Symbiont-specific responses in foraminifera to the herbicide diuron

Marine Pollution Bulletin 65 (2012) 373–383

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Symbiont-specific responses in foraminifera to the herbicide diuron Joost W. van Dam a,b,c,⇑, Andrew P. Negri b, Jochen F. Mueller c, Sven Uthicke b a

The University of Queensland, Centre for Marine Studies, St. Lucia, QLD 4072, Australia Australian Institute of Marine Science, PMB 3 Townsville MC, QLD 4810, Australia c The University of Queensland, National Research Centre for Environmental Toxicology, 39 Kessels Rd., Coopers Plains, QLD 4108, Australia b

a r t i c l e Keywords: Great Barrier Reef Pollution Photoinhibition Phytotoxicity Microalgae Symbiosis

i n f o

a b s t r a c t The effects of the photosystem II (PSII) herbicide diuron was assessed on thirteen tropical foraminifera hosting diatom, dinoflagellate, red or green algae endosymbionts. Inhibition of photosynthesis (reduced DF=F 0m ) by diuron depended on both symbiont type and test ultrastructure, with greatest sensitivity observed for diatom- and chlorophyte-hosting species (24 h IC25 2.5–4 lg L1). Inhibition kinetics was slow (24–48 h until maximum inhibition) in comparison with corals, suggesting structural differences may influence herbicide uptake and transport. Although foraminifera were generally less sensitive to direct effects of diuron (inhibition DF=F 0m ) than other marine phototrophs, damage to PSII (reduction Fv /Fm) occurred at concentrations lower than observed for other organisms (24 h IC25 3–12 lg L1). Damage to PSII was highly light dependent and occurred at very low light intensities indicating limited photoprotective capacity. The high diversity, widespread occurrence and relative sensitivity make foraminifera good bioindicator organisms to evaluate phytotoxic stress on coral reefs. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Coral reefs worldwide are under increasing pressure from anthropogenic influences such as climate change and overfishing (Hughes et al., 2003). Furthermore, many inshore reefs are located near outflow areas draining regions of intense agricultural activity, exposing reefs to land-based toxicants and nutrients (Fabricius, 2005). For example, an increasing body of evidence documents widespread contamination of inshore waters of Australia’s Great Barrier Reef (GBR) with low concentrations of agricultural herbicides (Kennedy et al., 2010; Lewis et al., 2009; Shaw et al., 2010; Shaw and Mueller, 2005). The main source of herbicides into the GBR lagoon is terrestrial runoff and reflects agrochemical applications within particular river catchment regions (Lewis et al., 2009; Mitchell et al., 2005; Packett et al., 2009). The most commonly detected agricultural herbicides in GBR waters are chemicals that block photosynthetic electron flow, collectively known as photosystem II (PSII) herbicides (Kennedy et al., 2010; Lewis et al., 2009). This group of chemicals includes the s-triazines atrazine, simazine, ametryn and hexazinone and the urea-derivates diuron and tebuthiuron and are commonly used to treat pastoral lands, in cultivation of sugarcane, cotton, bananas and other crops, in orchards and for total weed control on railways, roadsides, irrigation drains and industrial areas (Lewis et al., 2007; Radcliffe, ⇑ Corresponding author at: Australian Institute of Marine Science, PMB 3 Townsville MC, QLD 4810, Australia. Tel.: +61 7 47534149; fax: +61 7 47725852. E-mail addresses: [email protected] (J.W. van Dam), [email protected] (A.P. Negri), [email protected] (J.F. Mueller), [email protected] (S. Uthicke). 0025-326X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.08.008

