagens in fish tissues was
adopted to address some of the obstacles associated
with effects-directed investigations of final effluents.
Parrott et al. [70] used caged fish to investigate the
uptake of aryl hydrocarbon receptor (AhR) ligands
from effluent from a bleached kraft mill. Ligands were
recovered in methanol and DCM and non-dioxin
ligands were found in tissue extracts using EROD
induction in H4IIE cells as the indicator. In these
investigations, the approach has been to focus on what
is bioavailable to the organism by using controlled
exposures to investigate bioactive substances in tissue
residues [71,72]. One of the advantages of focusing
effects-directed investigations on tissue residues is
that it takes into account additional modification
processes that may be involved in the responses, such
as modification after mixing of effluent process
streams, modifications during secondary treatment,
modifications after release into the receiving environment, and metabolic modifications after accumulation
[72].
In further applications of the accumulation
approach, both unexposed wild fish and fish collected
adjacent to the effluent outfall were held in a
concentrated effluent stream (50%, v/v) for 4 days
at a bleached kraft mill known to cause reproductive
dysfunction in wild fish [73]. Hepatic tissue extracts
from exposed fish were soxhlet extracted with DCM,
and fractionated according to lipophilicity using RPHPLC. In this level of fractionation, HPLC elution
conditions were optimized to achieve a linear
relationship between Kow and capacity factor (K0 ),
where K0 is the ratio of the reduced retention volume to
the dead volume of the elution conditions. In
generating such a calibration using a range of different
classes of environmental contaminants, fractions with
different Kow were tested for the presence of
bioavailable chemicals that function as ligands for
the AhR in H4IIE cells, rainbow trout hepatic estrogen
receptors (ER), goldfish testicular androgen receptors
(AR), and goldfish sex steroid binding protein (SSBP).
Using the Kow fractionation approach, Hewitt et al.
[73] showed that fish rapidly accumulate multiple nondioxin ligands across discreet ranges of Kow for the
AhR and fish sex steroid receptors after a 4-day
exposure. PCDD/DF equivalents measured by EROD
activity in H4IIE cells and by high resolution GCMS
218
showed that in all fish historically exposed to effluent,

the contributions to total toxic equivalents (TEQs)
from TCDD was >80%, and that nave fish held in
effluent accumulated 1,2,3,7,8-pentachlorodibenzofuran that accounted for a major portion of TEQ [73].
This study also showed that when fish normally
residing in the effluent plume leave for a brief period
to spawn in an uncontaminated stream, hepatic
burdens of ligands for the AhR and sex steroid
receptors decrease to the levels found in fish at
reference locations. For wild populations historically
exposed to bleached kraft mill effluent, this suggested
that a sustained exposure of non-dioxin, metabolized
AhR ligands is required to maintain tissue concentrations, which was consistent with field observations.
A follow-up study at a bleached sulfite/groundwood mill was conducted to determine if the
accumulation profiles (based on log Kow) of bioavailable substances were related to pulp production type
[74]. Wild fish again accumulated ligands for each
receptor after 4-day exposure to effluent, and the
pattern of accumulated substances was very similar to
that previously obtained at the bleached kraft mill. A
third study involved wild fish collected directly from
the receiving environment at another bleached kraft
mill [75]. This study was able to demonstrate that
under the annual-high conditions of spring dilution,
detectable levels of hormonally active substances were
present in hepatic tissues of wild fish. HPLC
fractionations of male and female hepatic tissues
showed the accumulation of biologically active
substances in the same ranges of Kow and that there
were gender-related differences in accumulated substances. Collectively, the bioaccumulation model is an
excellent foundation for use in effects-directed
investigations of substances of concern, with the
added advantage of beginning with a mixture of lesser
complexity.
Several researchers have investigated individual
waste streams within the papermaking process to
determine the source(s) of hepatic EROD induction
and compounds affecting steroid levels in fish. Black
liquor was the subject of investigations involving
EROD activity and hormonal endpoints. The pulping
process digests lignin, the complex phenolic polymer
that binds cellulose fibers together. The spent cooking
liquor, known as black liquor, contains the degradation
products of lignin and cellulose as well as wood
extractives such as resin and fatty acids. Zacharewski

et al. [76,77] found that the methanol extract of black
liquor particles and colloids >0.1 mm from a bleached
kraft mill contained AhR ligands which also displayed
anti-estrogenic effects via the AhR in vitro. Hodson
et al. [77] investigated the potential of black liquor
from hardwood and softwood pulping at a bleached
kraft mill to induce EROD activity in rainbow trout
and found significant activity. More-potent liquor was
associated with alcohol digestion of wood chips as
well as solvent extracts of wood.
In the late 1990s, an extensive investigation was
conducted at a bleached kraft mill in New Brunswick,
one of a handful of pulp mills in Canada that does not
employ secondary treatment. This work successfully
resulted in the identification of chemical recovery
condensates as a primary source of substances that
depress circulating sex steroids in fish and focused
subsequent bioassay-directed studies. Minimal high
molecular weight material was found in the condensates, facilitating bioassay-directed fractionation
studies [78]. Using steroid depressions in mummichogs, a solid-phase extraction method was developed
which completely recovered the active chemicals from
the condensates in two fractions [78]. In this study, a
combination of two SPE cartridges in series (styrene
divinylbenzne and reversible graphitized carbon) was
ultimately successful at isolating polar, bioavailable
compounds affecting steroid levels. GCMS profiles
of both fractions revealed relatively simple mixtures
of <20 chemicals and the mass spectra of several
unknowns appeared to be consistent with lignin
degradation products and terpenoids originating from
the wood furnish [79].
While studies have focused largely on the effects on
wild fish populations, other non-mutagenic effects of
final effluents from pulping facilities have been
investigated. Higashi et al. [80] used early embryonic
development in marine echinoderms and mollusks to
direct manipulations of bleached kraft mill effluents in
northern California. Final effluents were pH adjusted,
filtered and lyophilized, and the residues sequentially
extracted with DCM followed by acetonitrile. The
solvent extracted residue was processed through an
ultrafiltration membrane and the retentate (>10 kDa)
was lyophilized and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDSPAGE) [81]. These investigations have determined
that lignin derived macromolecules bind to the plasma

