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Marine Pollution Bulletin 51 (2005) 495–506

Review

The ecotoxicological effects of Photosystem II herbicides on corals

Ross Jones *

Centre for Marine Studies (CMS), Seddon Building (No 82C), St. Lucia Campus, The University of Queensland, Brisbane, QLD 4072, Australia

Abstract

The recent discovery of contamination of the tropical marine environment by Photosystem II (PSII) herbicides used in agricul-ture and antifouling paints has led to concerns regarding the effects on corals and their symbiotic dinoflagellate algae. In reviewingthe ecotoxicological studies conducted so far, PSII herbicides appear able to readily penetrate the coral tissues and rapidly (withinminutes) reduce the photochemical efficiency of the intracellular algal symbionts. The dinoflagellates appear at least as sensitive toPSII herbicides as other phototrophs tested so far, with photosynthesis being affected at exceptionally low concentrations (i.e. in theng l�1 range). At these levels and over short exposure periods, the effects can be fully reversible (i.e. when corals are returned to cleanseawater) and vary according to type of herbicide; however, when exposed to higher concentrations in the light or over longer expo-sure periods, it results in a long-term sustained reduction of the photochemical efficiency of the algae (symptomatic of chronic pho-toinhibition). This can result in the dissociation of the symbiosis (bleaching) which is a common but nevertheless significant sublethal stress response requiring many months to recover from. It is argued that the reliance of corals on an endosymbiotic photo-autotrophic energy source, together with predilection for the symbiosis to dissociate when photosynthesis of the algae is affected,renders coral particularly susceptible to changes in environmental conditions—and especially phytotoxins such as PSII herbicides.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Diuron; Herbicide; Coral; PAM; Photosynthesis, bleaching

1. Introduction

One of the keys to the success of scleractinian coralsin shallow tropical waters has been establishment of amutualistic symbiosis with dinoflagellate algae belong-ing to the genus Symbiodinium (Stanley and Swart,1995; Veron, 1995; Stanley, 2003). The symbiois is char-acterized by re-cycling and the exchange of nutrientsbetween the partners, including the translocation of sub-stantial amounts of photosynthetic products—sugars,amino acids, carbohydrates and small peptides (Trench,1979; Muscatine, 1990; Lesser, 2004). These compoundsprovide the coral with energy for respiration and growth

0025-326X/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2005.06.027

* Present address: Bermuda Biological Station for Research, Inc., 17Biological Lane, Ferry Reach, St. George�s, GE 01, Bermuda. Tel.:+441 297 1880x235; fax: +441 297 8143.

E-mail address: rjones@bbsr.eduURL: http://www.bbsr.edu/index.html

(Muscatine, 1990; Lesser, 2004), endowing the host withorders of magnitude more energy than would otherwisebe available to heterotrophic organisms (Hallock, 2001).As a result of the symbiosis, and by a mechanism that isstill not fully understood, scleractinian corals are capa-ble of extracting massive amounts of calcium carbonatefrom seawater and secreting it as skeleton. Togetherwith carbonate deposition from other �hypercalcifiers�such as algae and sponges, this largely accounts forthe reef framework formation (Stanley and Hardie,1999).

Contrasting with the success of corals in the longerterm has been the present day decline in coral reef envi-ronments, recently referred to as the coral reef crisis

(Wilkinson, 2002; Gardner et al., 2003; Bellwoodet al., 2004). The causes of the decline have been vari-ously ascribed to over fishing (Hughes, 1994; Jacksonet al., 2001), disease (Harvell et al., 2002), and climatechange (Hoegh-Guldberg, 1999; Hughes et al., 2003),

496 R. Jones / Marine Pollution Bulletin 51 (2005) 495–506

acting either singularly or in combination. While therehas been much analysis and discussion over physicaland biological stress contributing to this decline, com-paratively little has been given to chemical stress, actingdirectly or in combination with biological and physicalstress. One class of marine contaminants that is increas-ingly causing concern for the tropical marine environ-ment is agricultural pesticides.

Currently, nowhere are pesticides more valued than indeveloping countries, particularly those in tropical re-gions seeking to enter the global economy by providingoff-season fresh fruits and vegetables to countries in moretemperate climates (Ecobichon, 2001). Food productionin such areas, and the production of two or even threecrops per year, cannot be achieved without the increaseduse of pesticides (Forget et al., 1993). This partly reflectsthe shift from labour- to more machine-intensive agricul-tural practices following both the need for increased pro-duction and an agricultural-to-industrial shift in theworkforce (Ecobichon, 2001). Although varying on a re-gional basis, global sales figures for pesticide use in thetropics shows greater use of herbicides than any otherclass of pesticide (Aspelin et al., 1992; Ecobichon, 2000).

Entry of herbicides to the marine environment canoccur via off-site transport from agricultural or non-cultivated land (i.e. road sides and railway track beds,sporting fields etc), via storm runoff and irrigationtail-water releases, spray drift and accidental spill. Thepotential offsite movement of pesticides and pesticideresidues depends upon many factors, including the nat-ure and magnitude of pesticide application, biophysicalproperties of the ion or molecule, volatilization, thecomposition and properties of the soil, sorption-desorp-tion of the pesticide onto soils, the hydrology of thelandscape, the environmental conditions, chemical andbiological degradation and plant uptake (Kookanaet al., 1998). In addition to terrestrial sources, more re-cently herbicides such as diuron (DCMU, [3-(3,4-dichlorophenyl)-1,1-dimethylurea]) and Irgarol 1051have been incorporated into antifouling paint formula-tions where they function to prevent the growth of algaeon boats, buoys and marine structures. Their use in anti-fouling paints stemmed from restrictions on the use oftri-n-butyltin (TBT) in the 1980s, and the realizationthat in some instances antifouling could not be achievedby relying solely on copper as a biocide. These recentdevelopments and the potential environmental conse-quences have been reviewed by Voulvoulis et al. (1999)and Evans et al. (2000) and most recently by Konstanti-nou and Albanis (2004).

