Marine Pollution Bulletin 53 (2006) 3048 www.elsevier.

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Review

The eects of biological fouling control at coastal and estuarine power stations
Colin J.L. Taylor
British Energy, Barnett Way, Barnwood, Gloucester GL4 3RS, UK

Abstract The use of low levels of oxidants as antifouling agents in coastal and estuarine power station cooling water circuits can give rise to concerns over the likely production of chlorination byproducts (CBPs) including organohalogens, of largely cryptic identity, loading and impact. In order to review the current understanding of the degree of impact involved this paper describes a recent collaborative programme of work on CBPs within the wider context of a number of allied studies on power station cooling water entrainment and discharge eects. Bromoform was the single most important CBP found in coastal power station euents. Although bromoform was found to accumulate in the liver of the sea bass Dicentrarchus labrax (L.) continuously exposed to a chlorinated euent stream, no otherwise untoward impact on continuously exposed individuals was discerned. Populations of the common blue mussel Mytilus edulis L., likewise exposed, exhibited two responses: a high degree of genoplasticity and the evolution of stress proteinsalthough both may have been due to thermal rather than toxic stress. Experiments on plankton that simulated the conditions of entrainment within cooling water systems isolated the inuence of oxidant usage, with mortality varying between taxa and life stage. The results suggest a very limited impact of oxidant use and the associated CBPs on receiving waters both in terms of plume toxicity or any more widespread ecotoxicological inuence. Instead, it is in combination with applied temperature (DT), pressure and the other stressors involved in entrainment within the power station cooling water circuits themselves that an impact is most marked. 2006 Elsevier Ltd. All rights reserved.
Keywords: Power stations; Antifouling substances; Entrainment; Low-level chlorination; Chlorination byproducts (CBPs); Environmental impact

1. Introduction The exposed inner surfaces of seawater cooled power stations (Fig. 1) provide an ideal refuge for a variety of epifaunal species that may, if left to grow unhindered, readily compromise plant eciency, reliability and safety. There are many ways of controlling this biological foulingsome are well known to electricity generators, whilst others are variously promising, speculative, erroneously conceived or entirely impracticable (Jenner et al., 1998). The procedure most favoured by operators on north-western European coasts and estuaries is continuous low-level chlorination (Turnpenny and Coughlan, 1992; Jenner et al., 1998; BREF, 2000). This procedure follows the guidance

E-mail address: colin.taylor@british-energy.com 0025-326X/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2006.01.004

of Lewis (1984) who carried out long-term applied toxicity studies on the primary target species, the mussel Mytilus edulis L. It involves a continuous low dose of oxidants to the cooling water (CW) stream, sucient to restrict settlement and growth through chronic toxicity but not sucient to have an acute impact within the once-through cooling water circuit itself. The oxidant is introduced either through the electrolysis of seawater or the injection of sodium hypochlorite solutionin seawater this engenders a bromine-based chemistry (Jenner et al., 1998). This use of oxidants has long been understood to have an impact on entrained plankton (Heinle, 1976) but its dilimitation is obscured by the inuence of the other stressors involved: pressure, temperature, turbulence/shear and impact. Oxidant use can also give rise to concerns over the likely release of chlorination byproducts (CBPs) in the euent stream, including organohalogens, of largely

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Fig. 1. Basic elements of the cooling water system of a direct cooled thermal power station, typical of coastal plant. Direct cooled thermal stations reject $50 cumecs (cubic metres per second) of cooling water per 1000 MW g (megawatts generation) with a DT of +10 C, whilst indirect cooled thermal stations (most riverine or inland power stations) reject $5 cumecs of purged water from recirculating systems per 1000 MW g, with a DT of +10 C or higher. Arrows show direction of cooling water ow in both open (seawater) and closed (boiler water) circuits. A: oshore cooling water intake; B: coarse bar screens; C: 10 mm mesh rotating drum screens (4 of, in parallel); D: main cooling water pump (4 of, in parallel); E: reactor; F: steam generators; G: turbines and electricity generators; H: steam condensers (4 of, in parallel); I: 2 mm mesh pressure strainers (4 of); J: auxiliary and emergency system oil coolers (16 of); K: intake and outfall surge shafts; L: oshore outfall; X: biocidal injection points.

cryptic identity, loading and impact. The scientic literature prior to the studies reviewed provides only a limited understanding of the chemical dynamics involved in coastal waters, much speculation on the likely byproducts (mostly derived from experience in fresh water; see Jolley et al., 1990 and earlier volumes in the same series), and almost no eld data from coastal and estuarine waters that can be relied upon. In 1994 a collaborative programme (Box 1) was established to refresh the issue of CBP production in coastal waters and improve understanding of the impacts involved. The programme was led by Nuclear Electric (NE, successor company post-privatisation of the UKs Central Electricity Generating Board (CEGB), now British Energy), N.V. KEMA of the Netherlands (for the Dutch Electricity Pro de France (EDF). duction Companies) and Electricite

parallel tracks followed, exploring limits of of practicability and presuming that one or more would fail; management measures put in place to identify such failures early and redirect resource; Study sites selected across Europe for (rst) suitability for each planned experiment, (second) to reect range of receiving waters involved and (third) convenience to analytical facilities; Reference laboratory established dedicated to CBP analysis.

Box 1. The NE, KEMA and EDF Joint Utilities CBP Programme 19942000 Core activities: Task 1: Biological Field Eects; Task 2: Environmental Fate in Seawater; Task 3: Sources of Toxicity; Task 4: Natural Organohalogen Production. Working basis: Certain earlier studies identied as foundational; Parallel local eort by each party to explore assessment methods and develop understanding in specic areas; No xed endpoint established for the core eort in the rst instance: subsequent development based on review; where methodologies recent or novel several

This paper reviews the outcome of work in each of the task areas within the wider context described by some of the preliminary and parallel studies, also mentioned here. Inevitably much of the material reviewed is grey literature historically produced in-house by the electricity utilities involved, or under contract to them, and indeed the value of a review such as this is to render such material and the context within which it was accomplished visible. Where such references are cited here they are available directly from the author on request. More detailed descriptions of individual studies and their results will be found in separate publications. 2. Preliminary investigations 2.1. Early community studies There have been very few instances of acute toxic eects being observed beyond a power station CW outfall where low-level chlorination is used (Langford, 1983). One instance in 1985 involved the rapid and otherwise unexplained death of lobsters being stored in oating

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keep-boxes o Cockenzie power station (Firth of Forth, Scotland), which were found on examination to be in an advanced state of necrosis (pers. obs.). The keep-boxes had been moored in close proximity to the cooing water outfall and, oating at the surface within the thermally bouyant plume, would have been exposed to signicant levels of residual oxidant. Such circumstances are exceptional. Coastal power stations in the UK commonly follow the guidance originally developed within the CEGB by Lewis (1984) where the level of in-circuit chlorination is deliberately maintained at a threshold that will not cause the death in mussels in the circuit itself but, instead, only retard growth (see Fig. 2). To exceed this threshold continuously would be wasteful of resources and, if applied in an ad hoc manner would run the risk of large quantities of loosened debris being ushed on to condenser faceplates or coolers, causing operational diculties. Adherence to this protocol means that, at worst, chronic rather than acute levels of toxicity might be observed in the receiving water in close proximity to the discharge. In practice the sequential oxidant demand upon dilution that occurs as the euent plume mixes with fresh seawater rapidly negates the chronic toxicity of the oxidant itself beyond the point of discharge (Davis and Coughlan, 1983). The potential for synergies between chlorine toxicity and enhanced temperature is well known (Langford, 1983, 1990) but due to rapid oxidant loss upon dilution is more likely to be signicant in causing an impact on organisms entrained within the CW itself than in the area of the dispersing euent plume; a series of studies on entrainment impacts is reviewed later within this paper. A priority involved in initial power station site selection is to nd a location that allows for eective CW dispersal, minimising re-entrainment and associated eciency losses

(Robson et al., 1994). As a result the opportunity to study a gradient of eects caused by long-term exposure to chlorination residues and the more obvious stressor of thermal input above ambient (DT) is limited to rare congurations of outfall, shoreline topography and nearshore bathymetry where, very locally, initial dilution is limited. Two sites where such studies have been possible are at Kingsnorth power station (Kent, UK) and Wylfa power station (Anglesey, North Wales, UK). A study of the soft sediment benthos in the constrained cross-shore outfall channel of Kingsnorth power station (Bamber, 1985) showed that the CW euent had a significant inuence, although the response varied markedly between species. For example, the isopod Cyathura carinata (Kroyer) showed increased rates of growth and reproduction; the tubicid oligochaete Tubicoides amplivisatus Erseus proved opportunistic, whilst its congener Tubicoides benedeni (Udekem) was forced out; many amphipod species were able to recruit to the warmed areas but their adult populations could not tolerate higher temperatures; the opposite was observed with the bivalve mollusc Petricola pholadiformis Lamark, whose adults survived successfully but whose larvae did not apparently settle or survive. These various impacts could be explained by the DT regime alone and were not attributed to any inuence of chlorination (Bamber, 1985). At Wylfa power station a 60 cumecs (m3/s) of chlorinated CW euent is discharged to the head of a funnelshaped rock gully that, after 150 m, quickly widens to the open coast. Euent dilution and dispersion are very limited in the gully itself but the local hydrographic regime serves to entrain the plume that leaves it, sweeping it abruptly oshore. The site is thus ideal in operational terms but it also provides a short length of shore and seabed

Fig. 2. Predicted time to death of mussels (Mytilus edulis) within a cooling water circuit at various levels of chlorination (as mg/l Total Residual Oxidant) and cooling water circuit temperatures. The level of residual oxidant chosen for the coastal power station operational procedure of low-level chlorination is normally at or around 0.2 mg/l TRO. After Lewis, 1984.

