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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
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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210
Fig. 1. General analytical approach for conducting effects-directed investigations of effluents. Adapted from [12,127].
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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
213
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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
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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.
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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
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