The potential environmental significance of herbi-cidal contamination of the coral reef environment hasnot gone unnoticed (for example Glynn et al., 1984).However, only recently has there been an increasedawareness of the potential environmental problems ofherbicides, since the discovery of diuron and Irgarol

1051 in the tropical marine environment (Scarlettet al., 1999; Haynes et al., 2000; Connelly et al., 2001).More recently analysis of coastal waters and sedimentin and adjacent to the Great Barrier Reef Marine Park(Australia) have indicated frequent contamination byPSII herbicides—see Bengtson Nash et al. (2004),McMahon et al. (2004), Mitchell et al. (2004) and Dukeet al. (2004). Here I review and evaluate the known eco-toxicological effects of one of the main classes of herbi-cides, the Photosystem II (PSII) herbicides (whichinclude diuron and Irgarol 1051) on scleractinian corals.Some emphasis in this review is placed on the use ofchlorophyll fluorescence techniques (for theoreticaland practical reviews, see Krause and Weis, 1991; Schre-iber et al., 1994; Maxwell and Johnson, 2000; Schreiber,2004), since the recent introduction of compact, porta-ble, and in some cases submersible pulse amplitudemodulation (PAM) chlorophyll fluorometers has al-lowed rapid analysis and insights into the ecotoxicolo-gical effects of PSII herbicides.

2. Herbicide classification and mode of action

Herbicides can be classified according to their targetsites, modes of action, similarity of induced symptoms,or chemical classes (Retzinger and Mallory-Smith,1997). Modes of action are varied, including inhibitionof light processes, cell metabolism or growth, and celldivision. Specific target sites include, amongst others,inhibition of: acetyl CoA carboxylase, carotenoid bio-synthesis (so called �bleaching� herbicides), EPSP syn-thase, glutamine synthetase, microtubule assembly,mitosis/microtubule organization, cell wall (cellulose)synthesis, lipid synthesis and auxin transport; as wellas uncoupling (membrane disruption), and diversion ofelectrons at Photosystem I (for reviews see Fedtke,1982; Oettmeier, 1992). However, over 50% of commer-cially available herbicides act by reversibly binding toPhotosystem II (PSII) (Ashton and Crafts, 1981), amembrane-protein complex in the thylakoid membraneswhich catalyses the oxidation of water and the reductionof plastoquinone (Draber et al., 1991). Based upon bind-ing properties and chemical structure, PSII herbicidesfall into two distinct groups: (1) phenylurea and tria-zines, (2) phenols (Trebst and Draber, 1979). Withinthese PSII groups there are also different chemical clas-ses, which include, amongst others, the triazines (includ-ing ametryn, atrazine, cyanazine, simazine), triazinones(hexazinone, metribuzin), uracils (bromacil, terbacil),phenyl-carbamates (desmedipham, phenmedipham),ureas (i.e. diuron, fenuron, fluometuron, linuron, sidu-ron, tebuthiuron), amides (propanil, pentanochlor),nitriles (bromoxynil, ioxynil), benzothiadiazinone (bent-azon), phenylpyridazines (pyridate, pyridafol) (for areview on PSII herbicides see Oettmeier, 1992).

R. Jones / Marine Pollution Bulletin 51 (2005) 495–506 497

PSII is a pigment-protein membrane complex madeup of the reaction center D1/D2 heterodimer carryingthe main functional groups of PSII, the chlorophyll-pro-teins CP47 and CP43 acting as inner antennae, a and bsubunits of cytochrome b559 and the oxygen-evolvingcomplex (Mattoo et al., 1989). The reaction center ofPSII carries out photochemical reactions, includingprimary charge separation and the subsequent electrontransfer from water to plastoquinone (PQ). Hencethis protein complex can be called water-plastoquinoneoxido-reductase. In normal photosynthesis, excitationenergy from light absorption in the light harvestingcomplex surrounding the reaction centre is transferredto a special chlorophyll species in the reaction centreknown as P680, forming P680* (excited singlet state).P680* then reduces a nearby pheophytin (pheo) form-ing the P680+ pheo� state (primary radical pair), whichin turn, reduces a bound PQ called QA, the first sta-ble electron acceptor, and extracts an electron fromthe water-splitting enzyme system. The electron isthen passed from Q�

A to QB, on the D1 protein. Afteranother successive electron transfer from QA, QB isreduced to a plastoquinol with the concomitant uptakeof 2H+ and is exchanged for PQ from the plastoquinonepool.

