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 4A6b 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

www.elsevier.com/locate/reviewsmr

Community address: www.elsevier.com/locate/mutres

Mutation Research 589 (2005) 208–232

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 non-

mutagenic 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 (GC–MS) and/or other spectrometric techniques for identification.

LC–MS methods have been developed for difficult-to-analyze polar substances identified from effects-directed studies, but the

potential for LC–MS 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) 208–232 209

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

2. Effluent and drinking water mutagenic investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

3. Non-mutagenic effluent evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

3.1. Pulp mill effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

3.2. Sewage effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

3.3. Other effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

3.4. Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

3.5. Runoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

4. Endpoint considerations in bioassay-directed investigations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

5. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

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 informa-

tion 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 end-

point to direct chemical manipulations of a mixture to

separate active components from inactive ones

(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 [5–7] 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 non-

mutagenic 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232210

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 con-

ducted 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 211

de novo synthesis of biotransformation enzymes is

induced by intraperitoneal injection of a PCB mixture.

In the case of effects-directed mutagenic investiga-

tions, 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 sub-

stances 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 adsor-

bents 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

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 effluent’s 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 extrac-

tion, 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 spectro-

metry (GC–MS), 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 extrac-

tion 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 pre-

servation of the active substances from microbial

degradation once they are contained in a solvent and

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232212

removed from the effluent matrix, and (iii) concen-

tration 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 2–4

bed volumes/min [15]. While convenient and non-

responsive 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 XAD-

2 resins to extract dissolved mutagenic substances

sequentially at neutral and acidic pH from influents

and effluents from a municipal sewage plant proces-

sing 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) fractio-

nation 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 concen-

trates 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. Com-

parisons 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 com-

pounds were present in drinking waters led to the

general acceptance of chlorinated humic material to

cause the formation of 3-chloro-4-(dichloromethyl)-5-

hydroxy-2L(5H)-furanone, or (MX), initially discov-

ered 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.

GC–MS 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 20–50% 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 213

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 ozona-

tion, DeMarini et al. [24] found high frameshift

frequencies in TA98 associated with MX and ‘‘MX-

like’’ compounds. The authors suggested that halo-

genated 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 investi-

gations, 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 analo-

gous chemicals [27–29]. 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 muta-

genic activity (21% of the total) against Salmonella

typhimurium was identified as 2-[2-(acetylamino)-

4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-

amino-7-bromo-4-chloro-2H-benzotriazole, or PBTA-

1. Employing the blue rayon passive sampling

procedure, a total of eight PBTA isomers were

subsequently discovered [27–29]; these compounds

account for approximately one-third of the total

effluent mutagenicity [30], and are thought to be

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-acetyl-

glucosamine) powder bearing covalently linked CPT

residues [31]. The chief disadvantage of rayon or cotton

supports lies in their limited ability to passively batch-

treat 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]. CPT–chitin columns have been

used to investigate mutagens in river water contami-

nated with sewage by elution with methanol–ammonia

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 CPT–chitin columns, hanging blue-rayon and XAD-

2 columns for mutagens in two Japanese rivers known

to contain mutagens [33]. The results showed that the

CPT–chitin 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 activ-

ity [29].

The CPT–chitin technique has since been inves-

tigated 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232214

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

Methanol–ammonia, followed by dichloromethane,

were found to provide adequate recoveries of 22 PAHs

with three to six rings and NO2–PAHs 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, includ-

ing 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 dye-

processing plant discharge and a reservoir contami-

nated 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 sug-

gested 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 70–90% of the mutagenic

activity from chlorination bleachery effluents. The

majority of the recovered activity was removed by

partitioning with aqueous NaHCO3. Preparative thin-

layer chromatography (TLC) was used as a first step in

isolation, followed by C8 RP-HPLC, further pre-

parative TLC, C18 HPLC, and a final TLC step that

allowed the isolation of MX. As this compound was

not amenable to GC–MS analyses, methylated,

acetylated and trimethylsilyl derivates were synthe-

sized and analyzed to facilitate structural interpreta-

tion.

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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 215

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 direct-

acting 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 parti-

tioning 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 octanol–water 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

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 implemen-

ted, and the industry has responded to these regulations

resulting in significant reductions in acute environ-

mental 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232216

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 com-

plexity 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, differ-

ences 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, includ-

ing tetrachloroguaiacol. Subsequent exposures con-

firmed 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 filtra-

tion, 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 lignin-

derived pterostilbene structure was postulated for an

unknown compound strongly associated with induc-

tion [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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 217

associated with induction present in the primary-

treated 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. Con-

taminated TMP steam condensates were identified as

the major process source of EROD-inducing sub-

stances. Using conventional liquid/liquid extraction,

silica gel fractionation and preparative thin-layer

chromatography procedures, an EROD-inducing frac-

tion 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 dysfunc-

tion 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 fractio-

nations. 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.

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 environ-

ment, 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 RP-

HPLC. 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 non-

dioxin 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 GC–MS

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232218

showed that in all fish historically exposed to effluent,

the contributions to total toxic equivalents (TEQs)

from TCDD was >80%, and that naıve fish held in

effluent accumulated 1,2,3,7,8-pentachlorodibenzo-

furan 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 concentra-

tions, which was consistent with field observations.