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where a discernible gradient of change in sessile species associated with plume-related water quality can be described (Bamber, 1991). The assessment tool used in this instance (Bamber, 1991) was the mapping of invertebrate assemblages associated with Laminaria hyperborea (Gunnerus) Foslie holdfasts. The nding was of a species list that was progressively impoverished with increasing proximity to the point of discharge. An apparent bleaching of the intertidal area close to the discharge point itself was noted (pers. obs.)probably an impact on epilithic algal populations by residual oxidants. Bamber (1991) concluded that the primary determinands of the distribution of the Laminaria holdfast assemblages were scour and DT. As chlorination residues would also have been directly correlated with both, the involvement of this particular source of stress could not be discounted. The chlorinated CW outputs of Sizewell A and B power stations (Suolk, UK) amount to some 85 cumecs. In contrast to the CW euents of both Kingsnorth and Wylfa power stations these discharges arise from oshore seabed outfall structures on an open coast, with no restrictive shoreline topography and only a limited bathymetric constraint to mid-eld dilution and dispersion. From 1990 to 1995 extensive numerical, eld calibration and validation studies were completed in order to model the behaviour of the thermal plumes and the dispersion, demand and decay of residual oxidants associated with chlorination (e.g. Phillips and Gmitrowicz, 1994; Dixon, 1994). Extensive marine benthic infaunal studies were repeated over the course of several years (19801998) (see, for example, Bamber, 1993; Elliott et al., 1998). Although not specically directed towards an assessment of the eects of residual oxidants, far less CBPs, the benthic studies served to conrm that in a moderately dispersive environment no combined impacts of the CW stressors could be discerned in terms of species, abundance or biomass distributions. Thus with a single possible exception none of these community studies identied an inuence that could be attributed to chlorination residues alone. That single exception was at the Wylfa power station site where although some inuence of chlorination residues might have been presumed the more obvious stressors were DT and scour. Specic inuences could not be discounted from these studies: (i) Oxidant stress on sh larvae and shrimp on entrainment through the CW system; (ii) Ecotoxicological impacts of CBPs on eurythermal species associated with the plume area; (iii) Any subtler and potentially more widespread inuence of the complex mixture of CBPs involved, such as through bioaccumulation. It had thus become apparent that if the potential for such inuences were to be investigated it would have to be in a manner other than community studies in the eld.

2.2. Early bioaccumulation studies In 1992 an experimental rig was established by Nuclear Electric (Davis, 1993a) to test a variety of dosing strategies against that originally suggested by the work of Lewis, 1984. As with the experimental layout used originally by Lewis, 1984, the rig incorporated a once-through ow system and did not recirculate treated water, thus avoiding an unrealistic toxic build-up of CBPs such as bromoform. Separate populations of the test organism, Mytilus edulis, were exposed to continuous chlorination residuals of 0.1 mg/l and 0.2 mg/l TRO over a period of 22 weeks. At the end of the trials the opportunity was taken to take tissue samples. These were analysed by gas chromatography coupled with mass spectrometry in search of trihalomethanes (THMs), as likely CBPs (Davis, 1993b). No measurable concentration of THM was found in the tissues. A hypothesis arose (Davis, 1993b) that the mussels had been exposed to chlorinated water within 1520 s after injection of sodium hypochlorite and that a longer reaction time would have been necessary to produce the target CBP species. An alternate hypothesis, subsequently considered more likely, was that there had been some diculty with sample treatment or the analytical procedure used. Empirical observations at power station sites have since shown that the reaction pathway to bromoform is completed very quickly, with 99% conversion to bromoform within 1015 s (see Section 2.3). Further, more recent bioaccumulation studies have shown that unless very considerable care is taken CBPs can very easily be volatilised to air during pre-treatment procedures such as maceration.

2.3. Review of chlorine chemistry in seawater Field studies of CBPs in coastal waters prior to the recent collaborative eort described here are rare and were limited either in extent or by available analytical technologies or both. As a result there has been a general presumption that the nature of CBPs and their loading was broadly similar to that of disinfection byproducts (DBPs) associated with the treatment of potable water and the disinfection of sewage. The oxidant chemistry and DBPs associated with potable water have been particularly well studied (see Jolley et al., 1990 and previous volumes in that series). Early eorts by electricity generators sought to develop a greater understanding of the behaviour of chlorine through experiments with dosed articial seawater at near to freezing temperatures. Decay and demand models were then developed to better understand the decline in the eective biocidal residual as it passes through the power station CW systems themselves and these models were successfully validated both on power station CW circuits and in the euent plume areas (Davis and Coughlan, 1983, 1992). Both laboratory and eld studies demonstrated that natural seawater imposed a considerable demand on

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oxidisable substrate when rst added. This was already known to have operational signicance, as typically on British coastal power stations using low level chlorination a tenfold dose of hypochlorite solution was required to obtain a sucient residual oxidant level in the CW stream. But in terms of environmental impact it also meant that when a chlorinated euent was discharged and then sequentially diluted by fresh seawater a very signicant oxidant demand was applied: this is why little sign of measurable residual oxidant could be found beyond the point of discharge. Fig. 3 summarises the current understanding of chlorine chemistry associated with coastal power stations, based on studies carried out both within the collaborative programme and subsequently (Khalanski, 2002). Seawater contains $68 mg/l bromide at full salinity: when chlorine is added it oxidises the bromide ions yielding hypobromous acid, HOBr. This reaction is rapid, with 99% conversion within 10 s at full sea water salinity and within 15 s even at half sea water salinity. The free oxidants formed by the chlorination of seawater are thus predominantly composed of HOBr and the hypobromite ion OBr. At the ambient pH of seawater this free oxidant is essentially undissociated and consequently more ecient as a biocide than the dissociated OCl that would result from applying chlorination within the same pH range in freshwater.

Several categories of non-oxidising CBPs generated by substitution reactions with organic matter have been dened (after Jenner et al., 1998): (i) Organohologens (OX) = organochlorinated or organobrominated compounds, together constituting Total Organohalogens (TOX). (ii) Purgeable Organohalogens (POX) = volatile organohalogens, a signicant proportion being trihalomethanes (THM). (iii) Adsorbable Organohalogens (AOX) = organohalogens adsorbable on activated carbon, being the heavier (non-volatile) compounds + volatile compounds not eliminated before dosage. If the AOX is measured after elimination of the volatile compounds, AOX represents the non-volatile component such that TOX = AOX + POX. (iv) Extractable Organohalogens (EOX) = organohalogens extractable in an organic solvent; as with AOX some volatile compounds may persist in the extract. POX, AOX and EOX are global measurements using micro-coulometry; EOX was especially developed for measurement in solutions rich in chloride ions such as seawater. In practice global analysis of EOX and POX typically gives poor results at low concentrations (<40 lg/l) due to chloride interference, a more appropriate approach at such lev-

Sodium hypochlorite solution 500 to1000 mg/l

NH2Cl
(monochloramine)

Reactions + with NH4 NH2Br NHBr2 NBr3

(bromamines)

ClOH ClO(free chlorine)

Br (bromide)

BrOH

BrO-

(free bromine)

Dilution in sea water

Free oxidants Combined oxidants Organic CBP

Reactions with dissolved organic matter

THM
(volatiles) CHBr3 CHBr2Cl CHBrCl 2 CHCl 3

(semi-volatiles) Bromoacetonitriles Bromoacetic acids

(non-volatiles) Bromophenols Other CBPs

POX

EOX

TOX

Fig. 3. The chemistry consequent upon sodium hypochlorite or electrochlorination product injection in natural seawater. After Khalanski (2002).