PSII herbicides are plastoquinone analogs that inhi-bit photosynthesis by reversibly binding to the QB bind-ing site on the D1 protein (Tischer and Strotmann,1977), often called the �herbicide-binding� protein (Mat-too et al., 1981; Lavergne, 1982). In the presence of aPSII herbicide such as diuron (or DCMU), QA becomesfully reduced upon illumination and cannot be oxidizedby QB, thus photosynthetic electron flow is blocked afterthe one-electron reduction of the bound QA (Bowyeret al., 1991). This inhibition of linear electron transportto the cytochrome b6/f-complex results in a shortage ofreduced nicotinamide-adenine dinucleotide phosphate(NADPH), which is essential for the reduction of carbondioxide. Starvation is however not the primary cause ofherbicide-induced plant death; instead, mortality is be-lieved to be caused by chlorophyll-mediated photodam-age (Bowyer et al., 1991). This process is thought to becaused by the formation of a chlorophyll triplet state inthe heart of the reaction centre that is capable of react-ing with triplet oxygen (3O2) to form the reactive singletoxygen (1O2) (Rutherford and Krieger-Liskay, 2001).Singlet oxygen can damage adjacent chlorophyll-bearingproteins, disconnect these chlorophylls from their en-ergy transfer systems and also from protective pigments(carotenoids), and thus cause further photogenerationof singlet oxygen (Rutherford and Krieger-Liskay,2001). In this way, illumination acts as an �amplifier� thatmay cause significantly more inhibition than the primarybinding of a PSII herbicide.

From a chlorophyll fluorescence standpoint, when aPSII herbicide is bound to the D1 protein and the PSII

reaction centres become �closed� upon illumination, theconversion of excitation energy into chemical energy isblocked and the maximal fluorescence yield is reached,provided the herbicide is bound to all reaction centresand illumination is sufficiently strong (see e.g. Maxwelland Johnson, 2000; Schreiber, 2004). The latter pointis important, as QA can also become re-oxidized bythe back reaction (the recombination of charge pairsstored in PSII). Assessing the effects of herbicide bindingis thus best achieved by chlorophyll fluorometry undera low level constant illumination (approximately5 lmol quanta m�2 s�1, at which the rate of centreclosure by charge separation exceeds the rate of the backreaction, Schreiber et al., 2002). In practice, the steady-state fluorescence yield, F, is compared with the maxi-mal fluorescence yield, F m

0, induced by a pulse of satu-rating light, which closes all reaction centres that arenot yet blocked by the PSII inhibitor (saturation pulsemethod, for a recent review see Schreiber, 2004). Basedon the F and F m

0 values the effective quantum yield ofphotochemical energy conversion in PSII can be calcu-lated: UPSII ¼ ðF m

0 � F Þ=F m0 ¼ DF =F m

0 (Genty et al.,1989). The maximal potential quantum yield is observedafter dark-adaptation, when all reaction centers areopen, the minimal fluorescence yield, F0, is measuredand a saturation pulse induces the maximal fluorescenceyield, Fm. In this case �variable fluorescence� is maximaland (UPSII)max = (Fm � F0)/Fm = Fv/Fm. A lowering ofFv/Fm is indicative of a loss in photochemical energyconversion efficiency and/or damage at the level of PSIIreaction centers, e.g. by photoinactivation. While alowering of ðF m

0 � F Þ=F m0 is an immediate response to

herbicide binding, a lowering of Fv/Fm is the result ofsecondary damage, e.g. via the effect of active oxygenoutlined above.

3. Ecotoxicological effects of Photosystem II herbicides

Scleractinian corals are mutualistic symbioses be-tween a cnidarian host and dinoflagellate of the genusSymbiodinium. Recent molecular data suggests Symbi-

odinium is highly speciose, and presently has beendivided into seven highly divergent clades, designatedA–G based on nuclear and plastid rDNA sequences (Ba-ker, 2003). In the host cell the dinoflagellates typicallyreside within gastrodermal cells, in a perialgal vacuoleof phagosomal origin. The vacuole is delineated by mul-tiple membranes (Taylor, 1968), of which the outermostmembrane originates from the host, and all of the innermembranes probably originate from the dinoflagellates(Wakefield and Kempf, 2001). From an ecotoxicologicalperspective, PSII herbicides could affect the host (ani-mal) the algal symbionts, or both. It follows that whileherbicide contact with the host (animal) is direct, multi-ple membranes will need to be traversed before the

498 R. Jones / Marine Pollution Bulletin 51 (2005) 495–506

herbicides reach the intracellular algae. Recognizing theindividual components of the symbiosis, experimentalexaminations of the effects of PSII herbicides on coralshave either tested the effects on algae freshly isolatedfrom the host using maceration techniques (termedin vitro), on algae still within the tissues of corals(in symbio), or on the animal (host).

Herbicides that compete with plastoquinone for bind-ing to the thylakoid membrane have been useful probesin studies of plant physiology for exploring structure-function relationships on the reducing side of PSII. PSIIinhibitors have also been used to probe aspects ofthe coral-algal symbiosis (Vandermeulen et al., 1972;Barnes, 1985). These studies demonstrated reduced cal-cification in corals when photosynthesis of the symbioticdinoflagellates was arrested by diuron; however, sincethese studies were not intended as ecotoxicologicalinvestigations, the concentrations used were exception-ally high. More recent ecotoxicological investigationshave used significantly lower concentrations and in-cluded an examination of PSII herbicides on a rangeof life history stages, including oocytes, larvae, recentlysettled juvenile and adult corals, and on symbiotic dino-flagellates isolated from corals (Table 1). These studieshave also included analyses of sublethal and lethal end-points, including inhibition of fertilization, behaviouralresponses, dissociation of the symbiosis (coral bleach-ing—see below), and the effects on photosyntheticoxygen evolution, C14 ðH14CO�

3 Þ incorporation andchlorophyll fluorescence parameters (Table 1).