2002). For the greatest part of the year and at most sites along the GBR, herbicide concentrations detected are in the ng L1 range (Kennedy et al., 2010; Shaw et al., 2010). However, heavy wet season rainfall and subsequent flooding events can contribute to a significantly higher influx of agrochemicals, leading up to concentrations of lg L1 for some herbicides, within the reef lagoon (Lewis et al., 2009). Although exposure profiles are site-specific and should be assessed as such, concentrations of herbicides such as diuron, atrazine and tebuthiuron can exceed ecological guideline trigger values and/or known effect concentrations at some inshore reefs during flooding events. While these concentrations would indicate a risk to inshore areas of the GBR lagoon, risks of exposure to PSII herbicides may be exacerbated over wider spatial scales due to the demonstrated additive toxicity of mixtures of these herbicides (Bengtson-Nash et al., 2005; Magnusson et al., 2010). Probably the most significant of agrochemicals detected within the GBR is the PSII inhibiting herbicide diuron, a relatively water soluble compound with a high potential to reach coral reefs, high toxic potency and common prevalence, often contributing to over 90% of the total PSII-inhibiting toxicity of samples collected at inner reefs along the GBR (Kennedy et al., 2010). While diuron is heavily used in Queensland agriculture it is also used as a herbicide ‘booster’ in antifouling paint formulations, where it functions to prevent unwanted growth of a wide range of algae and invertebrates on boats or other marine structures. Diuron is a plastoquinone analog which disrupts photosynthesis by temporarily binding to the QB binding site on the D1 protein within the chlorophyll P680 complex (Oettmeier, 1992). This inhibits oxidation of the primary electron acceptor QA in PSII, thereby blocking photosynthetic electron flow

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and CO2 assimilation (Jones, 2005). Because of the highly conservative character of the D1 protein in phototrophic organisms, compounds developed to interact with the D1 protein potentially interfere with a wide range of non-target species. PSII herbicides can be of particular ecological concern when found on inshore reefs, due to their ability to impact upon a most crucial biological process on coral reefs: photosynthesis of endosymbiotic microalgae. Coral reefs are primarily formed by phototrophic calcifying organisms, providing a structural habitat for all kinds of reefdwelling life forms. The high biological diversity and productivity of coral reefs, remarkable within such oligotrophic conditions, can be largely explained by the photosynthetic contribution of endosymbiotic microalgae to the host tissue of symbiotic organisms (Hallock, 1981a; Muscatine, 1990). The highly specific symbiosis between host and microalgal symbiont serves to optimize nutrient cycling, enables efficient mixotrophy (the ability to practice both heterotrophy and autotrophy) and is the central feature of reef ecosystems (Muscatine and Porter, 1977). As a result of the symbiosis, the host is endowed with substantially more energy than would otherwise be available to the heterotrophic host animal, thus enabling the holobiont to extract vast amounts of calcium carbonate from surrounding waters and secrete it as a skeleton (Gattuso et al., 1999). Interference with photosynthetic electron flow by PSII inhibitors such as diuron can reduce photosynthetic yield in the symbiotic microalgae, while chronic blockage of PSII at high inhibitor concentrations or intense light can cause secondary cellular damage through the formation of highly reactive singlet oxygen O 2 (Bowyer et al., 1991). Reduced electron transport through PSII and oxidative stress potentially lead to flow-on effects for the host animal, including impaired reproductive output through reduced energy uptake (Cantin et al., 2009, 2007), and may ultimately result in the expulsion of symbionts (bleaching) (Jones et al., 2003; Negri et al., 2005). While the majority of research on herbicide toxicity in tropical marine organisms has been performed on scleractinian corals, other photosynthetic species may also be impacted. Benthic foraminifera are mobile, single-celled calcifying protists and widely distributed through the world’s oceans; however, most symbiotic species are found in tropical waters. Symbiotic foraminifera can contribute significantly to total calcification on coral reefs (Hallock, 1981b) and in contrast to corals that only host dinoflagellates of the genus Symbiodinium, foraminifera can host a variety of microalgal groups, including dinoflagellates (dinophyta), diatoms (bacillariophyta), red algae (rhodophyta) and green algae (chlorophyta) (Lee and Anderson, 1991). In addition, some foraminifera are able to maintain chloroplasts harvested from algal food (Hallock, 2000). Foraminifera are often found in cryptic habitats and as such are adapted to reduced light intensities. These organisms extract calcium from surrounding seawater to build calcite shells (tests), an important process in the formation of sediment (Hallock, 1981b), and are widely utilised tools in paleoenvironmental research. As living specimens, foraminifera are known to be sensitive to changes in environmental conditions, and as such have been proposed as valuable bioindicators to assess water quality (Alve, 1995; Hallock et al., 2003). For example, water quality (suspended sediments and nutrient concentrations in the water column) directly correlates with foraminiferal community composition (Uthicke and Nobes, 2008). At an organismal level, growth and abundance of symbiotic foraminifera are inversely related to dissolved inorganic nitrogen (Uthicke and Altenrath, 2010). Furthermore, foraminiferal assemblages have been used as indicators in trace metal pollution studies (Carnahan et al., 2008). However, their sensitivity to PSII herbicides has not been investigated and may be an additional factor influencing their distribution. In this paper the phytotoxic effect of diuron on the photochemistry of a range of different symbionts of benthic foraminifera in