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els being chromatography. A CBP loading to coastal waters will also include polar compounds, such as the haloacetic acids, that are not measured by these means; in part these can also be identied and measured by gas, liquid or ion chromatography. Detailed reviews of the chemistry involved as it applies to CBP generation and power station operation may be found in Jenner et al. (1997), Jenner et al. (1998) and Khalanski (2002). 2.4. Toxicological review of potential CBPs Following a review of both the external literature and various earlier reports to the electricity generators, a shortlist of the most likely CBPs that could occur in coastal waters was developed. A study was then established to review what was known of the toxicity of these substances and explore existing water quality standards (Murgatroyd and Smith, 1993). The gaps in these ndings then led to the relevant quantitative structureactivity relationships being explored. Verhaar and Hermens (1991) state that for chemicals classied as belonging to Class I (inert chemicals), eect concentrations can be predicted by QSAR equations given for minimum (baseline) toxicity. This class of substances is considered to cause eects by the non-specic mechanism of narcosis. For chemicals identied as Class II (less inert chemicals), a range of eect concentrations between 0.2 and 0.5 times the baseline toxicity eect concentrations could be expected. If a chemical is classied as belonging to Class III (reactive chemicals) or Class IV (specically acting chemicals), the actual eect concentration would

be expected to be 10104 times lower than the baseline eect concentration. For chemicals that cannot be classied as belonging to any of the dened classes, no prediction can be made. The results of the toxicological review and resultant QSAR predictions, after Johnson et al. (1994), are given in Table 1. Comparison of these stated and derived reference standards with levels of the substances measured in the eld in subsequent studies (reported below) suggested that, amongst the suspected CBPs, only bromoform might cause adverse eects in the estuarine or marine environment.

2.5. Ecotoxicological method development Anticipating the need to establish an ecotoxicological understanding of chlorination procedures and in particular the impact of CBPs on coastal and estuarine receiving waters a study was established to review and advise on the potential suitability of available and developing methodologies (Elliott et al., 1993). This led directly to the extensive use of sentinel organisms (Mytilus edulis; the sea bass Dicentrarchus labrax (L.)), the development of laboratory mesocosms (Entrainment Mimic Unit (EMU) and reaction vessel studies using raw seawater) and the search for practicable surrogate measures of CBP impact (potential toxicity (pT) and semi-permeable membrane devices (SPMDs)). Each of these was associated with the NE/KEMA/EDF collaborative programme and the results are described below.

Table 1 Coastal water quality standards and proposed reference levels for presumed CBPs Substance by QSAR classication Class I (inert chemicals) Chloroform Bromoforma Dibromochloromethanea Dichlorobromomethanea Class II (less inert chemicals) 2,4-Dibromoaniline 2,4-Dibromophenola 2,4,6-Tribromophenola 2,4,6-Trichlorophenol Class III (reactive chemicals) Dibromoacetonitrile Unclassiablepotential Class IV (specically acting chemicals) 1,2-Dibromophenola 1,2,3,4-Tetrachlorobenzene Water quality standard 12 lg/lAA Proposed reference level 5 lg/lMAC 5 lg/lMAC 5 lg/lMAC

E P P

IDA IDA 12 lg/lMAC 12 lg/lMAC IDA

E/P E

20 lg/l (total DBPs)P 5 lg/lMAC E

Toxicity classications after Verhaar and Hermens (1991) and results after Johnson et al. (1994). IDA: Insucient data available to complete QSAR analysis. AA Annual average concentration. E Proposed reference level derived from experimental data. MAC Maximum allowable concentration. P Proposed reference level derived from QSAR predictions. a Subsequently found as a CBP.

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3. Task 1: Biological eld eects of chlorination residues 3.1. Development and use of a potential toxicity (pT) methodology Given that the toxicity of a chlorinated CW euent was intentionally controlled so that its toxicity was chronic rather than acute, an appropriate assessment approach was to combine a means of extraction and concentration of CBPs with a standard toxicity screening test. Such a technique was found to exist in the form of the pT-Microtox method developed in the Netherlands by the National Institute of Public Health and Environmental Protection (RIVM) for use in fresh water (De Zwart and Sloo, 1993). Pre-treatment involves the 500-fold concentration of raw seawater samples using XAD resins and subsequent elution with acetone. Prior to toxicity testing with a standard Microtox test the acetone is removed by overblowing the concentrate with nitrogen and topping up with Milli-Q water. With the further possible benet that the extraction and concentration method using XAD resins might subsequently allow bioassay-mediated characterisation of toxic fractions derived from eld samples, a study was established to explore the use of the pT-Microtox method in seawater (De Potter, 1994). This was then developed so that the relative signicance of results could be standardised across fresh and coastal waters. pT-Microtox measurements subsequently became a routine feature of the long-term exposure study on sea bass (see Section 3.2) and the studies on CBP fate (see Sections 4.3 and 4.4) and characterisation (see Section 5.1). 3.2. Studies on wild and farmed seabass Although much information is available on marine and estuarine invertebrates, as potential fouling species exposed to chlorination procedures, there is little knowledge of the likely impacts on marine vertebratesespecially those that might also form part of the human food chain. A review of biomarkers and populations potentially hostage to the inuence of CBPs in coastal waters (Elliott et al., 1993) pointed to the sea bass, Dicentrarchus labrax, as a likely sentinel species. In north-west European coastal waters the sea bass is commonly found in the vicinity of power station CW discharges where it is known to actively forage for food within the turbid plume. Zoogeographically, local populations become more limited to these plume areas and are apparently progressively more dependent upon them in higher latitudes, most probably due to the lower thermal limit of tolerance of this species. Such populations frequently form the basis of a local, high value, sporting or commercial shery. In addition this species is frequently farmed and some such farms use chlorinated CW from power stations. By basing an investigation around a coastal power station site where there was both a wild sea bass population

and a dependent sea bass sh farm, ve long-term exposure scenarios were potentially available: farmed/exposed, farmed/unexposed, wild/exposed/intake, wild/exposed/ outfall and wild/unexposed. Such a study (unpublished) was established at EDFs Gravelines power station, north-west France and the closely associated sh farm. This study established control sites at similar sea bass farms at Theoule, Canet, Cartagena and the Ebro delta on the Mediterranean coasts of France and Spain, none of which utilised a chlorinated seawater supply. In addition, wild reference sh were collected o the Costa del Morte on Spains Atlantic coast and on the North Sea coast of the Netherlands at Kop van Goeree. The various components of work developed over the course of three years were: (i) CBP inputs: Water samples from all locations routinely analysed for: chloroform, dichlorobromomethane, chlorodibromomethane, bromoform, dichloroacetonitrile, dibromoacetonitrile. Analytical method derived from US EPA (1995). (ii) CBPs in sea bass: Tissue samples from all locations routinely analysed for the same substances. Analytical method derived from US EPA (1995). (iii) Histopathology: Following Vethaak (1993) on the use of sh liver tumours as biomarkers for chronic toxicity studies, appropriate samples were taken at each location and screened. (iv) 7-ethoxyresorune-O-deethylase assay (EROD): Following Peacall (1992) and Ha nninen et al. (1991) on the use of the mixed function oxidase (MFO) system as an indicator highly responsive to chemical pollution, EROD activity in sh tissue was measured routinely. (v) Semipermeable Membrane Devices (SPMDs): SPMDs are a passive sampling tool incorporating a thin layer of neutral lipid (in this case triolein) acting as a sequestration medium believed to mimic bioconcentration in living organisms (Axelman et al., 1999). Such collection devices were routinely deployed at each site. The total burden of organohalogens (TOX) was determined in the SPMDs, sea bass fat and muscle tissue as an adjunct to the CBP determinations. The results of this study are summarised in Box 2.

Box 2. Biological eects of long-term exposure of sea bass to CBPs The survival rates of all life stages of sh raised in chlorinated water compared favourably with those at other commercial farms without a chlorinated water supply.