3.1. Effects of PSII herbicides on symbiotic dinoflagellates

(in symbio and in vitro)

Given their mode of action, it is not surprising that themost sensitive parameter measured is a reduction in pho-tosynthetic efficiency of the algal symbionts (Table 1).Where multiple PSII herbicides have been tested underthe same or similar conditions, and using a reductionin the effective quantum yield in symbio as an effectparameter, Jones and Kerswell (2003) indicate 10 hEC50 values differed by over three orders of magnitude:Irgarol 1051 (0.7 lg l�1) > ametryn (1.7 lg l�1) > diuron(2.3 lg l�1) > hexazinone (8.8 lg l�1) > atrazine (45 lg l�1) >simazine (150 lg l�1) > tebuthiuron (175 lg l�1) > iony-nil (>1000 lg l�1). Under similar conditions the 10 hEC50 values for a reduction of effective quantum yieldin symbio for the staghorn coral Acropora formosa weresimilar to those for Seriatopora hystrix: Irgarol 1051(1.3 lg l�1) > diuron (2.8 lg l�1) > atrazine (37 lg l�1)(Jones et al., 2003; Jones and Kerswell, 2003). Owenet al. (2003), working with isolated symbiotic dinoflagel-lates fromDiploria strigosa,Madracis mirabilis and Favia

fragum, reported similar relative toxicities: diuron =Irgarol 1051 > atrazine > simazine > desethyl atrazine(a primary metabolite of atrazine, Owen et al., 2003).

Overall, the EC50 values quoted for the effects of Irga-rol 1051, diuron and atrazine on the symbiotic algae ofcorals are similar to or perhaps slightly lower than com-parable studies with other algae (see also Jones et al.,2003). For example, the 10 h EC50 for a reduction in pho-tochemical efficiency in algae in A. formosa, Montiporadigitata, Porites cylindrica, S. hystrix and isolated algaefrom Stylophora pistillata (range 2–6 lg l�1 diuron,Jones and Kerswell, 2003) are directly comparable tothe EC50 for the marine microalgal diatom Phaeodacty-

lum tricornutum (1 h EC50 = 3.8 lg l�1 diuron), and forlyophilised lettuce thylakoids (1 h EC50 = 5.5 lg l�1 diu-ron) (Schreiber et al., 2002). These 10 h EC50 values forthe symbiotic algae of corals are lower than the 4 dEC50 values for the freshwater chlorophyte Selenastrum

capricornutum (12 lg l�1 diuron) reported by ElJayet al. (1997). Chesworth et al. (2004) report a 10 d EC50

for a reduction in quantum yield of photosynthesis ofthe seagrass Zostera marina of 1.1 lg l�1 Irgarol 1051and 3.1 lg l�1 diuron, similar to the values for symbioticdinoflagellates of corals—although over a longer expo-sure period. Also using chlorophyll fluorescence tech-niques, Ralph (2000) indicates similar rankings in therelative toxicity of three PSII herbicides to the seagrassHalophila ovalis as those observed with the symbioticalgae of corals, with toxicity decreasing in the orderdiuron > atrazine > simazine. Studies with marineperiphyton communities indicate Irgarol 1051 is 4–8times more toxic than diuron and 70 times more toxicthan atrazine (Dahl and Blanck, 1996). Similarly, Jonesand Kerswell (2003) noted that for a reduction in quan-tum yield in symbio in S. hystrix, Irgarol 1051 was �3times more toxic than diuron and �65 times more toxicthan atrazine.

PSII herbicides have a very rapid effect on the algalsymbionts. Using chlorophyll fluorescence techniques,Jones et al. (2003) report a >50% reduction in photo-chemical efficiency of algae isolated from S. pistillatawithin 30 s of exposure to a 12 lg l�1 diuron solution.In symbio the effects of PSII herbicides are equally fast,with effective quantum yield in S. hystrix decreasing to<50% of control values within �15 min following expo-sure to a 3 lg l�1 solution of diuron or Irgarol 1051 (JonesandKerswell, 2003), andwithin 15–20 min after exposureto 0.2 or 0.3 lg l�1 solutions. For the corals A. formosa,M. digitata and P. cylindrica, the effective quantum yieldof photosynthesis was reduced within 10 min of exposureto a 10 lg l�1 diuron solution (Jones et al., 2003). Simi-larly, Owen et al. (2002) report a reduction in therate of C14 ðH14CO�

3 Þ incorporation in algae isolatedfrom M. mirabilis when exposed to 0.063, 0.125 and0.250 lg l�1 Irgarol 1051 for between 6 and 8 h. At higherconcentrations the effect was more immediate, with noC14 incorporation being measurable in algae exposed to10 lg l�1 Irgarol 1051 when the first measurement weremade after 2 h of herbicide exposure (Owen et al., 2002).