hospite was investigated in laboratory bioassays. The aims were to identify the sensitivity and toxic threshold concentrations of diuron for symbiotic foraminifera. Chlorophyll fluorescence techniques were used to test and compare the sensitivity of multiple representative foraminiferal species hosting prominent symbiont groups to diuron. These consist of species hosting diatoms, dinoflagellates, red algae and green algae. In addition, we explored whether test ultrastructure may influence diuron biokinetics and species’ vulnerability. Results were compared with studies on other phototrophic reef species such as reef-building corals to examine the efficacy of symbiotic foraminifera in toxicity testing or as proxies for coral reef health. 2. Materials and methods All experiments were undertaken at the Australian Institute of Marine Science (AIMS), Townsville, Australia, unless otherwise stated. 2.1. Collection and maintenance Tropical foraminifera were collected by hand using SCUBA from various inshore sites of the GBR between August 2008 and April 2010. Following collection and sorting, species were kept separated in 500 mL plastic beakers while on board vessels and transferred into 20 L flow-through aquaria containing aerated, 0.5 lm filtered seawater upon return to the laboratory. Rapid light curves (RLCs) were established for 6 different species utilizing pulse-amplitude modulated (PAM) chlorophyll fluorescence techniques to calculate optimal light intensities for maintenance and toxicity testing (see below). Accordingly, light intensities were adjusted (mostly < 10 lmol quanta m2s1 PAR). Maintenance and dosage experiments were conducted under 12 h: 12 h diurnal light–dark cycles using 10,000 K compact fluorescent globes (Catalina). A total of 13 different species of foraminifera (Table S1 of the Online Supplementary Material (OSM) available on the Elsevier website) were used in dose–response toxicity bioassays. These species host at least four different taxa of endosymbiotic microalgae, with one species tested carrying retained chloroplasts. Marginopora vertebralis was collected at both inshore and midshelf reefs, while Heterostegina depressa exists in two different reproductive stages. These varieties were tested individually. 2.2. Symbiont composition Former investigations into the ultrastructure of different symbiont types has indicated a specifically exclusive relationship exists between endosymbiotic microalgae and foraminiferal host (Leutenegger, 1983). However, these analyses have always been performed morphologically. Photopigment composition was used here to confirm the principal symbiont type and whether additional symbionts were present. To achieve this, four individual specimens of each species (Table S1) were crushed by Teflon pestle in 2 mL centrifuge tubes, before addition of an appropriate volume (at least 10 volume of organism) of methanol at 4 °C (HPLC Grade, Merck). Resulting suspensions were sonicated on ice for 10 s (50 W) and allowed to extract in the dark at 20 °C for 60 min. Suspensions were then centrifuged for 5 min at 15,000  g and the supernatant transferred to a 2 mL cryovial and stored at 80 °C. Evaluation of pigment content was performed by high performance liquid chromatography (HPLC) as described by Magnusson et al. (2008). Pigments were separated over a two-solvent gradient: Solvent A – 70:30 (v/v) methanol: 28 mM TBAA (tetrabutyl–ammonium acetate, CAS # 10534-59-5, Sigma–Aldrich, Australia); Solvent B – 100% methanol. For sufficient resolution of pigment peaks all