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Histopathological studies of exposed and reference sh did not reveal any sign of liver tissue damage that could be attributed to CBP exposure. Bromoform was found at levels up to 1.7 mg/kg in the fat of sh raised in chlorinated CWa bioconcentration factor of 70160-fold above levels in the CW itself. This burden disappeared rapidly when chlorination ceased. Bromoform concentrations in SPMDs located in the power station CW intake, outfall and at the associated sh farm were comparable to those encountered in sh fat collected at the same locations. Analyses of muscle tissue revealed no bioconcentration of bromoform in sh exposed to chlorination. Bromoform concentrations in both the fat tissue and the muscle of wild reference sh not exposed to chlorination were below detection levels (<3 lg/kg fat). The CBPs (other than trihalomethanes) frequently found in sh fat and SPMDs in association with chlorinated CW were dibromoacetonitrile and dichloroacetonitrile. EROD activity measured in sea bass exposed to CBPs was comparable to levels measured in wild reference sh from relatively clean, non-chlorinated waters. An understanding of the potential relationship between EROD activity and CBP exposure appeared to be hampered by the temperature dependent activity of the enzyme system itself. TOX levels in sh fat were not positively correlated with CBP exposure. TOX levels in the fat of wild reference sh from relatively uncontaminated waters were comparable to those in sh from chlorinated water. TOX in the SPMDs was higher in sh farm waters compared to those placed outside the CW intakes (in theory non-chlorinated but in practice exposed to re-entrainment residues at certain tidal states). Bromoform only explained 0.5% of this increase, the remainder was unexplained. The range of results indicated that long-term exposure to CBPs produced by low-level chlorination did not impose any apparent ecotoxicological stress on the sea bass concerned.

ity generators on the likely fate of CBPs (De Potter et al., 1997) found that: Most of the available information on CBP fate and degradation rates in the literature related to experiments and calculations for fresh water and active sludge, not the marine environment. In comparison with chlorinated analogues, brominated compounds were reported to be less volatile and less water soluble due to their higher molecular weight (i.e. bromide 79.7, chloride 35.3). For THMs like bromoform the main route of loss from the water phase was reported to be through volatilisation, with a recorded volatilisation half-life in a water body 1 2 m deep of 12 days. The reported biodegradation halflives under aerobic conditions were around 1 month. Adsorption to suspended matter and abiotic degradation (e.g. via hydrolysis) were reported as negligible. The half-lives of dihaloacetonitriles such as dibromoacetonitrile (DBAN) were reported as being of the order of several days, dependent on pH, with the primary route being gradual hydrolysis into dihalolacetic acids. Adsorption to solids was reported as negligible and no information on biodegradation was found. Haloacetic acids were reported to not easily evaporate from the water phase since they were completely ionised at ambient pH. Very little information was available on abiotic degradation and biodegradation but a degradation half-life for dibromoacetic acid was stated as 300 days. Halophenols were known to be partially ionised at ambient pH values. The volatilisation half-lives of 2,4dichlorophenol and 2,4,6-trichlorophenol were reported as 2 weeks. Halophenols were known to be susceptible to photodegradation and this was understood to be the major fate process, with a half-life of several days. Biodegradation half-lives were reported as being 7 days and the degree of adsorption to solid matter (and bioconcentration) was reported as moderate. 4.2. Establishment of dedicated CBP laboratory A basic component of the collaborative eort between NE, KEMA and EDF was the establishment of a reference laboratory for CBP analysis, together with appropriate quality assurance measures. The laboratory was established at Arnhem (Netherlands) by N.V. KEMA, sharing the appropriate cross-calibration and validation procedures with the national laboratories at TNO-Delft (Netherlands) and the Pasteur Institute of Lille (France). The laboratory was established to characterise specic substances within three groups of CBPs: haloforms, dihaloacetonitriles and halophenols. All CBP analyses associated with the studies described in Sections 35 of this paper were accomplished through use of this facility except where otherwise stated. The analytical protocols employed were (Jenner et al., 1997):

4. Task 2: Environmental fate of chlorination residues in seawater 4.1. Literature review A literature review of material available both in the published literature and in internal reports available to electric-

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Haloforms: Extraction method: purge and trap techniques; Analysis: gas chromatography with Electron Capture and Flame Ionisation detection (ECD/FID); detection limit <0.1 lg/l for chloroform, bromoform, dichlorobromomethane, dibromochloromethane. Haloacetonitriles: Extraction method: n-hexane extraction; Analysis: gas chromatography with ECD; detection limit <0.1 lg/l, for dichloroacetonitrile (DCAN), dibromoacetonitrile (DBAN). Halophenols: Extraction method: extraction with Solid Phase Extraction (SPE); Analysis: after derivation with acetic acid anhydride, gas chromatography with ECD; analytical limits are given here rather than detection limits: 2,4-dichlorophenol (0.4 lg/l), 2,4,6-trichlorophenol (<0.6 lg/l), 2,4-dibromophenol (<0.02 lg/l), 2,6-dibromophenol (<0.1 lg/l), 2,4,6-tribromophenol (<0.05 lg/l). 4.3. Small-volume laboratory experiments using natural seawater The majority of previous experimental studies reported on in the scientic literature or discovered in early internal reports to the electricity generators have relied on articial seawater (De Potter et al., 1997). None of these had employed articial seawater to which a natural organic component or an analogue to this had been added. Those few that employed natural seawater were apparently not associated with a reference laboratory dedicated to CBP analysis. In the rst series of experiments carried out on fate within the collaborative study (unpublished) closed glass bottles were used as reaction chambers. These were lled with seawater and dosed at a series of chlorination levels (measured as total residual oxidant (TRO)). Samples were then withdrawn at intervals and measured for TRO and THMs. Controls were established using seawater taken from the vicinity of the Borselle and Maasvlakte power stations on the North Sea coast of the Netherlands. Others involved synthetic seawater and seawater from two reference nutrient-poor open sea locations: one over the northern Dogger Bank in the North Sea and the other from the Sargasso Sea. Two experiments were carried out. The rst involved sealed bottles containing water subject to no dilution, simulating likely conditions within the CW system prior to discharge; after dosing, samples were taken at xed intervals and sent directly for analysis. In the second, simulating water quality conditions in a dispersing plume, a volume in a sealed bottle was dosed and aliquots were removed at intervals and diluted 10-fold in further closed vessels; sampling and analysis for TRO and THMs then proceeded as with the closed vessels in the rst experiment.

Some of the seawater and synthetic seawater samples in both experiments were spiked with doses of a standard commercially available humic acid (Aldrich H1, 675-2) prior to oxidant dosing. The results are summarised in Box 3. Box 3. CBP fate in seawater: sealed vessel study A dose typical of low-level chlorination applied to a bottle containing natural coastal seawater yielded an amount of bromoform comparable to that found in the eld. Doubling the applied dose in natural coastal seawater led to a proportional increase in bromoform concentration. No such doubling occurred in the nutrient-poor open seawater. Doubling TRO doses in synthetic seawater without humic acid being added led to an equally minimal bromoform concentration. When the synthetic seawater was spiked with humic acid bromoform evolution rose signicantly. THM yields were a function of total organic matter (TOC), TRO dose, and the type of organic material involved. A positive correlation was found between bromoform yield and DTRO:TOC, i.e. the amount of TRO used per unit of organic matter. Predilution of a hypochlorite solution prior to dosing did not reduce THM formation. A reduction in THM formation could be achieved by lowering the pH of the CW but this did not inuence the total loading of CBPs involved. 4.4. Large-volume laboratory experiments using natural seawater In the second series of experiments carried out on fate within the collaborative study (unpublished) large (150 l) reaction vessels were designed and built in order to explore the half-lives of the main CBPs found in the eld and any evolution in potential toxicity, pT. The vessels were maintained open to the air or closed as appropriate to need, temperature and light conditions controlled, and their volumes continually recirculated. A variety of experiments were carried out using fresh natural seawater collected from near the Maasvlakte power station on the North Sea coast of the Netherlands, each lasting 1 week; the results are outlined in Box 4. Box 4. CBP fate and residual toxicity in seawater: large vessel study In open reaction vessels concentrations of THMs in chlorinated seawater decreased from 15 lg/l to <0.25 lg/l over 7 days:

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In closed reaction vessels only a small decrease of THMs in chlorinated water was observed. The following rst order rate constants (k) and half-lives (t0.5) were determined: Bromoform: total loss: k = 0.027 h1 t0.5 = 26 h; net volatilisation loss: k = 0.024 h1 t0.5 = 28 h. DBCM: total loss: k = 0.040 h1 t0.5 = 17 h; net volatilisation loss: k = 0.031 h1 t0.5 = 22 h. DCBM: total loss: k = 0.080 h1 t0.5 = 9 h. In both open and closed reaction vessels >90% dibromoacetonitrile was lost in 40 h giving k = 0.050 h1 and t0.5 = 14 h. The potential toxicity (pT) of open vessels of chlorinated seawater declined to non-chlorinated intake levels within 23 days. Thereafter the pT remained relatively constant. In a closed system no signicant decrease in pT was observed. Although samples were tested for genotoxicity the results were equivocal: local inputs, probably from other industrial activities, were sucient to mask any inuence of low-level chlorination.