Table 1The effects of photosystem two herbicides on isolated symbiotic algae, coral oocytes, larvae, recruits and adult colonies

Coral species Duration Experimental conditions Herbicide LOEC (lg l�1) Effect parameter Reference

Isolated symbiotic algaeMadracis mirabilis 8 h Constant light and temperature Irgarol 1051 0.06 Reduced 14C incorporation Owen et al. (2002)

M. mirabilis 6 h 25.5 �C, 33 lmol PAR Irgarol 1051 2 Reduced 14C incorporation Owen et al. (2003)Diploria strigosa Diuron 2Favia fragum Simazine 100

Atrazine 100

Stylophora pistillata 10 min 25 ± 1 �C, 3 lmol PAR Diuron 0.25 Reduction in DF =Fm0 Jones et al. (2003)

OocytesA. millepora 4 h 28 �C, <10 lmol PAR Diuron 1000 No inhibition of fertilization Negri et al. (2004)Montipora spp. 6 h Diuron 1000 No inhibition of fertilisation

LarvaeA. millepora 24 h 28 �C, 120 lmol PAR Diuron 300 No inhibition of metamorphosis Negri et al. (2004)P. damicornis Diuron >1000

A. millepora, P. damicornis 4 d 28 �C, <120 lmol PAR Diuron >1000 No effect on survivorship Negri et al. (2004)

RecruitsP. damicornis 4 d 28 �C, 340 lmol PAR Diuron 30 Loss of algae Negri et al. (2004)

100 Tissue retraction

P. damicornis 4 d 28 �C, 304 lmol PAR Diuron 1 Reduction in DF =Fm0 Negri et al. (2004)

Adult coloniesPorites cylindrica 1 h 600 lmol PAR Diuron 10 Respiration Raberg et al. (2003)

S. hystrix 10 h 25 ± 1 �C, 20 lmol PAR Diuron 0.3 Reduction in DF =Fm0 Jones et al. (2003)

Acropora formosa 10 h 25 ± 1 �C, 120 lmol PAR Diuron 1 Reduction in DF =Fm0 Jones et al. (2003)

P. cylindrica Atrazine 3Montipora digitata

S. hystrix

M. digitata 96 h 24–28 �C, 1200 lmol PAR Diuron 100 Loss of algae Jones et al. (2003)

S. hystrix 2.5 h 25 ± 1 �C, 30 lmol PAR Irgarol 1051 0.05 Reduction in DF =Fm0 Jones and Kerswell (2003)

Diuron 0.2

S. hystrix 96 h 3 23–26 ± 1 �C, 50 lmol PAR Diuron 0 Reduction in DF =Fm0 Jones and Kerswell (2003)

Loss of algae

A. millepora 4 d 28 �C, 293 lmol PAR Diuron 1 Reduction in DF =Fm0 Negri et al. (2004)

P. damicornis Diuron 1

S. hystrix 14 d 23–26 ± 1 �C, 100 lmol PAR Diuron 1 Reduction in DF =Fm0 Jones (2004)

Diuron 10 Loss of algae(continued on next page)

R.Jones

/M

arin

ePollu

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51(2005)495–506

499

Table

1(continued)

Coralspecies

Duration

Experimentalconditions

Herbicide

LOEC(lgl�

1)

Effectparam

eter

Reference

S.hystrix

7d

23–26±1�C

,1100

lmolPAR

Diuron

1Reductionin

DF=Fm0

Jones

(2004)

Diuron

10Lossofalgae

S.hystrix

10h

25±1�C

,25

lmolPAR

Irgarol1051

0.3

Reductionin

DF=Fm0

Jones

andKerswell(2003)

Ametryn

0.3

Diuron

0.3

Hexazinone

3Atrazine

3Simazine

30Tebuthiuron

10Ioxynil

1000

Experimentalconditionsincludeincubationtemperature

(�C)andirradiance

intensity

(Photosynthetically

ActiveRadiation(PAR)400–700nm)in

lmolquantam

�2s�

1.LOEC=Lowest-

Observed-EffectConcentration.

500 R. Jones / Marine Pollution Bulletin 51 (2005) 495–506

Since PSII herbicides reversibly bind to the QB bind-ing site, their effects on photosynthetic electron trans-port can be reversed by placing the corals in cleanseawater. For example, following a 2.5 h exposure to a3 lg l�1 solution of diuron, the effective quantum yieldin symbio in S. hystrix was 10% of control values, butrecovered to within 5% of control values within 5 h oftransfer to a container receiving a supply of running sea-water (Jones and Kerswell, 2003). Similarly, in coralsexposed to the same concentration of Irgarol 1051 theeffective quantum yield was reduced to 0% of controlvalues, but recovered to 50% of values in control coralsafter 5 h in clean flowing seawater (Jones and Kerswell,2003). Jones et al. (2003) also report a rapid (within 6 h)near complete recovery of the effective quantum yield ofphotosynthesis of symbiotic algae from A. formosa,M. digitata, and P. cylindrica that were exposed to10 lg l�1 diuron for 4 h then transferred to clean run-ning seawater.

Clearly the s-triazine herbicides Irgarol 1051 andametryn and the phenylurea herbicide diuron are themost phytotoxic, exerting ecotoxicological effects at excep-tionally low concentrations (Table 1). In in vitro experi-ments, Owen et al. (2002) report no C14 ðH14CO�

3 Þincorporation in symbiotic dinoflagellates fromM. mira-

bilis after a 6 h exposure to 63 ng l�1 Irgarol 1051. In sym-bio, the photochemical efficiency of the symbiotic algae inS. hystrixwas significantly reduced following exposure toconcentrations as low as 50 ng l�1 Irgarol 1051 for acouple of hours (Jones and Kerswell, 2003). Similarly,exposure to 250 ng l�1 and 200 ng l�1 diuron solutionscaused a significant reduction in the photochemical effi-ciency of isolated algae in vitro (Jones et al., 2003) andin symbio respectively (Jones and Kerswell, 2003). Thetriazine herbicide ametryn (formerly ametryne) reducedthe photochemical efficiency of the symbiotic algae(in symbio) in S. hystrix at concentrations as low as300 ng l�1 (Jones and Kerswell, 2003).