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separations were conducted over 55 min. Pigments were identified by assessment of absorption spectra and comparison of retention times with pigment standards (DHI, Denmark). As great variance was observed in pigment content between taxa, concentrations were reported proportionate to chlorophyll a content. 2.3. Chlorophyll fluorescence techniques Chlorophyll fluorescence measurements were made using a MINI-PAM fluorescence meter (Walz GmbH, Germany) to evaluate light adaptation status and herbicide effects on photochemical pathways of symbiotic algae within the foraminiferal host tissue. An 8 mm i.d. fibreoptic probe was used to measure fluorescence through the base of experimental 6-well plates by subsequently placing the probe against the plastic directly underneath the specimen to be measured. During the dose response experiments, both effective quantum yield DF=F 0m in ambient light and potential quantum yield Fv/Fm after 30 min dark adaptation were measured by the saturation pulse method as described by Schreiber (2004). In dark adapted samples, all photosynthetic reaction centers are open and maximum photochemical quenching is observed (loss of excitation energy through heat dissipation is minimal) (Schreiber et al., 1994). Application of a saturating pulse in a dark adapted sample will yield maximal fluorescence. Dark-adapted quantum PSII yield Fv/Fm can be calculated according to Genty et al. (1989) and indicates potential energy conversion at PSII. A reduction in Fv/Fm indicates damage to PSII. When samples are illuminated, partial closure of photosynthetic reaction centers occurs to prevent photodamage (stimulating heat dissipation or non-photochemical quenching), leading to a decrease in PSII yield resulting in the effective quantum PSII yield for illuminated samples DF=F 0m (Genty et al., 1989), which is a measure of ‘open’ reaction centres and indicative of the amount of energy converted in photochemistry. 2.4. Light adaptation status RLCs measure yield of PSII as a function of irradiance and provide information on saturation characteristics of photochemical electron transport. RLCs can be used to evaluate photosynthetic performance and acclimation status of the photosynthetic apparatus over a range of ambient light conditions (Ralph and Gademann, 2005). Here, we determined RLC parameters for 6 of our 13 species (two species hosting dinoflagellates and four species hosting diatoms). RLCs were obtained as described by Nobes and coworkers (2008). In short, effective quantum yields attained during the RLC were used to calculate the relative electron transport rate (rETR) (Ralph et al., 2002), curves were fitted to the data according to the model of Platt et al. (1980) in Sigmaplot 11.0 (Systat Software, Inc.) and saturation parameters calculated following Kühl et al. (2001). For maintenance of our foraminifera and irradiance intensities during toxicity tests, we selected light conditions under which near-optimal photochemical energy conversion occurred without oversaturating photosynthesis, thus optimizing production without inducing photostress. This point in the curve is described by the minimum saturating irradiance Ek and can be calculated as per Ralph and Gademann (2005). In order to exclude any possible adverse effects on experimental organisms caused by overexcitation (and since foraminifera are mobile and can avoid high light conditions), we applied light intensities below Ek in all incubation experiments. 2.5. Dose–response experiments A range of short-term (6 96 h), static (periodic renewal) toxicity tests were conducted to assess the relative toxicity of diuron on the photosynthetic apparatus of endosymbiotic algae in hospite of 13