membrane of the sperm head of purple sea urchins,
thereby blocking the acrosome reaction and preventing fertilization [82].
The importance of effects-driven fractionation
studies conducted on pulp and paper mill effluents
and its incorporation into regulatory practice in
Canada is worth noting. Environmental Effects
Monitoring (EEM) programs in Canada have been
developed for the pulp and paper and metal mining
industries, where cyclical evaluations of the health of
biota in receiving environments determine whether
effects exist when facilities comply with existing
regulations. Investigation of cause (IOC) is a specific
stage in EEM that involves determining the sources
and causes of effects observed in the receiving
environment of a discharger. Several levels of effort
have recently been described that can be undertaken
for cause identification [83,84]. The framework
includes levels to define whether there is an effect,
whether it is related to the effluent discharge facility,
and whether response patterns in the receiving
environment are characteristic of a particular stressor
type. The next tier of the framework involves
investigating individual process wastes within the
mill to determine the components contributing to final
effluent effects. In contaminant-focused causal investigations, questions progress along a continuum which
first asks if the source within the mill can be identified,
and to effects-driven identification to the compound
classes and ultimately, the specific chemicals
involved. The fundamental question driving the
investigations is whether sufficient information has
been generated to define the effect such that a
mitigative solution can be found. Effects-directed
questions within the framework have been tailored so
that the investigation may be halted when that
information is attained [84].
3.2. Sewage effluents
In the late 1980s and early 1990s, work on sewage
effluents focused on acute toxicity. In the US, these
investigations arose from the development of comprehensive toxicity-based approaches outlined by the
US EPAs Toxicity Identification Evaluation (TIE)
procedures [57]. The TIE approach uses the
responses of organisms or an appropriate bioassay
219
to detect the presence of toxic agents. This approach

characterizes the active substances of interest in a
complex matrix in three phases [57], which was
developed for municipal sewage investigations in
concert with toxicity reduction evaluations (TREs) to
ameliorate effluent acute and chronic toxicity [8].
Phase I of a TIE involves (i) determining the
characteristics of the active agents and (ii) establishing
whether or not the effect is caused by the same
substances [5]. Failure to establish effect variability
related to the active substances could lead to erroneous
conclusions and control measures that do not eliminate
the effect. The physical/chemical properties of the
active substances can be described using effluent
manipulations coupled to a bioassay that either
duplicates the field effects or is mechanistically linked
to them. Each test is designed to alter the substances
themselves or change their bioavailability so that
information on the nature of the substances can be
obtained. Repeating these tests over time on the same
sample will provide information on the consistency of
the substances to cause the effect. Examples of effluent
manipulations include filtration, pH adjustments,
addition of oxidizing agents and chelating agents,
temperature adjustments, aeration, and SPE.
Phase II involves specific methods to isolate active
chemicals and propose structures for their identification (isolation techniques, HPLC fractionation;
Fig. 1). In this step, active components are further
separated from inactive substances for their identification and confirmation [6]. These methods are
specific to the classes of chemicals outlined above and
utilize bioassay responses to evaluate the success or
failure of extraction, separation and concentration of
bioactive substances. Separation of the sample into
acid, base, and neutral fractions can be accomplished
here, usually with some knowledge gained from
Phase I manipulations as to which category of
compounds are involved. Acid/base neutral partitioning can also be applied to fractions generated from
HPLC fractionation. Until the mid-1980s, normal
phase HPLC was frequently the method of choice in
fractionating extracts in effects-directed investigations, employing elution conditions of increasing
polarity with solvents ranging from hexane to
dichloromethane, acetonitrile and finally methanol
[4]. Application of reverse phase columns has since
been the method of choice in HPLC fractionations,
220
since stable, bonded C18 phases have been commercially available at reasonable cost.
The question of whether one or more bioactive
substances are involved in the overall response is a
complicated one, often the solution is to focus on the
active component that is easiest to identify. Examples
of isolation techniques include C18 SPE and solvent
extraction (Fig. 1). Chemical isolation steps proceed
in an iterative fashion, directed by bioassay responses
until either further isolations are not possible, or
candidate chemicals are identified (Fig. 1). Once there
is strong evidence that one or more candidate
chemicals are associated with the response, the last
phase can be initiated.
Phase III involves techniques that confirm the
proposed substances are in fact responsible for the
observed toxicity (Fig. 1). This is usually accomplished through a weight of evidence assemblage of
information that collectively establishes the identity of
the active compounds [7]. It is also equally important
to establish that the cause of the effect is consistent
over time so that amelioration efforts can adequately
address the effect. Some judgment can be exercised in
terms of the extent to which confirmatory tests are
carried out, which reflects the authenticity of the
results. For example, if a suspected substance can be
removed by inexpensive pretreatment or process
modification, a higher level of uncertainty may be
acceptable than if an expensive treatment plant is
required. Confirmatory approaches include correlations between concentrations of suspected agents and
the bioassay response, symptom comparisons between
effluent exposures and those of pure substances and
spiking experiments to determine if the effect can be
reproduced in the matrix being studied.
The TIE approaches described above have been
applied mostly to sewage investigations, as they were
originally designed. Investigations in the early 1990s
focused on identification of acute and chronic
toxicants in primarily freshwater ecosystems but
marine environments were also investigated with
similar results [85]. Amato et al. [86] employed a TIE
approach to identify diazinon in final municipal
effluents as acutely toxic to Ceriodaphnia dubia. In
this study, C18 SPE cartridges were used to
successfully recover the activity, which was confirmed
by GCMS as diazinon, a pesticide widely used both
indoors and outdoors for insect control.
Surfactants were identified in 1992 as primary