3.2. Effects of PSII herbicides on the coral (animal)

Photosystem II herbicides, generally have been re-ported as having a low but measurable toxicity to aqua-tic animals. Over a 7–10 d exposure period, the NOAEL(No Observable Adverse Effect Levels) for diuron forsurvival and reproduction of Daphnia pulex, and sur-vival and growth of the amphipod Hyalella azteca, themidge Chironomus tentans, juvenile and embryo/larvalfathead minnows, Pimephales promelas, annelid worms,Lumbriculus variegatus, and snails, Physa gyrina, rangebetween 3000 and 23,000 lg l�1 (Nebeker and Schuy-tema, 1998). Similarly, Irgarol 1051 only affects cleavageand pluteus formation in the urchin Anthocidaris cras-

sispina at 1000 lg l�1 (Kobayashi and Okamura, 2002).Negri et al. (2004) noted no effects on fertilization inthe corals Acropora millepora and Montipora aequituber-

R. Jones / Marine Pollution Bulletin 51 (2005) 495–506 501

culata at 1000 lg l�1. Metamorphosis of symbiont-freeA. millepora larvae was only significantly inhibited at300 lg l�1 diuron, whilst Pocillopora damicornis larvae(which contain symbiotic dinoflagellates) were able toundergo metamorphosis after 24 h exposure to diuronat 1000 lg l�1 (Negri et al., 2004).

So far the most obvious direct affect of PSII herbi-cides on the coral (animal) is their ability to cause achange in coloration of the tissues or �coral bleaching�.Coral bleaching is synonymous with the loss of symbi-otic dinoflagellates from the tissues i.e. the dissociationof the coral-algal symbiosis (for recent reviews seeHoegh-Guldberg, 1999; Douglas, 2003). Since the sym-biotic dinoflagellates are pigmented with photosyntheticand accessory pigments, loss of algae from the tissuescauses the corals to pale or discolor (hence �bleach�) asthe white skeleton of the coral becomes progressivelymore visible through the relatively transparent animaltissue. Photographs of this bleaching response of coralsfollowing exposure to the herbicide diuron are providedin Jones (2004) and Negri et al. (2004). Coral bleachinghas been reported in M. digitata, P. damicornis and S.hystrix in response to diuron (Jones et al., 2003; Negriet al., 2004), and S. hystrix in response to Irgarol 1051(Jones, 2004), and M. digitata in response to atrazine(Shaw, 2002).

Coral bleaching is considered a sub lethal responseand has been reported in response to a diverse arrayof altered environmental conditions and chemical inhib-itors (see Brown and Howard, 1985; Meehan and Ostra-nder, 1997). For example, elevated copper ions (Jones,1997, 2004), cyanide (Jones and Hoegh-Guldberg,1999), hydrocarbons (Jones and Heyward, 2003)decreased salinity (Kerswell and Jones, 2003), heat stress(Jones et al., 1998), cold stress (Saxby et al., 2003) andlight shock (Jones and Hoegh-Guldberg, 2001) are allknown to cause coral bleaching. As discussed in thesepublications, a common theme associated with theresponse is that impairment of dinoflagellate photosyn-thesis by the inhibitor (or altered environmental condi-tion) precedes the dissociation. Bleaching induced byPSII herbicides such as diuron is also consistent withthis model. As discussed in Jones (2004), exposing coralsto diuron in darkness or reduced light does not inducebleaching, but exposing corals to the same diuron con-centrations at higher light levels, which causes a subse-quent long-term reduction in the maximum potentialquantum yield (measured via Fv/Fm after dark adapta-tion) associated with chronic photoinhibition, resultsin rapid tissue bleaching. Thus, whilst PSII herbicidessuch as diuron and Irgarol 1051 are unlikely to have di-rectly toxic effects on the coral (animal) component ofthe symbiosis until much higher concentrations arereached, they do have significant indirect effects on thecoral by causing dissociation of the symbiosis. Loss ofsymbiotic dinoflagellates has been reported from S. hys-

trix in response to pulsed exposures of 30 lg l�1 Irgarol1051 (Jones and Kerswell, 2003), and in S. hystrix andP. damicornis in continuous exposure to 10 lg l�1 con-centrations of diuron (Jones, 2004; Negri et al., 2004).Recovery of algal symbionts by bleached corals is possi-ble, but is a slow process taking many months (Jonesand Yellowlees, 1997).

3.3. Interspecific variability in the response of

corals to PSII herbicides

Any differential responses of symbiotic dinoflagellatesto PSII herbicides in symbio would be indicated by high-er EC50 levels (i.e. the concentration required to inhibitthe rate of electron transport by 50%) and potentiallymay be associated with differences in target-site delivery,genetic differences in the algae, and in particular themolecular architecture of the D1 protein. However, theD1 protein, which is encoded for by the psbA gene in allalgal phyla (Pakrasi, 1995) is amongst the most highlyconserved of photosynthetic proteins (Barber, 1992).The molecular basis for herbicide resistance to PSII her-bicides is associated with a single nucleotide substitutionin the chloroplast psbA gene that results in an aminoacid substitution in the D1 protein.