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species of foraminifera. Analytical grade diuron (N0 -[3,4-dichlorophenyl]-N,N-dimethylurea, CAS# 330-54-1, Sigma–Aldrich, Australia) was used to prepare stock herbicide solutions in Milli-Q water with dimethyl sulfoxide (DMSO) as carrier (final concentrations in experimental media < 0.05% (v/v)). Experimental organisms were exposed to concentrations of 0.3, 1, 2, 3, 10, 30 and 100 lg L1 diuron in aerated 0.5 lm filtered seawater for up to 48 h. PSII yield was measured in exposures and tested against a solvent control (carrier only). Experiments were conducted under a 12 h:12 h light–dark cycle. Following 2 h of illumination test specimens were transferred to12 mL polystyrene tissue culture plates containing 10 mL freshly prepared diuron solutions. Riedl and Altenburger (2007) recommend exposure concentrations be measured for compounds with a octanol/water partition coefficient (log Kow) > 3, as guidelines suggest potential loss of toxicant due to adsorption to test vessels is expected at high hydrophobicities (log Kow > 4) (OECD, 2000). Diuron is not volatile and has a Kow of 2.6 (Tomlin, 2000), therefore absorption to the test vessels was unlikely and nominal concentrations were deemed accurate for the chosen exposure regimes. Incubations took place at 25 ± 1 °C and diuron solutions were refreshed daily after fluorescence measurements had been taken. As diuron half-life in aerated aqueous environments exceeds 30 days (USEPA, 2007), loss through photolysis or biological degradation in the 24 h between media changes was considered minimal. Wells containing exposure concentrations were randomly assigned within plates. Ideally, 3 or 5 (dependent on size) specimens were assigned per well; or, where tests were performed on very small species (Alviolinella quoyi, Calcarina mayorii, Peneropolis planatus and Peneropolis antillarum), 16–25 specimens were assigned per well and randomly divided into three fractions to enable triplicate measurements. Where collection had yielded insufficient specimens (4 of 13 species), lower replication was used (with a minimum of 6 specimens per treatment). Specimens were divided randomly over 3–5 replicate plates, bringing the total number of specimens (or collections thereof) per exposure up to 9 (15 for the complementary set of incubations, see below). Two sets of dose–response experiments were performed (Table 1). One set was aimed at investigating diuron uptake, transport, effect on PSII and recovery over time. A subset of 7 species of foraminifera (H. depressa, Amphistegina radiata, C. mayorii, A. quoyi, M. vertebralis – collected inshore and offshore – and P. planatus, Table 1) were exposed to diuron for 48 h, followed by a 48 h recovery period after washing and transfer to uncontaminated seawater. Incubations were performed under 5 lmol photons m2s1 PAR. Chlorophyll fluorescence parameters were measured immediately before transfer to herbicide solutions and following 2, 6, 24, 48, 72 and 96 h incubation to determine effective quantum PSII yield DF=F 0m and potential quantum PSII yield Fv/Fm after 30 min dark adaptation. In order to gain additional information under slightly higher light intensities and for more species, further dose– response exposures were conducted on all 13 species (Table 1). These incubations consisted of a 24 h diuron exposure under 20 (dinoflagellate hosting species) or 10 (other species) lmol photons m2s1 PAR), after which Fv/Fm was measured. Inhibition of PSII yields DF=F 0m and Fv/Fm was calculated by comparing foraminifera exposed to diuron with control organisms: % inhibition = 100  (1  Ysample/Ycontrol). 2.6. Data analysis Symbiont composition was confirmed through evaluation of the pigment profile in a sample. Relative pigment concentrations were calculated as ratios of total chlorophyll a and a principal component analysis (PCA) was undertaken to examine whether foraminifera species would group according to their presumed symbionts and

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Table 1 Experimental conditions and setup for the two dose–response bioassays. aNumber of replicate measurements per treatment. bTwo or more specimens pooled to obtain sufficient signal. Experimental setup Exp 1

–2 –1

Light intensity 5 lmol quanta m s PAR Fluorescence measurements (DF/F 0m and Fv/Fm) taken after 0; 2; 6; 24; 48; 72 and 96 h incubation Diuron concentrations used 0.3; 1; 2; 3; 10; 30 and 100 lg L1, tested against solvent control

Symbiont type

Species used

Na

Diatoms

H. depressa macro A. radiata A. quoyib C. mayoriib M. vertebralis inshore M. vertebralis offshore P. planatusb

9 9 9 6 9 9 9

H. depressa macro A. radiata A. quoyib C. mayoriib O. ammonoides H. depressa micro M. vertebralis inshore M. vertebralis offshore S. orbiculus P. planatusb P. antillarumb P. marginalis Elphidium sp.

15 15 12 24 15 9 15 15 8 8 6 5 6

Dinoflagellates Red algae Exp 2

Light intensity: 10 lmol quanta m–2s–1 PAR: diatoms, red algae, green algae, retained plastids 20 lmol quanta m–2s–1 PAR: dinoflagellates Fluorescence measurements taken after 6 (DF/F 0m ) and 24 h (Fv/Fm) incubation Diuron concentrations used 0.3; 1; 3; 10 and 30 lg L1, tested against solvent control

Diatoms

Dinoflagellates

Red algae Green algae Retained plastids

identify which pigments are main contributors to separate those groups. Photosynthetic yield data were arcsine-transformed and tested for normality of distribution (Kolmogorov D-test) and homogeneity of variance (Levene’s test). Extreme outliers (determined as < mean-2stdev or > mean + 2stdev) were excluded from the dataset. Data passing all criteria were compared across treatments using one-way analysis of variance (ANOVA; a < 0.05) and Tukey– Kramer’s post hoc test was used to identify treatment groups significantly different from control groups. All univariate statistical analyses were performed using NCSS 2007 software (Hintze, J. (2008) NCSS, LCC. Kaysville, Utah, USA). PCA was conducted using the Vegan package in the R platform (R Development Core Team, 2008). Phytotoxicity of diuron to symbiotic algae in hospite foraminiferal host tissues was determined by evaluation of the lowest-observed effect concentration (LOEC, lowest tested concentration causing an effect) and the effective concentrations eliciting 10% or 25% inhibition of photosynthetic yield (IC10 or IC25, respectively). IC10 and IC25 were calculated by fitting a four-parameter logistic regression to the dose-inhibition data in Sigmaplot 11 (Systat Software, Inc.). The obtained equation was subsequently solved for 10% or 25% inhibition of photosynthetic yield and reported as nominal concentrations based on initial dosages.

