Analytical methods in environmental effects-directed investigations of effluents

Mutation Research 589 (2005) 208–232 www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres

Review

Analytical methods in environmental effects-directed investigations of effluents L. Mark Hewitt a,*, Chris H. Marvin b a

Aquatic Ecosystem Protection Research Branch, National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ont., Canada L7R 4A6 b Aquatic Ecosystem Management Research Branch, National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ont., Canada L7R 4A6 Received 2 September 2004; received in revised form 31 December 2004; accepted 10 February 2005 Available online 21 March 2005

Abstract Effluent discharges are released into aquatic environments as complex mixtures for which there is commonly either no knowledge of the toxic components or a lack of understanding of how known toxicants interact with other effluent components. Effects-directed investigations consist of chemical extraction and iterative fractionation steps directed by a biological endpoint that is designed to permit the identification or characterization of the chemical classes or compounds in a complex mixture responsible for the observed biological activity. Our review of the literature on effects-directed analyses of effluents for nonmutagenic as well as mutagenic endpoints showed that common extraction and concentration methods have been used. Since the mid-1980s, the methods have evolved from the use of XAD resins to C18 solid-phase extraction (SPE). Blue cotton, blue rayon, and blue chitin have been used specifically for investigations of mutagenic activity where polycyclic compounds were involved or suspected. After isolation, subsequent fractionations have been accomplished using SPE or a high-pressure liquid chromatography (HPLC) system commonly fitted with a C18 reverse-phase column. Substances in active fractions are characterized by gas chromatography/mass spectrometry (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: [email protected] (L.M. Hewitt). 1383-5742/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2005.02.001

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Contents 1. 2. 3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effluent and drinking water mutagenic investigations . . . . . Non-mutagenic effluent evaluations . . . . . . . . . . . . . . . . . 3.1. Pulp mill effluents . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Sewage effluents . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Other effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Runoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endpoint considerations in bioassay-directed investigations. Conclusions and future directions . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Several million kilograms of genotoxic and other biologically active substances are released into the environment each year [1]. Most substances are released as components of complex mixtures, such as liquid effluents, airborne emissions and solid wastes. There are also additional unknown toxicants released in these mixtures and those produced through biotic and abiotic processes. Because of the complexity of the emissions, standard target-chemical analyses are limited in their ability to generate adequate information on the toxic potential on a chemical-specific basis. Non-target analysis of complex mixtures allows the detection of a broader range of compounds; however the results are often difficult to interpret since toxicological data for compounds detected are not available, especially as they exist in the matrix of a given effluent. While bioassay assessments of industrial wastes provide a means to evaluate and compare effluents without detailed knowledge of their chemical compositions [2] they are limited in their predictive capacity of effluent effects. The coupling of bioassay assessments with chemical analysis yields more information than either of these assessments individually. This is evident in studies where combined chemical and biological evaluations of complex environmental mixtures show measured levels of priority pollutants are a poor indicator of toxicity [3]. Effects-directed analysis allows a biological endpoint to direct chemical manipulations of a mixture to separate active components from inactive ones

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(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 nonmutagenic research on point-source effluents from pulp and paper mills, sewage plants, chemical manufacturing and non-point source sources such as runoff.

2. Effluent and drinking water mutagenic investigations A wide range of industrial effluents have been associated with mutagenic effects, including those from organic chemical manufacturers, metal refining

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

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

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

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

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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 extraction, but more often than not this information is not reported. This is also the case with evaluating activity following extraction or fractionations steps. For example, Cerna et al. [13] examined genotoxicity using the Ames test on water collected from the Labe River in the Czech Republic in a study that involved effluent and river water extractions using macroporous polystyrene gel Separon SE resin columns. Acetone column extracts were then subdivided into five fractions based on polarity, acidity, and volatility, and screened by gas chromatography/mass spectrometry (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 extraction and fractionation. For example, alterations during solvent extraction as well as acid/base partitioning can induce chemical changes in the compounds of interest, resulting in apparent losses. Nevertheless, most studies have adopted successful extraction approaches to investigate mutagenic substances, as well as non-mutagenic substances (see below). Advantages of the extraction approach include (i) the method of extraction provides immediate information on the types of chemicals involved in the effect being studied, (ii) the preservation of the active substances from microbial degradation once they are contained in a solvent and