Box 5. Power station CW euents: CBP charactisation Dominant CBPs: Only bromoform and dibromoacetonitrile were found in all chlorinated CW euents at concentrations signicantly higher than the detection limits. Range in euent bromoform concentrations: Highest at Heysham, with mean of 25.16 lg/l. Lowest at Dungeness and Hartlepool: 5.75 lg/l and 3.5 lg/l, respectively. Mean at Maasvlakte was 11.4 lg/l, and at Penly and Gravelines 14.2 lg/l and 16.7 lg/l, respectively. Seasonal variance: Higher concentrations of bromoform were found in the Heysham euent over the summer; a similar pattern was apparent at Maasvlakte. Progressive formation of bromoform downstream: At all stations the average concentration of bromoform in the nal CW euent was marginally higher than that directly in front of the main condensers. DBAN: The highest euent concentration was found at Heysham, with mean 2.44 lg/l and maximum 6.5 lg/l. Elsewhere concentrations were below 2 lg/l. Bromoform and DBAN production rates: Averaging across all sites, a chlorine dose of $1 mg/l TRO (as Cl2) resulted in a mean CW euent bromoform concentration of 16.32 2.10 lg/l. The equivalent mean DBAN concentration was 1.48 0.56 lg/l. Dichlorobromomethane and dibromochloromethane: Both consistently present in the CW euents but at relatively low levels. Mean euent concentrations of both compounds at Maasvlakte, Heysham and Gravelines were 0.8, 0.7 and 0.6 lg/l, respectively, whilst concentrations in the euents of other stations were all <0.3 lg/l. Chloroform: Below the detection limit of <0.1 lg/l in the euents of all but Hartlepool on the heavily industrialised Tees Estuary. A single spot sample was found to contain 1.5 lg/l. 2,4,6-Tribromophenol: Rarely observed, with means of: 0.26 lg/l at Heysham; 0.29 lg/l at Maasvlakte; 0.12 lg/l at Paluel. 2,4-Dibromophenol: Found infrequently and then only just above detection limits; maximum observed was 0.055 lg/l in a spot sample at Dungeness.

5. Task 3: Sources of toxicity 5.1. CBP characterisation studies at power stations CBP determinations were carried out upon CW euent samples collected at a number of power station sites over a 3-year period, with the specic intent of characterising the CBPs involved and any seasonal variance in production (Jenner et al., 1997). The sites involved were: Heysham 2 (Lancashire), Dungeness B (South Kent), Wylfa (Anglesey), Bradwell and Sizewell A (Suolk) and Hartlepool (Teeside) power stations on the UK coast; Maasvlakte power station on the Netherlands coast; Paluel, Penly and Gravelines power stations on the Atlantic coast of France. Samples were collected remote from each site, on the inuent CW stream, at various points within the CW systems, and from the CW euent. The range of results across all sites is shown in Table 2. More detailed results can be found in Jenner et al. (1997). The Heysham power station site was chosen for a very much more intensive study, investigating the degree of variation encountered over tidal, daylight and seasonal cycles, both within the CW circuit itself and the local receiving water. At both this site and Sizewell A, equivalent analyses for CBPs and TRO as well as DT were carried out on samples collected along the length of the tidally streaming and dispersing CW plumes in order to develop and validate dispersion models (see Section 7.4). The results (after Jenner et al., 1997) are summarised in Box 5.

It is probable that the character and relative concentrations of potential CBP precursors (see Fig. 3) was one of the primary factors in explaining the dierences in CBP characterisation and specic load between sites. The relatively low bromoform concentrations arising from the cleaner (low organic load) and less turbid French coastal sites were similar to those on the Netherlands coast and on

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Table 2 CBPs measured in cooling water intake and outfall samples at coastal power stations Concentration range (lg/l) Intake From core eort Chloroform Bromoform Dibromochloromethane DBCM Dichlorobromomethane DCBM Monochloroacetic acid MCAA Dichloroacetic acid DCAA Trichloroacetic acid TCAA Monobromoacetic acid MBAA Dibromoacetic acid DBAA Dibromoacetonitrile DBAN 2,4-Dichlorophenol 2,4-DCP 2,4,6-Trichlorophenol 2,4,6-TCP 2,4-Dibromophenol 2,4-DBP 2,6-Dibromophenol 2,6-DBP 2,4,6-Tribromophenol 2,4,6-TBP Total organohalogens extracted with petroleum ether acid/basic: EOX Total organohalogens adsorbed through extraction of XAD resins with petroleum ether acid/basic: XADOX From extended analyses Dibromoethane 1,2,3-Trichloropropane 2-Chlorotoluene 4-Chlorotoluene 1,2-Dibromo-3-chloropropane 1,2,3-Tribromobenzene <0.1 <0.11.0 <0.1 <0.1 <1 <1 <1 <1 <2 <0.1 <0.4 <0.06 <0.02 <0.1 <0.05 0.32.4 lg Cl/l 120 lg Cl/l Outfall <0.10.3 143 0.12.2 0.11.0 <1 <1 <1 <15 <210 0.15.0 <0.4 <0.06 <0.02 <0.1 <0.050.3 113 lg Cl/l 315 lg Cl/l

0.1 0.2 0.1 0.1 <0.1 <0.1

0.7 15.8 2.2 1.3 1.2 1.1

the open east and south coasts of the UK, whilst the elevated results from the virtually pristine locality of Wylfa were similar to the long-term mean at the estuarine Heysham site, ostensibly of much lower receiving water quality. It is likely that anthropogenic inputs to water quality were not the only determining factor of the available assemblage of organic precursors, which could equally well have been provided by the metabolites of macroalgae, resuspended microphytobenthos and plankton. A consideration of such chemistry is complex and was beyond the scope of the collaborative programme. 5.2. Bioassay mediated chemical analysis (BAMCA) A protocol of bioassay mediated chemical analysis (BAMCA) was utilised in order to identify more obscure toxic components in the complex euent streams (unpublished). Over a period of three years a total of 18 sampling visitscovering summer, winter and springwere completed at Gravelines (France), Heysham 2 (UK) and Maasvlakte (NL) power stations. At each, non-chlorinated water was sampled at the intake and chlorinated water at the outfall. Controls included the sampling of intake and euent water when the power station was not chlorinating its CW ow. Additional samples were taken from within the Aquanord sh farm at Gravelines (see Section 3.2).

All samples were transported at low temperature in coolboxes and analysed within 24 h of sampling. On arrival in the laboratory samples were initially analysed for TRO, Microtox and pT-Microtox, THMs, DBAN, haloacetic acids*, halophenols*, EOX, AOX*, dissolved and total organic carbon (DOC and TOC), total carbon (TC), ammonium, chlorophyll* and phaeophytin*. XAD resin extracts were also tested for genotoxicity using the MutatoxTM test. Towards the end of the rst sampling season further measurement of the parameters marked (*) above was omitted as these had been observed to add no value within the BAMCA study. The bioassay mediated chemical analysis (BAMCA) procedure for CBPs in chlorinated CW was developed in co-operation with the Netherlands National Institute for Coastal and Marine Management (RIKZ). It aimed to identify compounds responsible for an increase in pT (see Section 3.1) after chlorination. XAD resin and liquidliquid (pentane) extracts were completed, put to High Performance Liquid Chromatography (HPLC), and the resultant fractions tested using an organic solvent protocol with Microtox. It was found that XAD-resin extraction of water samples did not yield high enough concentration factors, so the BAMCA procedure was completed using the liquidliquid (pentane) extracts alone.

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Toxic peaks identied through Microtox testing of the HPLC fractions were then subjected to Gas Chromatograph/Mass Spectrometry (GC/MS) analysis. Assays for dioxin-like modes of toxicity using chemical activated luciferase expression (CALUX) were performed by the University of Wageningen in the Netherlands (co-developers of this method with the University of California, USA). Appropriate analytical intercomparisons using HPLC and GC/MS were carried out between KEMA (Arnhem, Netherlands), TNO-Delft (Netherlands), and Institute Pasteur de Lille (France). The results are summarised in Box 6.

Box 6. Bioassay mediated chemical analysis of chlorinated CW euent The low-level chlorination of seawater did not cause acute toxic eects in the Microtox test when Total Residual Oxidant levels were below 0.10 mg/l (as Cl2). Bromoform values appeared to be a reliable measure of the magnitude of the chlorination reaction in CW streams. EOX formed after low-level chlorination may have consisted predominantly of bromoform, but since the EOX method was by denition not suitable for volatile compounds it was found to be an inappropriate parameter for assessing the impact of low-level chlorination. The amount of non-volatile EOX formed during low-level chlorination (when doses ranged from 1.03.0 mg/l as Cl2) was very low, in the range 36 lg/l as Cl2. Genotoxicity tests carried out on XAD extracts did not give any information on the possible formation of genotoxic compounds by low level chlorination. The presence of compounds with a dioxin-like mode of action (such as tetrachlorodibenzop-dioxin (TCDD), poly-chlorinated bi-phenyls (PCBs) and poly-aromatic hydrocarbons (PAHs)) in seawater was apparently related to the amount of suspended solids present. There was no indication from the CALUX assay that chlorination of seawater led to formation of these kinds of compounds, echoing the negative results of a parallel attempt to identify them through analytical chemical means alone (Section 7.5). Low level chlorination increased the toxicity in XAD extracts in proportion to the magnitude of the chlorination action and levels of bromoform observed. Bioassay mediated chemical analysis (BAMCA) of liquidliquid (pentane) extracts revealed that rhombic sulphur (S8) was the most important toxic compound present in these extracts.