toxicants to fish and C. dubia exposed to sewage
effluents where manipulations such as the type of
storage container was found to affect the responses
[87]. Other studies used TIE protocols to identify
diazinon, ammonia and chlorine as toxicants to three
species of Daphnia [88,89]. SPE extracts of final
effluents were eluted with increasing proportions of
methanol. Activity was recovered in fractions with
7585% methanol. These fractions were combined
and fractionated indiscriminately by RP-HPLC into
1 min fractions. A weight of evidence approach
was used, combining all aspects of sample manipulations, spiking experiments, and correlations of toxicity
with fraction concentrations of suspected toxicants for
confirmation in Phase III [88].
The behavior of heavy metals was found to vary
significantly during TIE manipulations of metal
contaminated effluents and sediment pore water
[89]. When metals are suspected toxicants, addition
of EDTA as a chelating agent can be useful to verify
their presence [5], however the responses of metals to
manipulations are often variable, suggesting the
presence of additional toxicants. It is also possible
that the extraction procedure may result in charge state
changes in metals and thus affect their activity. In the
case of SchubauerBerigan et al. [89], passage of the
effluent sample through a C18 column caused a
reduction in toxicity later attributable to bioavailable
zinc, rather than non-polar organic compounds, as one
might first suspect. It was also found that bioavailable
metals can be removed by filtration and recovered by
extraction. Moreover, dilution water matrix effects on
toxicity can impede Phase III confirmation experiments. This can occur if matrix conditions such as
hardness, alkalinity and dissolved organic carbon
(DOC) are sufficiently reduced from their whole
effluent concentrations to affect metal bioavailability
in dilution experiments. Finally, a combination of
EDTA, sodium thiosulfate and graduated pH tests
were used to distinguish copper toxicity from lead and
also metal toxicity in general to that for ammonia [89].
As the issues surrounding biologically active
compounds in pulp mill effluents evolved through
the 1990s, a similar story had begun to emerge for
municipal sewage effluents in the late 1990s. This
coincided with the development of the so-called
endocrine disruptor hypothesis, originally put forth by
Rachel Carson, and rejuvenated by the publication of

Our Stolen Future by Colborn et al. [47]. The first
indication that there could be hormonally active
compounds present in municipal sewage effluents
came with the discovery of feminized fish in the UK
[90].
Unlike pulp mill effluents, efforts to identify sex
hormone analogues in sewage effluents have proven to
be ultimately successful in that specific causative
agents have been identified and all biological activity
accounted for. This has been realized by applying TIE
protocols and approaches designed for acute toxicity.
Using SPE for extraction of hormonally active
substances from effluents and subsequent fractionation by RP-HPLC, the predominant estrogens in
municipal effluents were first identified as the natural
hormones 17b-estradiol and estrone as well as the
synthetic contraceptive hormone 17b-ethynylestradiol
[91]. Fractionations in sewage investigations of
estrogenic substances have been directed in large
part by in vitro tests using yeast assays transfected
with human estrogen receptors or fish cell tests. This
work has allowed for the complete toxicological
evaluation of these compounds and it has been
determined that environmentally relevant concentrations are sufficient to account for estrogenic responses
observed in wild fish [92]. The contribution of
xenobiotics including nonyl- and octylphenol and
bis-(2-ethylhexyl)-phthalate contribute minimally to
the overall estrogenic potency. As a result of the
success of these effects-driven investigations, analytical protocols for aqueous mixtures have been
developed using solid-phase extraction disks of
styrene divinyl benzene [93], and SPE followed by
LC coupled with tandem mass spectrometry (MS
MS) [94,95].
In an effort to improve the nitrification efficiency of
municipal effluent treatment systems, Svenson et al.
[96] employed bioassay-directed fractionations to
identify specific nitrification inhibitors in effluent
from a wood pressboard facility that was too toxic to
be passed onto municipal treatment. Fractionations
were directed by nitrification inhibition in Nitrobacter,
a bacteria isolated from sewage sludge. The authors
employed C18 SPE followed by RP-HPLC to isolate
activity in two fractions that were profiled by GCMS.
Activity in underivatized and methylated fractions
were shown to contain a series of unsaturated fatty
221
acids and resin acids. Linoleic acid was found to be the