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removed from the effluent matrix, and (iii) concentration of the active substances facilitates the tracking of the biological response by resolving it from background and additional mutagens (or other compounds of interest) that may be present. One of the most commonly employed methods of extracting and concentrating mutagens from sewage effluents and drinking water has been XAD resins. One of the first studies to utilize XAD resins in these types of applications was by Kool et al. [14]. It was found that a combination of XAD-4/8 was as effective in adsorbing mutagens from surface waters as XAD-2. Dimethylsulfoxide (DMSO) was found to be as efficient as acetone in eluting mutagens from XAD resins and provides adequate delivery of compounds to the Ames test. In surface waters, the majority of mutagens were found to be adsorbed at neutral pH. Optimal recoveries of mutagens in drinking water with XAD resins have been found using flow rates at 2–4 bed volumes/min [15]. While convenient and nonresponsive in mutagenicity assays, the use of DMSO as an elution solvent should be used with caution since, because of its high boiling point, further manipulation of extracts or solvent exchange is not possible. In a later study, Filipic and Toman [16] used XAD2 resins to extract dissolved mutagenic substances sequentially at neutral and acidic pH from influents and effluents from a municipal sewage plant processing both industrial and domestic wastes. The XAD resins were extracted sequentially with acetone and dichloromethane (DCM) and the extracts were tested with and without S9 activation using the Ames test. Ono et al. [17] used simple extractions of filtered samples with C18 solid-phase extraction (SPE) to detect error-prone DNA repair induced by chemicals in Japanese sewage and nightsoil. Following SPE, the authors used semi-preparative reverse phase-high pressure liquid chromatography (RP-HPLC) fractionation and fraction collection every 10 mL. While the endpoint biased this study for aromatic amines, ozonation treatment was found to be a treatment option that removed the activity. Takigami et al. [18] also used XAD-2 resin under neutral pH at the ratio of 18 L to 50 mL sorbent and a miniaturized Bacillus subtilis assay to examine genotoxicity in extracts of Japanese sewage, river water and tap waters. Resins were eluted sequentially with ethanol followed by

ethyl ether, but no mention was provided as to whether recovery of genotoxic substances was in any way quantitative. Quantitative recovery of mutagenic activity has been reported for drinking water evaluations using XAD-2 and XAD-8 resins with varying water pH [19]. The mmutagenicity of pH 2 drinking water concentrates were sevenfold higher than those of the pH 8 extracts, suggesting that acidic compounds accounted for the majority of the mutagenicity. The presence of residual chlorine did not affect mutagenicity. Comparisons of the mutagenic activity for the pH 2 versus pH 8 extracts prepared by lyophilization further indicated that the acidic mutagens were chlorine disinfection products [19], which proved that earlier results associating the formation of mutagens with residual chlorine and XAD-4 resin [20] were minor contributors. The indication that mutagenic compounds were present in drinking waters led to the general acceptance of chlorinated humic material to cause the formation of 3-chloro-4-(dichloromethyl)-5hydroxy-2L(5H)-furanone, or (MX), initially discovered in drinking water by Hemming et al. [21]. The identification of MX in drinking water was preceded by the effects-directed effort, which led to its initial discovery in spent bleaching liquors as the major mutagen in pulp mill effluents [22] (see below). MX was found to occur after chlorination of drinking water and natural surface waters containing humic material. In the drinking water study, MX was recovered by acidifying the samples to pH 2 and passing them through a column containing a 1:1 mixture of XAD-4 and XAD-8 [21]. After its discovery in drinking water, a quantitative effects-directed analysis of drinking waters was conducted by Kronberg et al. [23] where mutagenic compounds in XAD extracts of chlorinated humic water were separated in two stages of HPLC fractionation. XAD extracts were fractionated first by a preparative C18 column and mutagenic fractions were then sub-fractionated on a C6 analytical column. 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

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waters, accounting for the largest proportion of mutagenic activity. In an analysis of the mutation spectra of drinking waters treated under pilot-plant conditions of chlorination, cloramination and ozonation, DeMarini et al. [24] found high frameshift frequencies in TA98 associated with MX and ‘‘MXlike’’ compounds. The authors suggested that halogenated aromatics, such as halogenated polycyclic aromatic hydrocarbons account for much of the mutagenic activity and specificity of the non-volatile organics in drinking water. The discovery that chlorination of humic material could produce mutagenic substances led to efforts directed to the mutagenicity of natural waters and sewage effluents. In the late 1990s, an extensive series of studies on the mutagenicity of sewage effluents in Japan covered all aspects of effects-directed investigations, including extraction of mutagens from effluents, their isolation from inactive components, proposed structural identifications [25], confirmation with custom-synthesized authentic standards [26], and measurements in surface waters of additional analogous chemicals [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 mutagenic activity (21% of the total) against Salmonella typhimurium was identified as 2-[2-(acetylamino)4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5amino-7-bromo-4-chloro-2H-benzotriazole, or PBTA1. Employing the blue rayon passive sampling procedure, a total of eight PBTA isomers were subsequently discovered [27–29]; these compounds account for approximately one-third of the total effluent mutagenicity [30], and are thought to be