contained mainly violaxanthin, chlorophyll b and some b-carotene. A PCA for all pigments clearly separated species hosting different taxonomic groups of symbiotic microalgae, with over 80% of variance between species explained by the first two axes (Fig. 1). In addition, vectors indicating the pigments contributing to group differences correspond to pigments characteristic for the respective groups, i.e. fucoxanthin for diatoms, peridinin for dinoflagellates, zeaxanthin for red algae and chlorophyll b for green algae. 3.2. Light saturation status Light adaptation reflects the ability to deal with overexcitation (in the case of high light adaptation) or optimized use of low ambient light (for low light adapted species) and can be visualized by use of RLCs. Light curves (not shown) illustrated clear differences in light saturation characteristics between species. The minimum saturating irradiance (Ek) obtained for dinoflagellate hosting M. vertebralis was considerably higher (58 ± 5 lmol photons m2s1 PAR) than for the diatom hosting species H. depressa, O ammonoides, A. radiata and C. mayorii (21 ± 0.9; 17 ± 0.9; 20 ± 0.8 and 24 ± 1.8 lmol photons m2s1 PAR, respectively (Table 2), indicating optimal performance for M. vertebralis is achieved at higher light intensities than for the diatom hosting species.

3. Results 3.3. Dose–response experiments 3.1. Symbiont composition HPLC analysis of extracted pigments yielded distinct profiles for foraminiferal species. Pigment profiles of M. vertebralis and Sorites orbiculus were dominated by chlorophyll a, chlorophyll c2, peridinin and diadinoxanthin, indicating primary symbiosis with dinoflagellates (Table S1). Additionally, trace amounts of fucoxanthin (a signature pigment for diatoms) were found, indicative of possible low-level contamination with diatoms (epiphytic or ingested). Pigment profiles of species reported to host diatoms (Table S1: H. depressa, A. radiata, Operculina ammonoides, A. quoyi and C. mayorii) and the kleptoplastic Elphidium sp. were mainly dominated by chlorophyll a, fucoxanthin and chlorophyll c2, in addition to low levels of chlorophyll c1 and diadinoxanthin. Zeaxanthin and relatively high proportions of b-carotene were distinctive for the rhodophyte-hosting species (Table S1: P. planatus and P. antillarum), while the chlorophyte bearing Parasorites marginalis

Relative inhibition of effective quantum yield (DF=F 0m ) in the light and maximum quantum yield (Fv/Fm) after 30 min dark adaptation of symbiotic algae in hospite was used as a response parameter to assess sensitivity of different foraminifera to diuron exposure. All species demonstrated a similar general pattern of diuron-induced PSII inhibition over the 48 h exposure and recovery periods, although the response times and the extent of inhibition varied somewhat (Figs. 2 and 3). PSII yields of control organisms remained within 5% of initial values over the course of the experiments. Maximum inhibition of PSII yield was reached after 24– 48 h incubation for most species, with specimens exposed to concentrations P10 lg L1 diuron exhibiting the most rapid and pronounced responses. Test results over 96 h at 5 lmol photons m2s1 PAR demonstrated diatom hosting H. depressa (Fig. 2A) and A. radiata (Fig. 2B) to be slightly more sensitive to the effects of diuron than other species tested. For these species, maximum

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Zx P. antillarum P. planatus

Red algae

Fx

β-caro Chl. c1

Parasorites marginalis

O. ammonoides A. radiata H. depressa macro Elphidium sp. H. depressa micro A. quoyi C. mayorii

Chl. b Vx

Diatoms (and kleptoplasts)