Other more minor components of the same HPLC fraction were two unidentied phthalates and the compound di-o-hexylphtalate (DEHP), however subsequent experiments suggested that they made no signicant contribution to the toxicity measured in that fraction. The presence of rhombic sulphur was observed to be positively correlated with the presence of bromoform in CW euents (r2 = 0.84) with: (rhombic sulphur, lg/l) = 0.06 + 0.11 (bromoform, lg/l). The low-level chlorination of seawater increased the amount of rhombic sulphur in liquidliquid extracts. Sulphur is a ubiquitous compound in the marine environment and the concentrations of rhombic sulphur found (up to 4 lg/l) would not have been expected to result in adverse ecological eects. XAD extracts of chlorinated seawater did not contain any rhombic sulphur. This implied that as-yetunidentied compounds caused the increase in pT associated with low-level chlorination.

Given that these BAMCA studies suggested that there were further unidentied compounds associated with toxicity in the liquidliquid extracts, this work was extended through a further iteration. About 25% of the pT of the total liquidliquid extract was found to be caused by S8, and 26% of the increase in pT between intake (unchlorinated) and outfall (chlorinated) samples could be attributed to this same compound. Thus some 74% of the increase in pT associated with the liquidliquid extracts was caused by unknown CBPsin turn, 72% of this component had log Kow values in the range 48. A reasonable hypothesis, given the unexpected nding of S8, was that these unknown CBPs might not solely consist of organohalogens formed by the chlorination of humic material present in the CW but might include components from other sources formed by the chlorine oxidation process. Extended analytical procedures carried out on a very limited number of samples, in collaboration with TNODelft (NL), found a further six CBPs: these data are appended to the ndings of the earlier eld characterisation studies shown in Table 2. The additional ndings suggested that there remained much potential opportunity for further research as a number of other halogenated compounds, such as alkanes, toluenes and benzenes, might be formed during CW chlorination in coastal waters. 6. Task 4: Natural organohalogen production 6.1. Review of natural production in relation to anthropogenic sources Grimvall (1995) identied marine algae, fungi and higher plants as the dominant natural sources of halomethanes. Concentrations of bromoform of up to 500 ng/l

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have been observed (Nightingale, 1991; Nightingale et al., 1995) in association with oceanic beds of macroalgae but the level of open oceanic production by phytoplankton is reported as uncertain (see Liss, 1986; Penkett et al., 1985; Moore et al., 1993, 1995). As a result global estimates using data on algal biomass have used only the known rates of bromoform production associated with macroalgae: no separate understanding of phytoplanktonic production has been incorporated. Based on the results of Task 3 studies described above and reported in more detail in Jenner et al. (1997); an estimate of the global bromoform production due to seawater chlorination at coastal power stations was made with the following assumptions: Global electricity generation amounted to 12 1012 kilowatt hours (kW h); 40 m3/s CW was used for every 1000 megawatt (MW) generated (i.e. 144 l of water per kW h); All CW was subject to low-level chlorination resulting in an average bromoform concentration of 16 lg/l in the CW euent (i.e. seasonal dosing cycles and the potential use of intermittent dosing regimes were ignored). On this basis the estimate of bromoform produced was $2.6 104 tonnes/year. This was about ve times more organic bromine than estimated by Gshwend et al. (1985), who attributed 4.6 103 tonnes/year to the same sector. These two ndings suggest that the contribution from power station chlorination alone are approximately 1.53.0% of estimates of global natural production. A power station source of bromoform of 2.6 104 tonnes/year may be compared with the other industrial sources identied by Gshwend et al. (1985), being: (i) $5.0 104 tonnes/year of the fumigant methyl bromide (CH3Br) (now largely phased out as a known ozone depleter); (ii) $1.0 105 tonnes/year of the fumigant/petrol additive 1,2-dibromoethene (C2H2Br2); (iii) $1 103 tonnes/year of the ame retardant triuorobromomethane (CF3Br). In the narrower connes of the North Sea region, the main sources of bromoform have yet to be determined. Nightingale (1991) described a strong seasonality in the amount of bromoform present in that sea area but it is not clear to what extent this was inuenced by anthropogenic sources (e.g. seasonal CW chlorination) as compared to biogenic sources (e.g. seasonally dependent production by algae). 7. Parallel and subsequent studies 7.1. Entrainment mimic unit (EMU) The passage of material, including zooplankton and the larval stages of sh and crustacea, within the water body

passing through a power station is known as entrainment. Previously assessments of the combined eects of temperature, pressure, turbulence/shear and chlorination residuals on such organisms have relied on gross observations of the passage of organisms serendipitously available in the eld. In order to study the dierential stresses on a variety of target species a laboratory-based apparatus, the Entrainment Mimic Unit (EMU) was constructed at the behest of Nuclear Electric to mimic the conditions of entrainment passage through a CW system (Bamber et al., 1994; Bamber and Seaby, 2004). The core of the EMU was a power station condenser tube through which precisely dosed and heated seawater could be passed. A number of marine organisms having planktonic life stages were introduced into the apparatus and then exposed to a regime of pressure change, chlorine dose, heat and mechanical stress (both separately and in combination) in a series of experiments (Bamber and Seaby, 1993a,b, 1994a,b,c,d, 1995a,b). The particular conditions employed simulated and bracketed these predicted for a coastal power station then undergoing commissioning: Sizewell B (Suolk, UK). The results were subsequently employed in completing an assessment of the impact of the Sizewell B power station on regional sheries (Turnpenny and Taylor, 2000). The results of the EMU trials shown in Table 3 have been presented in order to isolate the impact of chlorination from that of DT. These results must be taken as indicative only as a complex range of interactions and thresholds between and against each of the variables (TRO, DT and pressure) was found, varying with the various taxa and life stages tested. The EMU experiments thus elaborated upon the degree to which low-level chlorination is associated with entrainment mortality. It is likely that this observed impact was a direct consequence of the presence of residual oxidants at their intentionally eective antifouling levels in the undiluted CW ow prior to discharge, directly analogous to the impact of these same residual oxidants observed by Lewis (1984) on sessile Mytilus edulis within a simulated CW circuit (see Fig. 2). It is equally likely that an equivalent proportional impact would not have been seen by secondary entrainment of larvae (i.e. those not directly entrained by the power station but drawn from the receiving water into the plume as it disperses), due to the non-conservative behaviour of the residual oxidants through sequential dilution and demand chemistry (see Sections 2.3 and 7.4). 7.2. Scope for growth (SFG) in mussels The same species that provides the reference fouling organism for power station operators in north-west European coastal waters, the common blue mussel Mytilus edulis, may be utilised as a water quality sentinel organism (Phillips, 1980; Widdows and Johnson, 1988; Elliott et al., 1993). The energetics of mussels exposed to a chlorinated cooling water stream were studied (unpublished) at

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Table 3 Mortality of individuals exposed to conditions and retention times typical of cooling water entrainment within Entrainment Mimic Unit (EMU): 11 C DT; 0.2 mg/l TRO; pressure to specic prole over range 0.2 to 2.4 At.; duration 10 min Data source Mytilus edulis L. larvae (spat) Crassostrea gigas (Thunberg) larvae (trochophores) Acartia tonsa Dana adults Crangon crangon L. larvaea Homarus gammarus (L.) larvaea Dicentrarchus labrax (L.) larvaea Solea solea L. larvaea Scophthalmus maximus L. larvaea Trial Interpolated Trial Trial Interpolated Trial Interpolated Trial Interpolated Trial Interpolated Trial Interpolated Trial TRO + P 0n.s. <10 100 8.72 3.69 $13 0n.s. $30 2.70 3.86b <10 49.8 16.3 $25 21.48 14.53 $27 70b
c n.s.

DT + P 28.8 16.9 $10 28.77 11.9 7.35 3.08 <10 5.59 8.36n.s. $20 13.4 9.69b <10 32.89 <10 12.87 1.61 $10 73.2b
c n.s.

TRO + DT + P 46.7 29.0 $36 94.9 7.25 19.12 9.97 $18 24.5 2.03 $35 13.25 2.15b <10 28.25 10.85 $30 91.66 2.95 $35 72.6b
c n.s.