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produced from azo dyes during effluent treatment [25]. In related works, a similar technique unique to mutagenicity investigations has been developed in a column-based preparation using chitin (poly-N-acetylglucosamine) powder bearing covalently linked CPT residues [31]. The chief disadvantage of rayon or cotton supports lies in their limited ability to passively batchtreat water samples, whereas the chitin based solid phase preparations allow for construction of SPE columns in Sep-pak cartridges. Constructed cartridges can then be handled under laboratory conditions, with smaller sample volumes, and provide quantitative data. All sorbents bearing CPT have been found to be highly selective for polycyclic planar structures that can function as mutagens and thus offer a unique method of extracting them from aqueous matrices, food and human excreta [32]. CPT–chitin columns have been used to investigate mutagens in river water contaminated 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 XAD2 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 activity [29]. The CPT–chitin technique has since been investigated further and has found numerous applications in the studies of environmental mutagens, particularly those that are polyaromatic hydrocarbon (PAH)based. PAH mutagens have been evaluated in detail and their elution conditions from modified blue chitin

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

Beginning in the early 1980s and continuing into the mid-1990s, mutagenic studies of pulp mill effluents have employed extraction techniques based solely on XAD resins. Unlike studies with drinking water and other surface waters, CPT-based solid phases have not been employed during investigations of mutagenic compounds in pulp mill effluents. This is likely due to matrix effects, in particular the large amount of lignin material present in pulp mill effluents that would affect the adsorption of planar polycyclic mutagens. Early pulp mill studies used in vitro Salmonella assays to indicate mutagenic activity, and later studies investigated fish-specific mutagenic responses. Most of the pulp mill-derived mutagens are derived from polar compounds produced from individual waste streams, with corresponding weak evidence of final effluents containing mutagenic substances. Efforts were first directed towards chlorination stage effluents of mill bleach plants that provided the strongest activity and led to the association of chloroacetones with mutagenic activity [40]. Kinae et al. [41] detected genotoxins in livers of wild fish collected from areas receiving pulp mill wastes, indicating the potential for exposure and bioaccumulation. Holmbom et al. [22] used a combination of ethyl acetate and XAD-4 resins to quantitatively recover 70–90% of the mutagenic activity from chlorination bleachery effluents. The majority of the recovered activity was removed by partitioning with aqueous NaHCO3. Preparative thinlayer chromatography (TLC) was used as a first step in isolation, followed by C8 RP-HPLC, further preparative TLC, C18 HPLC, and a final TLC step that allowed the isolation of MX. As this compound was not amenable to GC–MS analyses, methylated, acetylated and trimethylsilyl derivates were synthesized and analyzed to facilitate structural interpretation. XAD resin extractions have been used in attempts to isolate compounds from pulp mill effluents that are mutagenic in fish-based assays [22,42] but the results indicate weak activity, and that fish are not affected by mutagens in final treated effluents. Rao et al. [43] was able to elute weakly acidic and polar mutagens from final effluent using an XAD-8 column eluted with NaOH or methanol. The authors also employed diethylaminoethyl (DEAE) cellulose to adsorb high molecular weight lignin interferences from final

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effluents, which was also effective in studies of aryl hydrocarbon receptor agonists (see Section 3.1 and [44]). Metcalfe et al. [42] found evidence of directacting mutagens in extracts of XAD-7 (methanol) and XAD-4 (diethyl ether) using a S. typhimurium fluctuation assay, but not with trout eggs or sac fry exposed in vivo; further characterizations were not undertaken. A spectrum of other municipal and industrial effluents has been studied for mutagenic activity, and these studies have employed different analytical approaches to isolate genotoxic substances, fractionate them and, in some cases, characterize unknown mutagens. White et al. [9] conducted a screening study using the SOS Chromotest to evaluate DCM extracts of effluents from a cross-section of industries. Samples were fractionated according to whether substances were dissolved or particulate-bound. Acid/base partitioning was used to further differentiate those substances that were water-soluble. S9 metabolic activation was found to almost exclusively decrease genotoxic potency. The highest loadings, expressed in benzo(a)pyrene equivalents, were from sewage plants, pulp and paper mills and metal refining processes. Higher loadings were usually associated with effluent particulate material, and generation of genotoxic sorption partition coefficients (Kd-genotox), were later found to generally agree with 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