In the few studies where the response of different coralspecies to individual herbicides has been examined, re-sults suggest the response is similar. For example, in sym-bio the concentration of diuron required to reducethe effective quantum yield of photosynthesis by 50%over a period of 10 h (i.e. 10 h EC50), is similar forA. formosa (5.1 lg l�1),M. digitata (5.9 lg l�1), P. cylind-

rica (4.3 lg l�1) and S. hystrix (2.3 lg l�1) (Jones et al.,2003; Jones and Kerswell, 2003). In response to atrazineexposure, the 10 h EC50 values are also similar betweenspecies: A. formosa (37 lg l�1), M. digitata (88 lg l�1),P. cylindrica (67 lg l�1) and S. hystrix (45 lg l�1) (Joneset al., 2003; Jones and Kerswell, 2003). These corals har-bour different clades of algae—A. formosa and S. hystrix

harbour clade C3 and M. digitata andP. cylindrica har-bour C15 (LaJeunesse et al., 2003). Owen et al. (2003) re-port that relative to control corals, net C14 ðH14CO�

3 Þincorporation was reduced by 20–50% in algal symbiontsisolated from M. mirabilis, D. strigosa and Favia fragum

following a 6 h exposure to 10 lg l�1 diuron and by 40–50% following a 6 h exposure to Irgarol 1051. TheseAtlantic coral species harbour the clade B phylotype (Sa-vage et al., 2002). Negri et al. (2004) report a 15–25%reduction in the effective quantum yield of photosynthe-sis in A. millepora and P. damicornis exposed to 1 lg l�1

diuron and a 75–85% reduction at 10 lg l�1. These coralsharbour clade D (A. millepora) and clade C (P. damicor-

nis, van Oppen et al., 2001; van Oppen pers. comm.).In the studies of Jones et al. (2003), Jones and

Kerswell (2003) and Negri et al. (2004), where similarchlorophyll techniques were used to examine the effects

502 R. Jones / Marine Pollution Bulletin 51 (2005) 495–506

of diuron on the symbiotic dinoflagellates (in symbio) ofadult corals, there was no evidence of systematic differ-ences in the response of 6 coral species that harbouredclades C, C3, C15 and D. Thus, there is little convincingevidence to date for intraspecific variability in the re-sponse of different corals to PSII herbicides caused byvariability in the clade of symbiotic dinoflagellate theyharbour. Given that differences in light levels can so pro-foundly affect the outcome of experiments (see below),we would prefer to emphasize the similarity between dif-ferent coral species in their response to the herbicidesrather than emphasize the differences. Nevertheless, amethodical examination of the effects of a range of her-bicides on cultured or isolated clades of algae may beworthwhile.

4. Variables affecting the phytotoxicity of

Photosystem II herbicides

Pollution of the coastal marine environment from theoffsite transport of agricultural herbicides is generallyassociated with heavy rainfall, i.e. associated with sea-sonal formation of turbid flood plumes during the wetseason (i.e. Mitchell et al., 1996). Hence temperature,salinity and light are the key environmental variablesthat could influence the toxicity of PSII herbicides fromagricultural sources. Jones et al. (2003) found thatreducing the salinity of seawater from 35 to 28 ppt didnot change the effects of either atrazine or diuron onthe effective quantum yield of photosynthesis of thealgae in symbio in the corals A. formosa andM. digitata.Jones and Kerswell (2003) noted that short term (8 h)exposure to diuron (3 lg l�1) caused a greater reductionof the maximum potential quantum yield of photosyn-thesis of the algae in symbio in S. hystrix at cooler(20 �C) rather than warmer (30 �C) temperatures,although the effect was slight. The effects of longer term(i.e. days to weeks) exposure to elevated herbicide con-centrations under reduced salinity seawater or slightlyelevated or cooler seawater should be studied, includingphysiological end-points that involve the host as well asthe symbiotic algae.

Arguably the most significant environmental variableaffecting PSII herbicide toxicity is the irradiance underwhich the experiments are conducted. To date, experi-ments have been conducted under both natural andartificial light, with maximum intensities generally mim-icking those found at the collection depth of the test cor-als (Table 1). As discussed above and in Jones (2004), bybinding to the site of the exchangeable quinone (QB) onthe D1 protein, PSII herbicides block the forward elec-tron transport beyond the one-electron reduction ofthe first stable electron acceptor, the bound quinoneQA (Bowyer et al., 1991). Under normal growth irradi-ances (or at higher light levels), a reduction or cessation

of photosynthetic electron flow can result in photoinac-tivation of the algal symbionts and a long-term reduc-tion in the maximum potential quantum yield ofphotosynthesis (indicated by a suppression of the chlo-rophyll fluorescence parameter Fv/Fm), (Jones, 2004).This is synonymous with damage to PSII reaction cen-tres (photoinactivation, sensu, Osmond et al., 1999)and as discussed in Jones (2004), long-term reductionsin the maximum potential quantum yield of the symbi-otic algae invariably result in their loss from the coraltissues associated with the bleaching response.