most important inhibitor after confirmation with
authentic standards. Phase I manipulations suggested
that volatile substances might also inhibit nitrification.
Using a purge/trap injector for GCMS analysis, a
series of monoterpenes were identified, which were
found to contribute approximately 13% to the total
nitrification inhibition [96].
While the majority of modern literature on
municipal sewage effluents has dealt with estrogenicity, recent studies in the UK have begun to investigate
androgenicity. Using effects-directed investigations,
Thomas et al. [97] coupled a yeast based androgen
screen to studies of surface waters and final effluents.
Similar to the case for estrogens, natural steroidal
compounds and metabolites are associated with the
observed responses. The study identified the natural
steroids and steroid metabolites dehydrotestosterone,
androstenedione, androstandione, 5b-androstane3a,11b-diol-17-one, androsterone, and epi-androsterone and quantitatively confirmed them to be responsible for 99% of the in vitro activity of municipal
effluents discharged into UK estuaries.
3.3. Other effluents
Bioassay-directed investigations have been utilized
in a variety of other point source industrial effluents to
determine the identities of biologically active compounds and their sources within industrial processes.
The majority of investigations have employed the TIE
approach in seeking to characterize compounds
associated with acute toxicity, and it appears that
adoption of this approach provides elevated chances
for success. Fractionations have commonly been
driven by toxicity to different species of Daphnia,
used for their case-specific suitability, reproducibility
and convenience to drive chemical fractionations.
DiGiano et al. [98] attempted to determine if TIE
protocols could be applied to textile mill effluents.
Phase I manipulations showed slight removal of acute
toxicity to C. dubia after C18 SPE. Anion exchange
introduced artifactual toxicity but chloride was
eventually confirmed as accounting for 2533% of
the toxicity. HPLC fractionation of 100% methanol
elutions of C18 SPE was able to isolate toxicity into
several fractions, which were profiled by GCMS.
This study was only able to weakly associate dyes, dye
222
intermediates and surfactants to the toxicity without

confirmation.
Castillo and Barcelo [99] used D. magna to
investigate the toxicity of textile effluents and landfill
leachates. Two different extraction and fractionation
methods were applied: samples were pre-concentrated
by sequential SPE using C18 and styrene divinyl
benzene solid phases. C18 cartridges were eluted to
capture broad compound classes into three fractions:
hexane (aliphatic polyethoxylates, Cn>15), DCM:hexane (4:1; aliphatic polyethoxylates, Cn<15) and
methanol:DCM (9:1; nonylphenol ethoxylates). Phenolic substances in the residual water were extracted
by styrene divinyl benzene cartridges that were eluted
with a solution that was 5 mM each in triethylamine
and acetic acid in methanol (9:1). Subsequent analysis
by LCatmospheric chemical ionization (APCI)-MS
and LCESI-MS enabled the characterization of polar
compounds present in toxic fractions. A broad range
of compounds in both the landfill leachate and textile
mill effluents were identified, such as phenolic
compounds, phthalates, aliphatic carboxylic acids,
aromatic carboxylic acids, amines, alkanes and linear
aliphatic alcohols. No further fractionations were
attempted as correlations with immobilization in
Daphnia were then used to implicate nonylphenol
isomers, nonylphenol ethoxylates, and several phthalates.
The diagnostic capability of cation-exchange
treatments of effluents to implicate hardness-dependent components was highlighted in textile effluent
investigations by Wells et al. [100]. Fractionations
were directed by acute toxicity in Daphnia pulex.
After standard approaches involving filtration and C18
SPE treatments, ion exchange treatments affected the
resultant toxicity, suggesting metals were involved. A
reduction in toxicity following anion exchange
indicated that zinc was present in an anionic form
in the effluent. Toxicity testing coupled with zinc,
calcium, and magnesium analyses confirmed zinc as
the primary toxicant. Subsequently, calcium was
determined to have a greater protective effect of zinc
toxicity to D. pulex than magnesium. In an evaluation
of different cation-exchange media for applicability in
TIEs, Burgess et al. [101] recommended columns
from two manufacturers, which removed 80100% of
five metals (Cd, Cu, Ni, Pb, and Zn) from spiked
seawater with equivalent recoveries. Anion and
mixed-bed (30:70 cation:anion) exchange were used

to identify hexavalent chromium as responsible for an
isolated acute toxicity event as well as chronic toxicity
in C. dubia exposed to effluent from an unidentified
industrial source [102].
Jin et al. [103] employed TIE approaches directed
by D. magna to effluent from a chemical plant effluent
in Nanjing, China. Phase I results showed toxicity was
removed completely by aeration and C18 SPE at pH 3.
In Phase II testing, toxicity was recovered by elution
with 80% methanol, which was subjected directly to
GCMS analysis. Confirmation of toxicity with
authentic standards of benzopyrone and phenol
showed a synergistic interaction. In a similar study,
also in China, Yang et al. [104] tracked toxicity to D.
magna using a simplified version of a TIE where C18
fractions of increasing proportions of methanol in
water from SPE were not sub-fractionated but
analyzed directly by GCMS. A toxic unit approach
was used to implicate a number of chloro-nitro
benzenes but this study stopped short of complete
Phase III confirmation tests.
In a particularly thorough study, Jop et al. [105]
used toxicity to fathead minnows (Pimephales
promelas) to investigate treated chemical plant
effluent. Approximately 70% of the toxicity was
attributed to un-ionized ammonia, as evidenced by
removal of toxicity following treatment with zeolite,
clinoptilolite, activated carbon, cation resin and a
combination of zeolite and cation fractions. Residual
toxicity was investigated using XAD resin coupled
with extractions of DCM at different pHs. Fractionation of the neutral DCM extract by RP-HPLC eluted
with a gradient of pH 3.6 acetate buffer and
acetonitrile isolated toxicity in a single fraction. A
combination of 1H-NMR, 12C-NMR, GC using
thermionic detection and GCMS was next employed
in fraction analysis. These analyses revealed the
presence of two components, one of which was
identified as 4-hydroxy-2-methylthiobenzothiazole,
which was confirmed chemically with authentic
standards, and biologically with spiking experiments
and single exposures.
Bioassay-directed approaches have been used to
also investigate produced water, but with little success.
Produced water is a mixture of injected water and
water from formations in which oil and gas is
recovered. Large volumes of effluent are discharged
into offshore waters and coastal areas without