A follow-up study at a bleached sulfite/ground-

wood mill was conducted to determine if the

accumulation profiles (based on log Kow) of bioavail-

able 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 sub-

stances. 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 con-

densates, facilitating bioassay-directed fractionation

studies [78]. Using steroid depressions in mummi-

chogs, 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. GC–MS 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 (SDS-

PAGE) [81]. These investigations have determined

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 219

that lignin derived macromolecules bind to the plasma

membrane of the sperm head of purple sea urchins,

thereby blocking the acrosome reaction and prevent-

ing 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 inves-

tigations, 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 com-

prehensive toxicity-based approaches outlined by the

US EPA’s Toxicity Identification Evaluation (TIE)

procedures [5–7]. The TIE approach uses the

responses of organisms or an appropriate bioassay

to detect the presence of toxic agents. This approach

characterizes the active substances of interest in a

complex matrix in three phases [5–7], 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 identifica-

tion (isolation techniques, HPLC fractionation;

Fig. 1). In this step, active components are further

separated from inactive substances for their identi-

fication 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 partition-

ing 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 investiga-

tions, 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,

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232220

since stable, bonded C18 phases have been commer-

cially 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 accom-

plished 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 correla-

tions 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 GC–MS 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

75–85% 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 manipula-

tions, 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 experi-

ments. 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 221

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 fractiona-

tion 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 concentra-

tions 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, analy-

tical 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 GC–MS.

Activity in underivatized and methylated fractions

were shown to contain a series of unsaturated fatty

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 GC–MS 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 estrogeni-

city, 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-androstane-

3a,11b-diol-17-one, androsterone, and epi-androster-

one and quantitatively confirmed them to be respon-

sible 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 com-

pounds 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 25–33% of

the toxicity. HPLC fractionation of 100% methanol

elutions of C18 SPE was able to isolate toxicity into

several fractions, which were profiled by GC–MS.

This study was only able to weakly associate dyes, dye

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232222

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:hex-

ane (4:1; aliphatic polyethoxylates, Cn<15) and

methanol:DCM (9:1; nonylphenol ethoxylates). Phe-

nolic 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 LC–atmospheric chemical ionization (APCI)-MS

and LC–ESI-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 phtha-

lates.

The diagnostic capability of cation-exchange

treatments of effluents to implicate hardness-depen-

dent 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 80–100% 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

GC–MS 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 GC–MS. 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. Fractiona-

tion 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 GC–MS 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 223

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. Bioassay-

directed 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 sub-

stances. Moderate success was achieved in the

identification of C1–C5 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 GC–MS

[112]. Synthesis of several isomers afforded an elution

series profile by GC, which confirmed the formulation

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 chromato-

graphic columns for GC and HPLC. For example, a

series of 14 O-ethyl-O-phenyl-N-isopropyl-phosphor-

amidothioate 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 inves-

tigated 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 for-

mulation 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 inhibi-

tors 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

GC–MS, UVand LC–MS. Activity in the fraction with

the highest toxicity was further isolated after

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232224

application to a silica gel column eluted in a gradient

with hexane and diethyl ether. GC–MS 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 manipula-

tions showed that substances acutely lethal to

Gammarus pulex were associated with sediments

[119]. Phase II experiments utilized soxhlet extrac-

tions of sediments to recover active compounds and

then preparative alumina–silica chromatography to

generate five fractions eluted with increasing propor-

tions of dichloromethane in n-pentane [120]. GC–MS

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 exam-

ined the relationship between manure-borne hor-

mones, as well as hormones used to treat animals pro-

phylatically. 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 GC–MS. Equol was further character-

ized using fish hormone receptor binding assays and

was found to be weakly (200–1000-fold < 17b-

estradiol) 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 environ-

ments 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 Henry’s

law constants >0.01 were withdrawn through a

septum of the distillation apparatus. Analysis of

volatiles was accomplished by GC–MS 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 GC–MS.

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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 225

preparation for bioassay testing as well. Miniaturized

test systems for D. magna and P. promelas have been

specifically developed for bioassay-directed applica-

tions where organisms are exposed to 1 mL solutions

in microtiter plates [125]. Factors for consideration are

the organism biomass–test 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 addi-

tional consideration involves the bioassay response

itself, its consistency, its reliability, adequate replica-

tion, 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 non-

toxic substances or due to incompatible physico-

chemical 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,

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. Develop-

ments in the use of genetically modified bacteria

transfected with reporter genes linked to toxicant-

specific 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 short-

term 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 mechan-

istically 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 investiga-

tions.

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 sustain-

able industrial practices and effects-directed investiga-

tions 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 effects-

directed 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

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232226

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 investiga-

tions. 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. identifica-

tion 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 effects-

directed 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 [29,31,37,39–41,44–46,55,76,80,89,93,

96,99,100,103–105,108,120,124,128–133]. 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 RP-

HPLC 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 fractiona-

tion, 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 condi-

tions 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 com-

plexities [104,121,125]. Instrumental techniques for

the characterization of unknowns have involved

traditional analyses by electron impact GC–MS. This

technique is ideal for non-polar and moderately polar

compounds and adequate chromatographic resolution

of components from a single HPLC fraction can

L.M. Hewitt, C.H. Marvin / Mutation Research 589 (2005) 208–232 227

usually be achieved [44,105,111,122]. The commer-

cial availability of chiral columns now affords the

opportunity to resolve between enantiomeric com-

pounds, 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 formulat-

ing 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 avail-

able 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

[25,26,61,91,97,111–113,121,122], owing to the dif-

ficulties 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 LC–MS 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 MS–MS 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/

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 asso-

ciated 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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