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

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

area of bioassay-directed compound identification has progressed on mechanisms that have been derived from effects assessments of wild fish populations, namely induction of P450IAI enzymes (measured as ethoxyresorufin-O-deethylase or EROD activity) and impacts on levels of gonadal sex steroids. Hewitt et al. [54] fractionated effluents before and after treatment, and after a maintenance shutdown at a bleached kraft mill in one of the first studies to address the role of secondary treatment in affecting EROD activity. Laboratory rainbow trout were exposed to treated and untreated effluent, whole and filtered (<1 mm) effluent, resuspended solids, and two fractions of effluent generated by nanofiltration (>400 Da, <400 Da). These analyses found correlations of EROD activity with several chlorophenolics, including tetrachloroguaiacol. Subsequent exposures confirmed that tetrachloroguaiacol did not cause induction, but Hewitt et al. [54] concluded that the correlations might indicate the potential source of the compounds is derived from lignin in the wood furnish. Burnison et al. [44] attempted to directly isolate chemicals inducing EROD activity in fish by following an effects-directed approach on final effluent from two bleached kraft mills located in Ontario. Using centrifugation, tangential flow filtration, and C18 solid-phase extraction, effluents after secondary treatment were investigated using a 4-day rainbow trout in vivo bioassay. It was determined that methanol extracts of particulates/colloidal material and SPE fractions contained active substances. Work focused on the particulate material and showed that activity could be isolated using methanol extractions. HPLC isolations determined that the active substances were present in a relatively non-polar region of the chromatographic separation, with a log Kow of 4.6– 5.1. As a result of follow-up studies using rainbow trout exposures and incubations with a rat hepatic carcinoma cell line (H4IIE), which directed HPLC fractionations of the methanol extract of the high molecular weight material, a chlorinated ligninderived pterostilbene structure was postulated for an unknown compound strongly associated with induction [55]. This was significant in that it showed a natural product, modified in the bleach plant, was eliciting the biological response. In a comprehensive study, Martel et al. [61] determined the source and identities of two substances

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associated with induction present in the primarytreated effluent of a newsprint thermomechanical pulp (TMP) mill. To determine the sources of activity within the mill, rainbow trout were exposed static for 96 h to TMP condensate, deinking, paper machine effluents, TMP whitewater, and various process effluents sampled throughout the mill. Exposure concentrations were based on the flow of these process streams in relation to final effluent. Contaminated TMP steam condensates were identified as the major process source of EROD-inducing substances. Using conventional liquid/liquid extraction, silica gel fractionation and preparative thin-layer chromatography procedures, an EROD-inducing fraction was isolated. The major constituents were identified by gas chromatography/mass spectrometry as juvabione, dehydrojuvabione, and manool, which are naturally occurring extractives in balsam fir. After extraction and isolation from balsam fir and TMP condensates using the methodology developed, trout exposed to juvabione and dehydrojuvabione exhibited significant hepatic EROD induction. Subsequent studies from the mid-1990s to the present day have attempted to address the more complex issue of reproductive effects in wild fish. This approach of focusing on in vivo effects of biota in the receiving environment and then working towards cause and effect solutions represents a unique and highly appropriate application of effects-directed studies. Although ecologically relevant, reproductive dysfunction in wild fish has represented a much greater challenge to address because the mechanisms involved are not understood. The responses have included effects on gonad size, depressed levels of circulating steroids [62], perturbations in the sex steroid biosynthesis pathway [63], and effects on gonadotropin production and peripheral sex steroid metabolism [64], indicating multiple mechanisms and chemicals are involved. In the late 1990s, development of suitable bioassays, such as fish-specific sex steroid receptor assays [65,66], life cycle tests [67] and short-term in vivo tests for steroid effects [68,69], has provided the opportunity to couple mechanistically linked endpoints to chemical fractionations. This has led to the ability to formulate questions regarding the characteristics of bioactive substances, their relationship to production type, and whether compounds associated with sex steroid depressions are related to other reproductive impacts.

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In the late 1990s, an approach similar to that used by Kinae et al. [41] for mutagens in fish tissues was adopted to address some of the obstacles associated with effects-directed investigations of final effluents. Parrott et al. [70] used caged fish to investigate the uptake of aryl hydrocarbon receptor (AhR) ligands from effluent from a bleached kraft mill. Ligands were recovered in methanol and DCM and non-dioxin ligands were found in tissue extracts using EROD induction in H4IIE cells as the indicator. In these investigations, the approach has been to focus on what is bioavailable to the organism by using controlled exposures to investigate bioactive substances in tissue residues [71,72]. One of the advantages of focusing effects-directed investigations on tissue residues is that it takes into account additional modification processes that may be involved in the responses, such as modification after mixing of effluent process streams, modifications during secondary treatment, modifications after release into the receiving environment, and metabolic modifications after accumulation [72]. In further applications of the accumulation approach, both unexposed wild fish and fish collected adjacent to the effluent outfall were held in a concentrated effluent stream (50%, v/v) for 4 days at a bleached kraft mill known to cause reproductive dysfunction in wild fish [73]. Hepatic tissue extracts from exposed fish were soxhlet extracted with DCM, and fractionated according to lipophilicity using RPHPLC. In this level of fractionation, HPLC elution conditions were optimized to achieve a linear relationship between Kow and capacity factor (K0 ), where K0 is the ratio of the reduced retention volume to the dead volume of the elution conditions. In generating such a calibration using a range of different classes of environmental contaminants, fractions with different Kow were tested for the presence of bioavailable chemicals that function as ligands for the AhR in H4IIE cells, rainbow trout hepatic estrogen receptors (ER), goldfish testicular androgen receptors (AR), and goldfish sex steroid binding protein (SSBP). Using the Kow fractionation approach, Hewitt et al. [73] showed that fish rapidly accumulate multiple nondioxin ligands across discreet ranges of Kow for the AhR and fish sex steroid receptors after a 4-day exposure. PCDD/DF equivalents measured by EROD activity in H4IIE cells and by high resolution GC–MS