Dim 2 29.88 %

Green algae Pdn

DDx

M. vertebralis inshore S. orbiculus M. vertebralis offshore

Dinoflagellates Dim 1 53.4 %

Chl. c2

Diatoms Dinoflagellates Green algae Red algae Retained plastids

Fig. 1. Biplot of a principal component analysis for pigment composition of foraminiferal species used in this study. Lines correspond to pigment vectors relative to chlorophyll a after extraction and HPLC analysis (as found in Table 1). Species hosting similar microalgal phyla are grouped. chl c1 = chlorophyll c1, chl c2 = chlorophyll c2, Pdn = peridinin, Fx = fucoxanthin, Vx = violaxanthin, DDx = diadinoxanthin, Zx = Zeaxanthin, chl b = chlorophyll b, b-caro = b-carotene.

inhibition of PSII yield was reached after 24 h incubation, while C. mayorii (Fig. 2C) exhibited an initial slower response in the high concentrations but was comparably affected after 48 h. DF=F 0m in these three species recovered quickly (90–100% within 24 h) following washing and transfer to uncontaminated seawater. A. quoyi (Fig. 2D) proved somewhat of an exception in comparison among diatom bearers, in that it exhibited a slower response, was less vulnerable to PSII inhibition by diuron and did not fully recover after 48 h in uncontaminated seawater. Like A. quoyi, both varieties of dinoflagellate hosting M. vertebralis (Figs. 2E and 2F) demonstrated relatively ‘slow’ response and recovery kinetics, with maximum inhibition observed after 48 h for the lower (<10 lg L1) diuron concentrations and incomplete recovery after 48 h. P. planatus (Fig. 2G), hosting red algae, was only affected at concentrations P10 lg L1 diuron and quickly recovered after transfer to clean medium. Diuron-induced inhibition of Fv/Fm elicited comparable, although less pronounced, response over time for all species tested (Fig. 3). Yield inhibition data after 24 h exposure at 5 lmol photons m2s1 PAR were fitted in dose–response curves grouped by symbiont type (Fig. 4). The leveling out of the various curves at higher diuron concentrations demonstrated that under these light conditions, inhibition of effective PSII yield DF=F 0m reaches a maximum somewhere between 30 and 70% (Fig. 4A–C), indicating that a high proportion of PSII reaction centres remain ‘open’ in spite of excessive diuron present. The diatom bearers H. depressa and A. radiata proved most sensitive to the phytotoxic effect of diuron, with 25% inhibition of DF=F 0m (24 h IC25 DF=F 0m ) occurring between 2.5 and 4 lg L1 diuron (Fig. 4A, Table 2). C. mayorii (Fig. 4A) and dinoflagellate hosting M. vertebralis (Fig. 5B) were less susceptible with 24 h IC25 DF=F 0m ranging between 5 and 9 lg L1 diuron. A. quoyi (Fig. 4A) and P. planatus (Fig. 4C) were only affected at higher diuron concentrations, with 24 h IC25 DF=F 0m from 10 to 20 lg L1 diuron. However, for all species tested (with the exemption of P. planatus), a significant (p < 0.05) depression of PSII yield was observed between 1 and 3 lg L1 diuron (24 h LOEC DF=F 0m – Table 2). All IC10, IC25 and LOEC values obtained in this study are listed in Table 2.