The specific cue that initiates the dissociation of thecoral-algal symbiosis in response to PSII herbicide expo-sure is unclear. For the algal cells, damage by PSII herbi-cides is thought to be due to light-induced oxidativestress caused by formation of singlet oxygen (Rutherfordand Krieger-Liskay, 2001). Singlet oxygen can damagethe D1 reaction centre protein of PSII triggering its deg-radation (Asada and Takahashi, 1987). Singlet oxygenhas been implicated in lipid peroxidation by reactionwith polyunsaturated fatty acyl residues in membranesand in general oxidative degradation of proteins (Halli-well, 1984). The dissociation of the symbiosis followingPSII herbicide addition could be viewed in terms of theexpulsion of photo-damaged algal cells (i.e. those thatno longer are a net benefit to the coral). However, oncethe algae are metabolically compromised and the mem-branes are physically disordered, export of reactive oxy-gen species (ROS) from the algae to the coral couldincrease. Oxidative stress has often been implicated asa mechanism for warm-water coral bleaching (for exam-ple Lesser et al., 1990; Lesser, 1996), and both the coraland the symbiotic algae possess a suite of antioxidant de-fense mechanisms to combat the physiological hyperoxiacharacteristics of the symbiosis (see Lesser et al., 1990;Lesser, 1997). The overwhelming of these defense mech-anisms is believed to be part of the cause of coral bleach-ing (Lesser et al., 1990; Jones et al., 1998; Lesser andFarrell, 2004) and differences in the antioxidant capabil-ities of the host tissue could underlie tolerance tocoral bleaching (Brown et al., 2002; see also Douglas,2003). While it remains unclear with respect to both her-bicide-induced bleaching and warm-water bleachingwhether export of reactive oxygen species from the algaeto the host is the cue that initiates the dissociation, nev-ertheless, in both instances bleaching could be viewed asa mechanism to mitigate the export of reactive oxygenspecies from the algae to the host (i.e. by expelling thedominant source, the algae).

Many PSII herbicides can be classified as moderatepolar chemicals and thus these chemicals can accumulatein sediments and in the dissolved phase within the watercolumn. So far all studies exposing corals to PSII herbi-cides have examined the effects of herbicides in the aque-ous phase. Several studies have shown that corals feed onsuspended and sedimenting particles (Anthony, 1999;

R. Jones / Marine Pollution Bulletin 51 (2005) 495–506 503

Mills and Sebens, 2004) and as noted by Owen et al.(2002) exposure to PSII herbicides by heterotrophicparticulate feeding may be an important route. Re-sus-pension of contaminated sediments during storms, ship-ping and boating activity and dredging operations couldresult in higher exposure to PSII herbicides. The effectsof contaminated, re-suspended sediments on coralsshould be examined as has already been performed withcrustose coralline algae (Harrington et al., 2005).

5. Conclusions

Significant progress has been made in recent years inunderstanding the ecotoxicological effects of PSII herbi-cides on corals. This has been motivated by the discoveryof contamination of the coastal tropical marine envi-ronment by agricultural herbicides such as diuron(Australia, Haynes et al., 2000), and herbicides used inantifouling paints formulations such as diuron and Irga-rol 1051 (Scarlett et al., 1999; Connelly et al., 2001).From the ecotoxicological studies conducted thus far, itis clear that PSII herbicides can readily penetrate the tis-sues of corals and rapidly (within minutes) reduce thequantum yield of the intracellular algal symbionts. Theavailable evidence suggests that the response of differentclades of symbiotic dinoflagellates to herbicide exposureis similar. The symbiotic dinoflagellates of corals are atleast as sensitive to PSII herbicides as other phototrophstested so far, and photosynthesis can be affected at excep-tionally low concentrations (i.e. in the part per trillionrange). At these levels and over short exposure periods,the effects are often reversible. However, the time takenpost exposure for full reversibility can vary dependingon the type of herbicide that the coral is exposed to. Lightintensity, rather than changes in salinity or temperature,appears to be the key environmental variable influencingphytotoxicity. Under elevated light levels, higher herbi-cide concentrations and over longer exposure periods,the binding of PSII herbicides will result in a long-termsustained reduction of the quantum yield of the algalsymbionts in the host (coral) tissues, symptomatic ofchronic photoinhibition. This in turn seems to act as acue for the corals to expel their algal symbionts i.e. forthe symbiosis to dissociate. This is a common sub lethalstress response of corals, and it may take many monthsfor corals to recover their full algal complement to nor-mal levels—i.e. for the symbiosis to re-establish.

PSII herbicides appear to have little direct effect oncoral (animal) host physiology, as also observed withother invertebrates, i.e. shrimps, amphipods etc. How-ever, by inducing the dissociation of the symbiosis, (i.e.coral bleaching) and hence causing corals to lose theirendosymbiotic energy source, PSII herbicides can indi-rectly have profound eco-physiological and ecologicallyrelevant effects on corals. While the formation of a mutu-

alistic symbiosis with dinoflagellates may have conferredan advantage to scleractinian corals from an evolution-ary perspective, from an ecotoxicological perspective itmay also represent an �Achilles heel�. That is, in the pres-ent day era of increased potential chemical stress fromxenobiotics, zooxanthellate corals (sensu Schuhmacherand Zibrowius, 1985) are animals that on account of aco-evolutionary dependence on endosymbiotic photo-trophs are highly susceptible to phytotoxins (chemicalspoisonous to plants and inhibiting plant growth).

Acknowledgements

This research received funding assistance from a Stra-tegic Partnership with Industry—Research and Training(SPIRT) award from the Australian Research Council(ARC), the Great Barrier Reef Marine Parks Authority(GBRMPA), The Townsville City Council, and the Nat-ural Heritage Trust (Australia) under the Anti-foulingProgram Project (#29298).

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