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

extractives such as resin and fatty acids. Zacharewski et al. [76,77] found that the methanol extract of black liquor particles and colloids >0.1 mm from a bleached kraft mill contained AhR ligands which also displayed anti-estrogenic effects via the AhR in vitro. Hodson et al. [77] investigated the potential of black liquor from hardwood and softwood pulping at a bleached kraft mill to induce EROD activity in rainbow trout and found significant activity. More-potent liquor was associated with alcohol digestion of wood chips as well as solvent extracts of wood. In the late 1990s, an extensive investigation was conducted at a bleached kraft mill in New Brunswick, one of a handful of pulp mills in Canada that does not employ secondary treatment. This work successfully resulted in the identification of chemical recovery condensates as a primary source of substances that depress circulating sex steroids in fish and focused subsequent bioassay-directed studies. Minimal high molecular weight material was found in the condensates, facilitating bioassay-directed fractionation studies [78]. Using steroid depressions in mummichogs, a solid-phase extraction method was developed which completely recovered the active chemicals from the condensates in two fractions [78]. In this study, a combination of two SPE cartridges in series (styrene divinylbenzne and reversible graphitized carbon) was ultimately successful at isolating polar, bioavailable compounds affecting steroid levels. 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 (SDSPAGE) [81]. These investigations have determined

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that lignin derived macromolecules bind to the plasma membrane of the sperm head of purple sea urchins, thereby blocking the acrosome reaction and preventing fertilization [82]. The importance of effects-driven fractionation studies conducted on pulp and paper mill effluents and its incorporation into regulatory practice in Canada is worth noting. Environmental Effects Monitoring (EEM) programs in Canada have been developed for the pulp and paper and metal mining industries, where cyclical evaluations of the health of biota in receiving environments determine whether effects exist when facilities comply with existing regulations. Investigation of cause (IOC) is a specific stage in EEM that involves determining the sources and causes of effects observed in the receiving environment of a discharger. Several levels of effort have recently been described that can be undertaken for cause identification [83,84]. The framework includes levels to define whether there is an effect, whether it is related to the effluent discharge facility, and whether response patterns in the receiving environment are characteristic of a particular stressor type. The next tier of the framework involves investigating individual process wastes within the mill to determine the components contributing to final effluent effects. In contaminant-focused causal investigations, questions progress along a continuum which first asks if the source within the mill can be identified, and to effects-driven identification to the compound classes and ultimately, the specific chemicals involved. The fundamental question driving the investigations is whether sufficient information has been generated to define the effect such that a mitigative solution can be found. Effects-directed questions within the framework have been tailored so that the investigation may be halted when that information is attained [84]. 3.2. Sewage effluents In the late 1980s and early 1990s, work on sewage effluents focused on acute toxicity. In the US, these investigations arose from the development of comprehensive toxicity-based approaches outlined by the US EPA’s Toxicity Identification Evaluation (TIE) procedures [5–7]. The TIE approach uses the responses of organisms or an appropriate bioassay

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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 identification (isolation techniques, HPLC fractionation; Fig. 1). In this step, active components are further separated from inactive substances for their identification and confirmation [6]. These methods are specific to the classes of chemicals outlined above and utilize bioassay responses to evaluate the success or failure of extraction, separation and concentration of bioactive substances. Separation of the sample into acid, base, and neutral fractions can be accomplished here, usually with some knowledge gained from Phase I manipulations as to which category of compounds are involved. Acid/base neutral partitioning can also be applied to fractions generated from HPLC fractionation. Until the mid-1980s, normal phase HPLC was frequently the method of choice in fractionating extracts in effects-directed investigations, employing elution conditions of increasing polarity with solvents ranging from hexane to dichloromethane, acetonitrile and finally methanol [4]. Application of reverse phase columns has since been the method of choice in HPLC fractionations,