The trial data presented are the mean % mortality of individuals (SD) in excess of control values at the test conditions most closely matching these typical entrainment conditions but in most instances varying across a wider range. To compensate for this bias interpolated % mortality values for the entrainment conditions stated are also presented, derived from bivariate spline analysis of all TRO, DT, and TRO + DT trial data. n.s. Trial data not signicantly greater than controls (t-test, P < 0.05). a These results incorporated in the Sizewell sheries assessment of Turnpenny and Taylor, 2000. b Predominantly attributed to mechanical stressors (impact, abrasion and shear) rather than TRO, DT or pressure. c Data are % damaged, non-viable larvae rather than % mortality.

Heysham 2 power station (Morecambe Bay, UK). This site was was selected for its particular outfall conguration: the CW euent was constrained within long training walls at low tide but progressively lifted clear of this culvert on a rising tide, oering a gradient of exposure to the chlorinated euent along its length. Various physiological measures were used to assess the health of mussels along the length of the culvert: Scope for Growth (SFG), mantle condition index, and body condition index (K factor). SFG is the measure of the energy status of an organism, quantifying the energy available for growth and reproduction by examining the basic physiological processes of feeding, food absorption, respiration and excretion (Warren and Davies, 1967). Body (somatic) condition indices are indicative of longer-term alterations in the nutritional status of the animal, or particular tissues within the animal. With the understanding that SFG was intentionally but marginally negative within the stations CW circuits themselves due to low-level chlorination (Lewis, 1984), the intention of this study was to elaborate upon the gradient of change in eect downstream. A downward trend in SFG was observed upstream along the length of the outfall culvert: ranges of +1.7 to +4.8 J/g/h for control site further out in Morecambe Bay, 4.8 to +1.8 J/g/h for the seaward end of the outfall culvert and 6.3 to +0.1 J/g/h for the upper end of the culvert. This trend was visible at all times of the year but the spread of results varied considerably through the spawning cycle.

7.3. Genoplasticity and stress proteins in mussels Studies of the common mussel Mytilus edulis are of particular interest both because of its potential for use as a sentinel species for monitoring environmental impact (Elliott et al., 1993) and because it is the single most important target species for fouling control in connection with coastal power stations in north-west Europe (Lewis, 1984; Jenner et al., 1998). Potential confounding factors in using Mytilus edulis as a sentinel species (or indeed in considering minimalist fouling control procedures) are the potential selection that may occur from within a genetically heterogeneous population with time at any particular locationsuch as within the Heysham outfall culvertor adaptation that may occur through dierences in gene expression, termed genoplasticity. A series of electrophoretic studies (unpublished) of cystolic malate dehydrogenase (cMDH) expression were established in parallel with the SFG study described in Section 7.2, sampling down the same gradient of change of exposure to the chlorinated CW euent. An early nding of these experiments was that the dierences between populations at the upper and lower ends of the outfall culvert were mostly at the level of gene expression. Subsequently, in in vitro conditions, the cMDH from mussels in the immediate outfall area was found to be more sensitive to temperature than the cMDH from mussels at the control site further out in Morecambe Bay. This suggested that in vivo other mechanisms operated in order

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to maintain the enzymes of the mussels in the outfall area in a functional state during prolonged exposures to high temperatures, most probably through synthesis of heat shock proteins. These observations suggested that the main strategy for coping with thermal stress was based on higher turnover rates in individuals in the aected population and/or higher production of heat shock proteins at the expense of growth. The population involved was thus found to be highly genoplastic. In parallel, gill and mantle tissue from individuals from within the same populations was tested in the laboratory using gel electrophoresis and the monoclonal antibody 5a5. Separate individuals were exposed to a variety of water quality regimes over a 14-day period: (a) no heating, no biocide dosing; (b) heat only; (c) biocide dosing only; (d) heating plus biocide dosing. Under regime (c) an increase in Stress Protein HSP 72 was observed. This was thought to be due to an increase in tissue anoxia brought about by a reduction in ltration rates associated with exposure to residual oxidants. Higher levels of HSP 72 evolved in gill tissue than in that of the mantle, supporting the hypothesis that chlorine may have been disrupting cell proteins exposing nascent cellular proteins to the toxicant. Still higher levels of HSP 72 were expressed under regime (d), where the water was both warmed and dosed. In general, elevated levels of HSP 72 indicated reversible protein damage from long-term sub-lethal exposure to chlorination residues; the degree of HSP 72 expression was positively correlated with that exposure. This investigation did not nd a means of isolating the impact of chlorination residuals on the populations concerned, although further eorts involving HSP 72 may yet hold promise. Instead it demonstrated two exible and apparently very eective responses to thermal and chlorination-induced stress by the species concerned. 7.4. Field dispersion studies and numerical modelling (TRO and bromoform) In order to improve the understanding of dispersion patterns of chlorination residues in the eld and validate the appropriate numerical models, CW euent plume studies were carried out o Sizewell (Suolk, UK) and Heysham (Lancashire, UK) power stations (Jenner, 1993; van Donk, 1994). At each location samples were taken beside a drogue as it drifted downstream, with the drogue being repeatedly reset to the centre point of that plume using temperature data. Bromoform concentrations were found to decrease along the length of both plumes with DBAN (found only at Heysham in that instance) behaving in a similar manner. Bromoform concentrations were strongly correlated with DT, with r = 0.99 for the Sizewell data and r = 0.97 for those from Heysham. In both instances bromoform concentrations dropped from around 14 lg/l in the immediate vicinity of the outfall to 1 lg/l by the last sampling point approximately 2 km directly downstream on the tide. The

decline in surface bromoform concentrations and DT along the long axis of the plumes was non-linear due to the Langmuir-like circulation (a downstream helical ow) typical of such buoyant, large volume, euent plumes (pers. obs.). Bromoform accounted for around 98% of the THMs generated by chlorination. Some 60% of that which was ultimately formed either in the CW circuit or the euent plume was present within 3 minutes of hypochlorite solution injection. The plume studies were employed to calibrate and validate numerical models of temperature, TRO and bromoform dispersion. The close correlation between bromoform concentrations and DT in these plumes suggested that bromoform, like DT, could be treated as a conservative parameter in the near and mid-eld. Such behaviour contrasted starkly with that of TRO, this parameter typically being non-conservative due to rapidly sequential demand upon dilution. In the far eld, atmospheric exchange due to volatility would have rendered bromoform nonconservative (see Section 4.4). 7.5. Screening for dioxins and furans The level of uncertainty over CBP evolution and, to a greater degree, allied understandings of sewage and water treatment-related disinfection byproducts (DBPs), have previously suggested that the formation of polychlorinated bibenzo-dioxins and furans could not be altogether discounted. As a result a limited study (Rechem, 1995) was established where eld samples were collected from the Heysham 2 power station intake (Lancashire, UK) and outfall areas and subsequently analysed. Extraction was performed using lter/XAD resin traps in a similar manner to that used in the pT-Microtox protocol. The traps were then extracted with toluene and concentrated by freeze drying prior to chromatography. The chromatography employed columns of silica gel slurried in hexane and sequential elutions with dichloromethane. The nal sample was collected, concentrated and solvent exchanged to nonane and analysed by GCMS using the United States Environmental Protection Agency method 1613 (US EPA, 1994). The detected concentrations of dioxins and furans had a Toxic Equivalent Value (TEQ) of 0.0114 ng/l at the inlet and 0.0121 ng/l in the nal euent. These concentrations were low and reported to compare well with other sea water samples previously analysed by Rechem. There was no signicant dierence between the (unchlorinated) intake sample and the (chlorinated) euent sample. These ndings would appear to be conrmed by the absence of any dioxin-like toxicity observed via the CALUX assay results described in Section 5.2. 8. Discussion and conclusions The motivation for many of the studies described here has been the understanding that the practice of low-level