treatment. It is a complex mixture known to contain
hydrocarbons, oil droplets, organic acids, phenols and
production chemicals [106] and exhibits both acute
and chronic toxicity in laboratory testing. Bioassaydirected studies have used toxicity in tropical mysids
[107], Ceriodaphnia, fathead minnows, and sea
urchins [108] and binding to yeast-based androgen
and estrogen receptors [106] to direct chemical
fractionations. The results between effluents from
different sites are highly variable with contributions
from non-polar organics, metals, ammonia and
volatile organic compounds, and pH-sensitive substances. Moderate success was achieved in the
identification of C1C5 and C9 alkylphenols as
contributing to the majority of estrogenic activity
[106]. The complexity and variability of the produced
water discharges appears to be the primary factor
affecting success of these investigations.
3.4. Pesticides
A series of effects-directed studies were used to
investigate the induction of EROD activity and steroid
responses associated with field formulations of the
lampricide 3-trifluoromethyl-4-nitrophenol (TFM).
Field formulations of TFM were shown to rapidly
cause pronounced induction in fish, however the active
ingredient itself did not cause induction [109].
Development of a C18 SPE protocol to isolate
formulation impurities showed the presence of dozens
of impurities [84,110]. Since TFM is a phenolic
compound, an aqueous alkaline buffer was used to
selectively elute it after application of the formulation
to SPE. Methanol was then used to elute and fully
recover bioactive formulation impurities. Methanol
extracts were subjected to RP-HPLC fractionations of
the impurities directed by EROD activity in fish, which
isolated activity in two distinct fractions [111]. The
fraction with the highest activity was subfractionated
using the same RP column under modified elution
conditions to provide enhanced chromatographic
resolution. Analysis of active sub-fractions revealed
the presence of three novel chloro-nitro-trifluoromethyl
substituted dibenzo- p-dioxin isomers, which were
tentatively identified using high resolution GCMS
[112]. Synthesis of several isomers afforded an elution
series profile by GC, which confirmed the formulation
223
dioxins were not laterally substituted, and therefore of a

lower risk to aquatic environments. To examine
formulation effects on fish sex steroid levels, the
authors were one of the first to couple effects-directed
fractionations to a competitive binding assay with
rainbow trout hepatic estrogen receptors. It was
subsequently determined that the active ingredient
itself, as well as two isomeric impurities, were
estrogenic [113].
Other effects-directed investigations have dealt with
chiral pesticides. These studies have arisen from the
concern over the toxic nature of the active enantiomer
on non-target organisms, and the stereo-selectivity of
microbial degradation. In the past, an enantiomer was
obtained by achiral synthesis or asymmetric synthesis,
and the product was then obtained by fractional
distillation and re-crystallization for investigation of
biological activity [114]. These investigations have
progressed with the development of chiral chromatographic columns for GC and HPLC. For example, a
series of 14 O-ethyl-O-phenyl-N-isopropyl-phosphoramidothioate enantiomers containing a phosphorus
atom as a chiral center have been separated by HPLC
on a Pirkle model chiral stationary phase [115]. Some
chiral organophosphorus pesticides also have been
separated by microcolumn liquid chromatography
using Chiralcel OD columns and a UV detector
[116]. The pesticide fenamiphos was recently investigated using toxicity to the non-target aquatic
invertebrate D. pulex and an HPLC fitted with a Pirkle
chiral column. It was found that both enantiomers
degraded at identical rates under environmental
conditions, but (+)-fenamiphos was about 20 times
more toxic to Daphnia than (+)-fenamiphos [117].
A related effects-directed study of another formulation of environmental significance relates to the
acute toxicity in aquatic invertebrates associated with
the release of aircraft de-icing/anti-icing fluids
(ADAFs). ADAFs are formulated to contain 50
90% ethylene, propylene and combinations of other
glycols. Concern over other formulation components
such as wetting agents, surfactants, corrosion inhibitors and thickeners led to an effects-directed
investigation directed by Microtox [118]. ADAF
formulations were subjected to semi-preparative
RP-HPLC and fractions were characterized using
GCMS, UVand LCMS. Activity in the fraction with
the highest toxicity was further isolated after
224
application to a silica gel column eluted in a gradient