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since stable, bonded C18 phases have been commercially available at reasonable cost. The question of whether one or more bioactive substances are involved in the overall response is a complicated one, often the solution is to focus on the active component that is easiest to identify. Examples of isolation techniques include C18 SPE and solvent extraction (Fig. 1). Chemical isolation steps proceed in an iterative fashion, directed by bioassay responses until either further isolations are not possible, or candidate chemicals are identified (Fig. 1). Once there is strong evidence that one or more candidate chemicals are associated with the response, the last phase can be initiated. Phase III involves techniques that confirm the proposed substances are in fact responsible for the observed toxicity (Fig. 1). This is usually accomplished through a weight of evidence assemblage of information that collectively establishes the identity of the active compounds [7]. It is also equally important to establish that the cause of the effect is consistent over time so that amelioration efforts can adequately address the effect. Some judgment can be exercised in terms of the extent to which confirmatory tests are carried out, which reflects the authenticity of the results. For example, if a suspected substance can be removed by inexpensive pretreatment or process modification, a higher level of uncertainty may be acceptable than if an expensive treatment plant is required. Confirmatory approaches include correlations between concentrations of suspected agents and the bioassay response, symptom comparisons between effluent exposures and those of pure substances and spiking experiments to determine if the effect can be reproduced in the matrix being studied. The TIE approaches described above have been applied mostly to sewage investigations, as they were originally designed. Investigations in the early 1990s focused on identification of acute and chronic toxicants in primarily freshwater ecosystems but marine environments were also investigated with similar results [85]. Amato et al. [86] employed a TIE approach to identify diazinon in final municipal effluents as acutely toxic to Ceriodaphnia dubia. In this study, C18 SPE cartridges were used to successfully recover the activity, which was confirmed by 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 manipulations, spiking experiments, and correlations of toxicity with fraction concentrations of suspected toxicants for confirmation in Phase III [88]. The behavior of heavy metals was found to vary significantly during TIE manipulations of metal contaminated effluents and sediment pore water [89]. When metals are suspected toxicants, addition of EDTA as a chelating agent can be useful to verify their presence [5], however the responses of metals to manipulations are often variable, suggesting the presence of additional toxicants. It is also possible that the extraction procedure may result in charge state changes in metals and thus affect their activity. In the case of SchubauerBerigan et al. [89], passage of the effluent sample through a C18 column caused a reduction in toxicity later attributable to bioavailable zinc, rather than non-polar organic compounds, as one might first suspect. It was also found that bioavailable metals can be removed by filtration and recovered by extraction. Moreover, dilution water matrix effects on toxicity can impede Phase III confirmation experiments. This can occur if matrix conditions such as hardness, alkalinity and dissolved organic carbon (DOC) are sufficiently reduced from their whole effluent concentrations to affect metal bioavailability in dilution experiments. Finally, a combination of EDTA, sodium thiosulfate and graduated pH tests were used to distinguish copper toxicity from lead and also metal toxicity in general to that for ammonia [89]. As the issues surrounding biologically active compounds in pulp mill effluents evolved through the 1990s, a similar story had begun to emerge for municipal sewage effluents in the late 1990s. This coincided with the development of the so-called endocrine disruptor hypothesis, originally put forth by

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

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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 estrogenicity, recent studies in the UK have begun to investigate androgenicity. Using effects-directed investigations, Thomas et al. [97] coupled a yeast based androgen screen to studies of surface waters and final effluents. Similar to the case for estrogens, natural steroidal compounds and metabolites are associated with the observed responses. The study identified the natural steroids and steroid metabolites dehydrotestosterone, androstenedione, androstandione, 5b-androstane3a,11b-diol-17-one, androsterone, and epi-androsterone and quantitatively confirmed them to be responsible for 99% of the in vitro activity of municipal effluents discharged into UK estuaries. 3.3. Other effluents Bioassay-directed investigations have been utilized in a variety of other point source industrial effluents to determine the identities of biologically active compounds and their sources within industrial processes. The majority of investigations have employed the TIE approach in seeking to characterize compounds associated with acute toxicity, and it appears that adoption of this approach provides elevated chances for success. Fractionations have commonly been driven by toxicity to different species of Daphnia, used for their case-specific suitability, reproducibility and convenience to drive chemical fractionations. DiGiano et al. [98] attempted to determine if TIE protocols could be applied to textile mill effluents. Phase I manipulations showed slight removal of acute toxicity to C. dubia after C18 SPE. Anion exchange introduced artifactual toxicity but chloride was eventually confirmed as accounting for 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