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chlorination is readily open to challenge due to a lack of knowledge, either by an environmental regulator in setting standards, or by an operator open to the suggestion of supercially attractive but operationally risky alternate approaches. The collaborative programme described here took advantage of improvements in analytical technology and ecotoxicological assessment methodologies to improve the understanding of the impacts involved. A parallel need was to refresh, and if necessary revise, understandings of the balance between the ecacy and cost (the latter both in commercial and environmental senses) of fouling control procedures: in operational parlance this is termed optimisation. Against the rst need, of developing an increased understanding and delimiting impacts, signicant progress was made. Two advances were artefacts of the collaborative engagement itself. Firstly, reports, documentation and the knowledge of both various eorts in hand and the range of concerns and uncertainties voiced across Europe concerning chlorination were shared. Secondly, a widespread network of practitioners and researchers from other generation companies in north-west Europe, the Baltic and Mediterranean coasts consolidated around the central team of collaborators, leading to the establishment of the Power Industry Cooling Water Specialists forum (PICWS); this forum continues to operate. Through the collaborative programme the understanding of the nature and behaviour of the chlorination residuals themselves and the eects of these residuals on populations either entrained or resident in the euent discharge area has been signicantly improved. Earlier literature (largely from fresh water studies) provided an initial approximation of what might have been expected at coastal sites but we now possess a good understanding of the identity of many of the CBPs actually involved and their variance within sites, between sites and within years. It became possible to describe the evolution of the CBP loading through a marine CW circuit and the likely fate and impacts of these CBPs beyond the point of discharge. The concentrations of CBPs measured in CW outfalls were found to be approximately 1000 times lower than the acute toxicity thresholds known for each. The mechanisms of sub-acute toxicity were elaborated upon in some instances and the impact of chlorinated euents at the population level delimited for the rst time for sea bass, and improved upon for Mytilus edulis. Signicant ndings were the isolation of taxa-specic and life-stage-specic eects of chlorination on organisms entrained through CW systems, and the associated additive eects and synergies with other stressors such as temperature and pressure. Bioassay mediated chemical analysis has led to an understanding of sources of toxicity. Some of the ndings were unexpected, such as that concerning rhombic sulphur, and may reward further investigation. The signicance of this latter nding was, at least in terms of the impact of low-level chlorination, found to be minor in ecotoxicological terms.

Data arising from the eld CBP characterisation studies armed that the primary CBPs of interest in the marine and estuarine environment, beyond the rapid demandinduced loss of residual oxidants, were the organohalogens, primarily bromoform. Subsequent results gained in collaboration with TNO-Delft, albeit involving a more limited dataset, suggested the opportunity for further academic eort on the involvement of substances such as alkanes, toluenes and benzenes. The attempts to nd an assay methodology that would distinguish between the impacts of DT and chlorination residues in a CW stream and beyond were less successful: although long sought, such a tool continues to be elusive. In this instance, the experience with stress proteins in Mytilus edulis clearly revealed that an entanglement common to these two stressors exists at a basic sub-cellular level within the organism. A surrogate tool for indicating the CBP burden beyond the point in an euent plume, where oxidant chemistry and its allied toxicity become insignicant, proved equally elusive. The use of bromoform might have appeared to be more appropriate for such a purpose than the currently widespread use of TRO, were it not for the fact that variance in the chemistry of the abstracted seawater was revealed as a primary factor. Against the nal need, that of reviewing the balance between ecacy and cost when considering fouling control methodologies, again signicant progress was made. A power plant operator or the relevant environmental protection authority can view current practice within the context of numerous studies on both impact and ecacy, with a variety of assessment tools available; an example of this is illustrated in Fig. 4. A direct consequence of the collaborative eort was the publication of a revised and updated good practice guide describing the biology and control of fouling (Jenner et al., 1998). Subsequently much of the guidance put forward in that guide was adopted within the European Integrated Pollution Prevention and Control regime (BREF, 2000). There has also been a resurgence of interest in intermittent dosing regimes. This has in turn converged with developments in instrumentation such as Redox-based microfouling sensors rendering direct feedback dosing control systems, responsive to the degree of fouling with time, more practicable than previously. This review, together with the work it describes, have sought to support a refreshed understanding of the balance between ecacy and environmental cost of biological fouling control procedures on large scale thermal power stations operating within the specic zoogeographical context of the north-west European coast. The overiding need has been to deal with chronic issues of comprehension both within and outside the electricity sector. Changes in the electricity sector across Europe since the early 1990s, primarily the dissolution and partial privatisation of nationalised state electricity companies such as the UKs CEGB, have resulted in a process of fragmentation and loss of the large research institutions that had previously

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Instrument standardisation; Trials of spot TRO monitors; Trials of semi-continuous TRO monitors

C.J.L. Taylor / Marine Pollution Bulletin 53 (2006) 3048

Trials of settlement monitors

Best practice manual

European expert group

Intsrumentation

Implementation mechanism

Operational Memorandum

Optimisation regimes

Low Level Chlorination Options Perception of harm

Current regulation Efficacy & cost benefit Intentional in-circuit toxicity (TRO) Known by products Presumed byproducts

Alternative chlorine regimes; Chlorine dioxide; Other agents: Actibrom, biodispersants, amines; Non-stick paints; Novel methods; 35 years operational experience

Future regulation

DOE Biocides Report; EA Cl EQS; EA/SNIFFER DTA; PARCOM/DIFF; Precautionary Principle; WFD & IPPC BAT & BREF

Toxicity review QSAR

Literature

Known estimated background (bromoform)

Plume toxicity (ecotoxicological appraisal)

DTA

Field studies: characterisation

Hey sham Gravelines Maasv lakte

Inferred applied load

pT Microtox Environmental correlates Dispersion studies

Pseudo-first order Cl decay model - all sites; 3D Cl & bromoform dispersion models - Sizewell, Gravelines AOX ? Bromoform / salinity?

Laboratory studies: Behaviour, decay biodegradation

Early dynamic studies Closed vessel studies

Population & community studies

Laminaria holdf ast comm. Sea bass; Mytilus SFG
Entrainment Mimic Unit; Genoplasticity ; SPMDs

Ecotoxicological apparisal -methods development

Fig. 4. The contextual framework within which the practice of low-level chlorination may be understood by an operator of direct cooled coastal or estuarine power plantdrafted from a UK perspective.

developed. As a consequence marine ecological practitioners within the electricity sector became rare at the same time as the most recent phase of large scale thermal plant construction in the region, and the associated and very distinctive needs of scoping and environmental assessment, came to an end. Much expertise has no doubt been lost and some of the operational experience that led to current practices rendered obscure, not least as much grey literature has never seen publication in the wider scientic literature. The risks to be countered are that current or future operators might specify plant, procedures or maintenance inappropriately, or that environmental or planning regulators might apply equally inappropriate controls.

In conclusion: The practice of low-level chlorination is deliberately specied to apply a chronic, not acute, toxicity to sessile species within the CW ow of a coastal power station. Only in very rare instances, where dilution and dispersion beyond the point of discharge are greatly constrained, has an inuence been observed potentially attributable to chlorination alone.

The chlorination procedure itself does cause the mortality of a proportion of planktonic organisms and the smaller seston entrained through a directcooled thermal power stations CW system; the degree to which this may be signicant varies with taxa, life stage and the thermal regime involved. The behaviour of chlorination residues and the formation of various chlorination byproducts both within the CW circuits and in terms of their fate beyond the point of discharge has been described. The dominant CBP observed is bromoform; unlike the residual oxidants themselves bromoform behaves as a conservative parameter in a diluting and dispersing euent plume and its eventual fate is volatilisation to atmosphere. The inuence of long-term exposure by chlorination residues on the sea bass Dicentrarchus labrax was found to be very limited. Although some bromoform bioaccumulation was noted during annual chlorination seasons this accumulation rapidly dissipated thereafter. No impacts on growth were observed and no damage to liver tissues. Populations of the common mussel Mytilus edulis exposed to the combined inuences of a CW ow were found to be highly genoplastic. Individuals were observed to generate stress proteins in response to both chlorination residues and DT.

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The toxicological signicance of CBPs in the complex euent associated with low-level chlorination has been explored through bioassay mediated chemical analysis; gaps remaining in the process of characterisation have been identied. Despite these apparent gaps in CBP characterisation the spread of studies reviewed here, covering observed inuences at the community, population, physiological, metabolic and genetic levels, suggest that the impact of the practice of low-level chlorination on a coastal receiving water is very limited. It is in combination with DT, pressure and the other stressors involved in entrainment within the power station CW circuits themselves that an impact of low-level chlorination is most marked.

Acknowledgements The author gratefully acknowledges the eorts of his immediate colleagues involved in the Joint Research Project on Chlorination Byproducts, namely Drs. Henk Jenner de (N.V. KEMA) and Michel Khalanski (Electricite France). The author also gratefully acknowledges the contributions of: Professor Mike Elliott and colleagues at the Institute of Estuarine and Coastal Studies, Hull; Drs. Roger Bamber, Peter Henderson, Martin Davis and Jack Coughlan, once of the Fawley marine laboratory of the CEGB. The assistance of the Aquanord Fish Farm, the laboratories of TNO-Delft and the Pasteur Institute of Lille, the Agricultural University of Wageningen and the Netherlands National Institute for Coastal and Marine Management (RIKZ) is also acknowledged with thanks. The core research programme described here was funded by British Energy and its predecessor, Nuclear Electric, together with the Dutch Electricity Production Companies de France. through N.V. KEMA, and Electricite References Copies of references marked * may be obtained on request from the author of this review paper.
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