with hexane and diethyl ether. GCMS analysis after
silica gel purification confirmed the activity was
associated with benzotriazole and tolyltriazole, both
corrosion inhibitors.
3.5. Runoff
Applications to non-point source stressors have
proven to be more challenging in that a supply of
activity is not readily available, as is the case with
most effluents. This has not affected the study of
pesticides, but is especially applicable to runoff.
Runoff from large highways has been investigated in
the UK for the identities of substances affecting
structure and functioning of benthic communities
[119]. Adopting the TIE approach, Phase I manipulations showed that substances acutely lethal to
Gammarus pulex were associated with sediments
[119]. Phase II experiments utilized soxhlet extractions of sediments to recover active compounds and
then preparative aluminasilica chromatography to
generate five fractions eluted with increasing proportions of dichloromethane in n-pentane [120]. GCMS
analysis of the most toxic (and least polar) fraction
showed it contained several higher molecular weight
PAHs. Phase III experiments with authentic PAH
standards showed that the majority of the toxicity
could be ascribed to pyrene, fluoranthene, and
phenanthrene [121]. Using a toxic unit approach,
the authors found that the contribution of these
compounds varied from 30 to 120% of the total
sediment-bound activity in sediments from different
sites over 5 months.
An emerging area in non-point source studies
concerns hormonally active substances in runoff from
livestock waste. Few studies have specifically examined the relationship between manure-borne hormones, as well as hormones used to treat animals prophylatically. Burnison et al. [122] investigated
estrogenic substances in hog manure using a
recombinant yeast estrogen screen assay to direct
fractionations of manure extracts. Manure extracts
were obtained by diluting concentrated aged manure
from a farm holding tank in southwestern Ontario,
Canada. Liquefied manure was extracted using SPE
and extracts were fractionated by RP-HPLC. The
endogenous estrogens 17b-estradiol and estrone, as
well as the phytoestrogen metabolite equol, were

confirmed by GCMS. Equol was further characterized using fish hormone receptor binding assays and
was found to be weakly (2001000-fold < 17bestradiol) estrogenic with a weak affinity also for
goldfish androgen receptors. The overall hormonal
activity of tile drainage following the first rain event
following manure application was minimal. It is clear
that in this emerging area more information on the
types and amounts of estrogens, androgens, gestagens,
growth promoters and antibiotics that exist in fresh
livestock excreta, and their fluxes to aquatic environments needs to be determined [123].
Finally, while nearly all bioassay-directed studies
mentioned in this review have dealt with those
substances present in effluents affecting aquatic biota,
Brack et al. [124] examined effects of volatile
bioactive substances from landfill leachates. The
bioassay used was based upon in vivo chlorophyll a
fluorescence of green algae, enabling the detection of
leachate chemicals interfering with photosynthesis.
Volatiles were isolated by distillation of leachates in a
closed system. Using gastight syringes, a concentrated
aqueous fraction containing compounds with Henrys
law constants >0.01 were withdrawn through a
septum of the distillation apparatus. Analysis of
volatiles was accomplished by GCMS with a
headspace sampler. Aliquots of distillate were placed
in 20 mL headspace vials containing 1 g CaCl2 to
increase the gas-phase concentrations of the volatiles
due to salting-out. Toxicity thresholds and toxic unit
approaches were used to compare toxicity values
from algae to concentrations measured by GCMS.
These comparisons implicated toluene, ethylbenzene,
m/ p-xylene, styrene, and 1,2,4-trichlorobenzene as
principal photosynthesis inhibitors.
4. Endpoint considerations in bioassay-directed

investigations
Since this review considers mutagenic endpoints
and non-mutagenic endpoints, we felt it necessary to
highlight some considerations surrounding endpoint
selection. Of primary importance is the scale of the
bioassay, which dictates the scale of the separations,
i.e. preparative or analytical scale. This will influence
not only fractionation method development, but
preparation for bioassay testing as well. Miniaturized

test systems for D. magna and P. promelas have been
specifically developed for bioassay-directed applications where organisms are exposed to 1 mL solutions
in microtiter plates [125]. Factors for consideration are
the organism biomasstest solution ratio, toxicity and
partitioning of exposure chamber materials, dissolved
oxygen in the test solutions, and dilution of test
solutions upon transfer of the organisms. An additional consideration involves the bioassay response
itself, its consistency, its reliability, adequate replication, rapidity of answer, etc. Also important and
related to the scale of the bioassay is its relevance to
the whole organism response being tested. Is it an in
vitro or an in vivo bioassay? Obviously an in vivo
assay has greater relevance to detecting an effect in an
organism, as opposed to an in vitro test, which
ultimately requires validation in vivo.
The scale of the bioassay has obvious effects on the
choice of chemical manipulations; micro-scale in vitro
assays require less material to test and less effluent to
be initially extracted. Larger scale in vitro bioassays
involve preparative techniques that can be quite
laborious and require large effluent volumes (>10 L)
to be processed. This can become quite tedious when
additional fractionation tiers are added to the
investigation and this can contribute substantially to
the cost of an investigation. An additional factor,
which comes into play here, is the consumption of
isolates relative to those required for chemical
analyses. Fractions can become quite precious in
terms of the time and effort allocated in their
generation and if it is possible to recover sufficient
amounts of the isolated material after bioassay testing
that can then be used for chemical analyses then this is
a definite advantage.
One of the primary concerns of in vitro test
applications should be cytotoxicity but often it is not
reported. The interference of cytotoxicity, due to nontoxic substances or due to incompatible physicochemical characteristics of an effluent sample, is a
highly relevant phenomenon in toxicity testing,
particularly for genotoxicity assays [10]. Dead cells
do not exhibit genotoxic effects, and cytotoxicity may
result in false negative data. It is therefore important to
evaluate cytotoxicity by testing a dilution series of the
sample or by providing some measurement of cell
viability (alkyl phosphatase activity, cell number,
225
optical density). In vitro tests offer distinct advantages