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intermediates and surfactants to the toxicity without confirmation. Castillo and Barcelo [99] used D. magna to investigate the toxicity of textile effluents and landfill leachates. Two different extraction and fractionation methods were applied: samples were pre-concentrated by sequential SPE using C18 and styrene divinyl benzene solid phases. C18 cartridges were eluted to capture broad compound classes into three fractions: hexane (aliphatic polyethoxylates, Cn>15), DCM:hexane (4:1; aliphatic polyethoxylates, Cn<15) and methanol:DCM (9:1; nonylphenol ethoxylates). Phenolic substances in the residual water were extracted by styrene divinyl benzene cartridges that were eluted with a solution that was 5 mM each in triethylamine and acetic acid in methanol (9:1). Subsequent analysis by 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 phthalates. The diagnostic capability of cation-exchange treatments of effluents to implicate hardness-dependent components was highlighted in textile effluent investigations by Wells et al. [100]. Fractionations were directed by acute toxicity in Daphnia pulex. After standard approaches involving filtration and C18 SPE treatments, ion exchange treatments affected the resultant toxicity, suggesting metals were involved. A reduction in toxicity following anion exchange indicated that zinc was present in an anionic form in the effluent. Toxicity testing coupled with zinc, calcium, and magnesium analyses confirmed zinc as the primary toxicant. Subsequently, calcium was determined to have a greater protective effect of zinc toxicity to D. pulex than magnesium. In an evaluation of different cation-exchange media for applicability in TIEs, Burgess et al. [101] recommended columns from two manufacturers, which removed 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. Fractionation of the neutral DCM extract by RP-HPLC eluted with a gradient of pH 3.6 acetate buffer and acetonitrile isolated toxicity in a single fraction. A combination of 1H-NMR, 12C-NMR, GC using thermionic detection and 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

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into offshore waters and coastal areas without treatment. It is a complex mixture known to contain hydrocarbons, oil droplets, organic acids, phenols and production chemicals [106] and exhibits both acute and chronic toxicity in laboratory testing. Bioassaydirected studies have used toxicity in tropical mysids [107], Ceriodaphnia, fathead minnows, and sea urchins [108] and binding to yeast-based androgen and estrogen receptors [106] to direct chemical fractionations. The results between effluents from different sites are highly variable with contributions from non-polar organics, metals, ammonia and volatile organic compounds, and pH-sensitive substances. Moderate success was achieved in the identification of 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

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dioxins were not laterally substituted, and therefore of a lower risk to aquatic environments. To examine formulation effects on fish sex steroid levels, the authors were one of the first to couple effects-directed fractionations to a competitive binding assay with rainbow trout hepatic estrogen receptors. It was subsequently determined that the active ingredient itself, as well as two isomeric impurities, were estrogenic [113]. Other effects-directed investigations have dealt with chiral pesticides. These studies have arisen from the concern over the toxic nature of the active enantiomer on non-target organisms, and the stereo-selectivity of microbial degradation. In the past, an enantiomer was obtained by achiral synthesis or asymmetric synthesis, and the product was then obtained by fractional distillation and re-crystallization for investigation of biological activity [114]. These investigations have progressed with the development of chiral chromatographic columns for GC and HPLC. For example, a series of 14 O-ethyl-O-phenyl-N-isopropyl-phosphoramidothioate enantiomers containing a phosphorus atom as a chiral center have been separated by HPLC on a Pirkle model chiral stationary phase [115]. Some chiral organophosphorus pesticides also have been separated by microcolumn liquid chromatography using Chiralcel OD columns and a UV detector [116]. The pesticide fenamiphos was recently investigated using toxicity to the non-target aquatic invertebrate D. pulex and an HPLC fitted with a Pirkle chiral column. It was found that both enantiomers degraded at identical rates under environmental conditions, but (+)-fenamiphos was about 20 times more toxic to Daphnia than (+)-fenamiphos [117]. A related effects-directed study of another formulation of environmental significance relates to the acute toxicity in aquatic invertebrates associated with the release of aircraft de-icing/anti-icing fluids (ADAFs). ADAFs are formulated to contain 50– 90% ethylene, propylene and combinations of other glycols. Concern over other formulation components such as wetting agents, surfactants, corrosion inhibitors and thickeners led to an effects-directed investigation directed by Microtox [118]. ADAF formulations were subjected to semi-preparative RP-HPLC and fractions were characterized using GC–MS, UVand LC–MS. Activity in the fraction with the highest toxicity was further isolated after