in that they do not kill large numbers of laboratory
animals, provide rapid responses with adequate
replication and are relatively inexpensive. Developments in the use of genetically modified bacteria
transfected with reporter genes linked to toxicantspecific mechanisms are continuing to evolve with
advances in the identification of receptor molecules
and genomics technologies [126]. These endpoints
have a unique utility if they are mechanistically linked
to the effect, i.e. they are an appropriate surrogate to
the physiological or ecological effect observed in the
field. This can be a difficult leap to make at times and
is the subject of an area of intense research to
standardize aquatic toxicity tests and develop shortterm tests predictive of long-term whole organism
responses (reviewed in [59]). If the mechanism
underlying the response is not known then using
hypothesis testing in the form of bioassays mechanistically linked to the response can be employed in a
screening effort to determine if the response in that
assay is present or not. This would serve to increase
the likelihood of successful effects-directed investigations.
5. Conclusions and future directions

Effects-driven investigations of effluents offer a
rational approach to the identification of active
substances of interest in real-world matrices having
impacts on aquatic ecosystems. The necessity for good
water quality has increased with demand for sustainable industrial practices and effects-directed investigations are increasingly attractive for providing solutions.
Although early studies on the acute toxicity of effluents
met with reasonable degrees of success (in terms of
identification of causative agents), the identification of
chemicals with mutagenic activity, toxicity and
endocrine disruption has proven to be a difficult
challenge, and many of these studies have been labeled
as ending with disappointment [127]. However, we
feel it is important to remember that each effectsdirected investigation is a hypothesis-driven research
project for which several uncertainties (e.g. extraction
method, complexity, sample stability, consistency of
active components, analyte detection) must be
addressed during the course of the study. It cannot
226
be emphasized enough that such endeavors are not

trivial and require significant resources including
infrastructure, personnel and a multidisciplinary team
of trained individuals. Experience in working with the
matrix being studied is also invaluable. As such,
relatively few organizations attempt such investigations. In addition to the resource requirements, one of
the main reasons for not conducting effects-directed
investigations on a more routine basis is the uncertainty
associated with the outcome of the investigation.
Organizations are reluctant to allocate the large
resources necessary when the outcome (i.e. identification of the specific cause) cannot be predicted with
much certainty. Funding typically dictates the depth of
effects-directed investigations. As shown in this review,
one of the biggest challenges associated with effectsdirected investigations today is the ability of analytical
techniques to overcome the obstacles (reactivity,
adsorbent recovery difficulties) related to the extraction
and analysis of polar compounds. For the purposes of
this review, we choose a more realistic definition of
successful investigations as those, which are able to
provide more information as to the nature and cause of
the effect than before the investigation began. In this
way, nearly all effects-directed studies provide some
measure of success, as in for example, the elimination
of suspected compounds as causative agents [37,54]
and the identification of compound classes involved in
the responses [29,31,37,3941,4446,55,76,80,89,93,
96,99,100,103105,108,120,124,128133]. This shifts
the emphasis from the bias that complete structural
confirmation of the chemicals causing the effect is
necessary for success. This realistic approach has
recently been applied to the Canadian EEM program
for the pulp and paper sector where the depth to which
investigation-of-cause projects advance is defined by
the attainment of sufficient information on the source
and nature of the effect so that remediation efforts can
be undertaken [84].
The analytical approaches to effects-directed
investigations of point source effluent and non-point
source stressors are relatively consistent across the
spectrum of endpoints that have been investigated
(mutagenicity, acute toxicity, endocrine disruption).
The approaches generally follow protocols developed
for the acute toxicity of sewage effluents (Fig. 1). One
of the first steps is to employ an isolation step to
separate the active substances from the effluent matrix
and then concentrate them for both analytical

fractionation and bioassay testing. XAD resins have
been the most commonly used extraction technique.
XAD resins have gradually been replaced with the
advent of more specific solid-phase extraction phases,
C18 being the most common sorbent used to recover a
broad range of dissolved substances from aqueous
effluent matrixes. As we have seen however, the
extraction efficiencies of such extraction methods are
rarely reported. Recent applications of a second
sequential SPE, using graphitized carbon or styrene
divinylbenzne, show promise to recover polar or ionic
active components in future studies [78,127].
A first series of rough fractionations with SPE
can be conducted using increasing proportions of
methanol [5]. As a next tier of fractionation, active
SPE fractions are recombined and subjected to RPHPLC for greater chromatographic resolution of
extracted components. A second tier of HPLC
fractionations may be employed using RP-HPLC or
other solid phases suited to the compound classes of
interest. Many studies blindly collect fractions at
1 min intervals (e.g. [6,88]), which limits the amount
of information that may be gleaned from this first
fractionation step. Frequently, a next tier of fractionation, involving recombinations of the 1 min fractions,
is necessary for effects-directed investigations to
progress (Fig. 1). One option is to conduct peak
collections [111,113], and several automated fraction
collectors have peak-collection options where valley
thresholds can be selected. Another option is to collect
fractions based on a physicochemical property.
Several studies have calibrated HPLC elution conditions against Kow so that information is immediately
obtained on the properties of the active chemicals,
even if further sub-fractionations are not undertaken
[44,73,74,111].
As was seen, characterizing toxicants associated
with mutagenic and other effects in extracts and
fractions is a daunting task and many researchers have
resorted to toxic unit approaches with known or
suspected contaminants to overcome effluent complexities [104,121,125]. Instrumental techniques for
the characterization of unknowns have involved
traditional analyses by electron impact GCMS. This
technique is ideal for non-polar and moderately polar
compounds and adequate chromatographic resolution
of components from a single HPLC fraction can
usually be achieved [44,105,111,122]. The commercial availability of chiral columns now affords the
opportunity to resolve between enantiomeric compounds, which has been shown for pesticides [117].
The confirmation of isolated candidate chemicals
proposed as causative agents is challenging not only in
the ability to isolate the active components from the
matrix being investigated but success also requires
matches from mass spectral libraries, or more likely,
spectral interpretation and structural postulation.
Other spectral techniques such as NMR and X-ray
crystallography may be necessary to aid in formulating structures of unknowns (e.g. [105]). Procurement
of authentic standards for proposed chemicals is
required for complete chemical and toxicological
verification. It is likely that authentic standards of
candidate structures will not be commercially available and custom synthesis or preparative isolation may
be required. Custom synthesis can be expensive, time
consuming and depending on the structure, difficult
to carry out. Structural confirmation of suspected
mutagens or other toxicants were only evident in
10% of the studies cited in this review
[25,26,61,91,97,111113,121,122], owing to the difficulties in carrying out these investigations to
completion.
As the issues surrounding some effluents (e.g.
pulp mills and sewage) have evolved, concerns have
shifted to compounds that survive existing treatment
regimes, which are by definition more polar and
have been traditionally more difficult to analyze.
Taking advantage of developments in the mass
spectrometry of bio-molecules, analytical methods
employing modern atmospheric pressure ionization
(API)-MS techniques have been developed to monitor
environmental levels of the active compounds, once
they have been identified [94,95]. While LCMS has
readily apparent applications in the analysis of more
polar compounds, it presently has a limited use in the
identification of unknowns. One reason for this is
the limited mass resolution under current modes of
routine operation. Even tandem MSMS systems
with collision-induced dissociation (CID) provide
only partial information on molecular compositions.
LC-quadrupole-time-of-flight (LC-QTOF) instruments
will go a long way to fill this gap as their purchase
costs decrease and availability increases. This should
increase the probability of the identification of polar/
227
high molecular weight substances in effects-directed

investigations.
Biological endpoints used to drive chemical
fractionations have also advanced significantly with
technological developments and as the issues associated with each effluent have evolved. Mutagenic
investigations have largely been based on the
Salmonella assay because of its historical successes
and convenience of application. Some strains have
proven more useful in the detection of different classes
of mutagens and this knowledge can be applied
diagnostically to determine the chemical classes that
may be involved [39]. Investigations of other effluents
have progressed from acute to chronic effects (e.g.
reproduction in wildlife [59]). In vitro assays have
become more sensitive and sophisticated in their
ability to be genetically or mechanistically linked to
higher level in vivo effects. The choice of endpoint and
its scale can dramatically affect which analytical
techniques are used in the investigation and the cost of
preparation of fractions for testing. Care must be
exercised to ensure that the bioassay results can be
meaningfully extrapolated to effects at the individual
and population levels.
Acknowledgement
The authors gratefully acknowledge the editorial
assistance of P.A. White in the invitation to submit the
manuscript and for its timely editing.
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Mutation Research 589 (2005) 208232

Analytical methods in environmental effects-directed

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

(Fig. 1). This approach allows analytical efforts to

2. Effluent and drinking water mutagenic

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

operations, dye manufacturers, petroleum refineries

Although a large number of genotoxicity assays

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

de novo synthesis of biotransformation enzymes is

substances can also occur during extraction and the

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

ethyl ether, but no mention was provided as to whether

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

waters, accounting for the largest proportion of

produced from azo dyes during effluent treatment

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

columns optimized for the study of river waters [34].

Beginning in the early 1980s and continuing into

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

effluents, which was also effective in studies of aryl

3. Non-mutagenic effluent evaluations

acute parameters and their causal factors have been

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

dispersants, juvenile insect hormone analogues and

area of bioassay-directed compound identification has

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

In the late 1990s, an approach similar to that used

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

showed that in all fish historically exposed to effluent,

extractives such as resin and fatty acids. Zacharewski

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

that lignin derived macromolecules bind to the plasma

to detect the presence of toxic agents. This approach

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

Surfactants were identified in 1992 as primary

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

Rachel Carson, and rejuvenated by the publication of

acids and resin acids. Linoleic acid was found to be the

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

intermediates and surfactants to the toxicity without

mixed-bed (30:70 cation:anion) exchange were used

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

into offshore waters and coastal areas without

dioxins were not laterally substituted, and therefore of a

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

application to a silica gel column eluted in a gradient

well as the phytoestrogen metabolite equol, were

4. Endpoint considerations in bioassay-directed

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

preparation for bioassay testing as well. Miniaturized

optical density). In vitro tests offer distinct advantages

5. Conclusions and future directions

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

be emphasized enough that such endeavors are not

and then concentrate them for both analytical

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

high molecular weight substances in effects-directed

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

methyl)-5-hydroxy-2(5H)-furanone in chlorinated drinking

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

[46] P.A. White, J.B. Rasmussen, C. Blaise, Genotoxic substances

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208232

[60] G. Van der Kraak, K. Munkittrick, M.E. McMaster, D.L.