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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 manipulations showed that substances acutely lethal to Gammarus pulex were associated with sediments [119]. Phase II experiments utilized soxhlet extractions of sediments to recover active compounds and then preparative alumina–silica chromatography to generate five fractions eluted with increasing proportions 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 examined the relationship between manure-borne hormones, as well as hormones used to treat animals prophylatically. Burnison et al. [122] investigated estrogenic substances in hog manure using a recombinant yeast estrogen screen assay to direct fractionations of manure extracts. Manure extracts were obtained by diluting concentrated aged manure from a farm holding tank in southwestern Ontario, Canada. Liquefied manure was extracted using SPE and extracts were fractionated by RP-HPLC. The endogenous estrogens 17b-estradiol and estrone, as

well as the phytoestrogen metabolite equol, were confirmed by GC–MS. Equol was further characterized using fish hormone receptor binding assays and was found to be weakly (200–1000-fold < 17bestradiol) estrogenic with a weak affinity also for goldfish androgen receptors. The overall hormonal activity of tile drainage following the first rain event following manure application was minimal. It is clear that in this emerging area more information on the types and amounts of estrogens, androgens, gestagens, growth promoters and antibiotics that exist in fresh livestock excreta, and their fluxes to aquatic environments needs to be determined [123]. Finally, while nearly all bioassay-directed studies mentioned in this review have dealt with those substances present in effluents affecting aquatic biota, Brack et al. [124] examined effects of volatile bioactive substances from landfill leachates. The bioassay used was based upon in vivo chlorophyll a fluorescence of green algae, enabling the detection of leachate chemicals interfering with photosynthesis. Volatiles were isolated by distillation of leachates in a closed system. Using gastight syringes, a concentrated aqueous fraction containing compounds with 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

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

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

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

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be emphasized enough that such endeavors are not trivial and require significant resources including infrastructure, personnel and a multidisciplinary team of trained individuals. Experience in working with the matrix being studied is also invaluable. As such, relatively few organizations attempt such investigations. In addition to the resource requirements, one of the main reasons for not conducting effects-directed investigations on a more routine basis is the uncertainty associated with the outcome of the investigation. Organizations are reluctant to allocate the large resources necessary when the outcome (i.e. identification of the specific cause) cannot be predicted with much certainty. Funding typically dictates the depth of effects-directed investigations. As shown in this review, one of the biggest challenges associated with effectsdirected investigations today is the ability of analytical techniques to overcome the obstacles (reactivity, adsorbent recovery difficulties) related to the extraction and analysis of polar compounds. For the purposes of this review, we choose a more realistic definition of successful investigations as those, which are able to provide more information as to the nature and cause of the effect than before the investigation began. In this way, nearly all effects-directed studies provide some measure of success, as in for example, the elimination of suspected compounds as causative agents [37,54] and the identification of compound classes involved in the responses [29,31,37,39–41,44–46,55,76,80,89,93, 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 RPHPLC for greater chromatographic resolution of extracted components. A second tier of HPLC fractionations may be employed using RP-HPLC or other solid phases suited to the compound classes of interest. Many studies blindly collect fractions at 1 min intervals (e.g. [6,88]), which limits the amount of information that may be gleaned from this first fractionation step. Frequently, a next tier of fractionation, involving recombinations of the 1 min fractions, is necessary for effects-directed investigations to progress (Fig. 1). One option is to conduct peak collections [111,113], and several automated fraction collectors have peak-collection options where valley thresholds can be selected. Another option is to collect fractions based on a physicochemical property. Several studies have calibrated HPLC elution conditions against Kow so that information is immediately obtained on the properties of the active chemicals, even if further sub-fractionations are not undertaken [44,73,74,111]. As was seen, characterizing toxicants associated with mutagenic and other effects in extracts and fractions is a daunting task and many researchers have resorted to toxic unit approaches with known or suspected contaminants to overcome effluent complexities [104,121,125]. Instrumental techniques for the characterization of unknowns have involved traditional analyses by electron impact 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

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usually be achieved [44,105,111,122]. The commercial availability of chiral columns now affords the opportunity to resolve between enantiomeric compounds, which has been shown for pesticides [117]. The confirmation of isolated candidate chemicals proposed as causative agents is challenging not only in the ability to isolate the active components from the matrix being investigated but success also requires matches from mass spectral libraries, or more likely, spectral interpretation and structural postulation. Other spectral techniques such as NMR and X-ray crystallography may be necessary to aid in formulating structures of unknowns (e.g. [105]). Procurement of authentic standards for proposed chemicals is required for complete chemical and toxicological verification. It is likely that authentic standards of candidate structures will not be commercially available and custom synthesis or preparative isolation may be required. Custom synthesis can be expensive, time consuming and depending on the structure, difficult to carry out. Structural confirmation of suspected mutagens or other toxicants were only evident in 10% of the studies cited in this review [25,26,61,91,97,111–113,121,122], owing to the difficulties in carrying out these investigations to completion. As the issues surrounding some effluents (e.g. pulp mills and sewage) have evolved, concerns have shifted to compounds that survive existing treatment regimes, which are by definition more polar and have been traditionally more difficult to analyze. Taking advantage of developments in the mass spectrometry of bio-molecules, analytical methods employing modern atmospheric pressure ionization (API)-MS techniques have been developed to monitor environmental levels of the active compounds, once they have been identified [94,95]. While 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/

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

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