While a 10% reduction of potential PSII yield Fv/Fm after 24 h (24 h IC10) was observed at diuron concentrations between 3 and 10 lg L1 for diatom- and dinoflagellate hosting species (Fig. 4D and E, Table 2), concentrations required for 25% reduction (24 h IC25) were found to be notably higher (>20 lg L1 diuron, Table 2). Even at concentrations as high as 100 lg L1 diuron, Fv/Fm of red algae in P. planatus remained unaffected (Fig 4F). Although a significant depression of Fv/Fm (24 h LOEC) was observed between 1 and 10 lg L1 diuron for symbiotic diatoms and dinoflagellates, 30 lg L1 was required to significantly impact Fv/Fm of red algae in P. planatus (p < 0.05, Table 2). Broadening the range of taxa tested, 13 species of foraminifera (Table 1 – 6 hosting diatoms, 3 dinoflagellates, 2 red algae, 1 green algae and one species containing retained plastids) were examined for inhibition of Fv/Fm after 24 h, at slightly higher light intensities (20 lmol photons m2s1 PAR for foraminifera hosting dinoflagellates and 10 lmol photons m2s1 PAR for other species) (Fig. 5). Inhibition of Fv/Fm by 10% occurred at diuron concentrations below 5 lg L1 for most species (24 h IC10 Fv/Fm, Table 2). Twenty-four hours IC25 Fv/Fm for diatom, dinoflagellate and chlorophyte hosting species was observed at concentrations between 3 and 12 lg L1 diuron, while red algae hosting Peneropolis species and the kleptoplast Elphidium were less sensitive (Fig 5, Table 2). Significant suppression of Fv/Fm (p < 0.05) was observed at concentrations 6 3 lg L1 diuron for symbiotic diatoms and between 3 and 10 lg L1 diuron for the other species (Table 2 and 24 h LOEC Fv/ Fm). For all species tested, 25% inhibition of Fv/Fm (photodamage) was observed at higher diuron concentrations for specimens incubated at 5 lmol photons m2s1 PAR (Fig 4D–F) than at higher irradiance intensities (Fig. 5, Table 2). Comparison of IC10-25 values between the different light conditions yielded further information on the influence of light on diuron-induced inhibition of the photosystem (Fig. 6). Species bearing diatoms exhibited an approximate two-fold greater sensitivity for 10% reductions in Fv/Fm after 24 h (24 h IC10 Fv/Fm, Table 2) at 10 lmol photons m2s1 PAR as compared to specimens incubated at 5 lmol photons m2s1 PAR. For a 25% reduction in Fv/Fm of symbiotic diatoms in hospite, light exhibited a larger influence again, with over four-fold greater

378 Table 2 Minimum saturating irradiance Ek, experimental light conditions and toxicological endpoints reported in this study. LOEC = lowest observed effect concentration; IC10 and IC25 = effective diuron concentration for 10% or 25% inhibition of PSII yield, respectively. Ek data are means () (SE), ICx data reported as means () (95% CI) (sometimes 95% CI could not be obtained). Ek (lmol quanta m2s1 PAR)

Exp. light conditions (lmol quanta m2s1 PAR)

24 h LOEC DF=F 0m (lg L1)

24 h LOEC Fv/Fm (lg L1)

24 h IC10 DF=F 0m (lg L1)

24 h IC25 DF=F 0m (lg L1)

24 h IC10 Fv/Fm (lg L1)

24 h IC25 Fv/Fm (lg L1)

Diatoms H. depressa macro

Hyaline

21(0.9)

A. radiata

Hyaline

20(0.8)

A. quoyi

Porcelaneous

–

Hyaline

24(1.8)

O. ammonoides H. depressa micro

Hyaline Hyaline

17(0.9) –

1 – 2 – 3 – 2 – – –

1 1 2 3 10 3 3 3 1 3

1.0 – 1.5 – 5.3 – 2.1 – – –

2.9 (2.6–3.2) – 3.5 (3.2–3.9) – 11.8 (10.8–12.9) – 6.6 (4.9–9.4) – – –

3.1 1.7 3.7 2.8 9.7 3.5 6.4 3.3 1.2 1.9

(2.6–3.6) (1.3–2.0) (2.9–4.8) (2.0–3.8) (8.7–10.8)

C. mayorii

5 10 5 10 5 10 5 10 10 10

20.4 (17.6–23.6) 4.7 (4.1–5.4) 25.9 (16.7–44) 7.1 (5.8–8.9) 53 (46–60) 11.1 53 11.3 (10.1–12.5) 3.0 (2.7–3.4) 6.0 (4.9–7.4)

Dinoflagellates M. vertebralis inshore

Porcelaneous

58(3.5)

Porcelaneous

58(5.0)

S. orbiculus

Porcelaneous

–

2 – 2 – –

10 10 10 3 3

2.4 (2.0–2.8) – 3.2 (2.8–3.6) – –

6.3 (4.7–8.8) – 6.6 (6.0–7.3) – –

6.3 7.9 9.9 3.7 1.9

(4.8–8.1)

M. vertebralis offshore

5 20 5 20 20

Red algae P. planatus

Porcelaneous

–

P. antillarum

Porcelaneous

–

5 10 10

10 – –

30 10 10

7.1 (5.6–8.8) – –

20.3 (17.6–23) – –

>100 4.1 (1.4–9.4) 5.6 (2.5–12.9)

>100 12.4 49.1

Green algae P. marginalis

Porcelaneous

–

10

–

10

–

–

3.8 (2.9–4.9)

8.6 (7.3–10.4)

Retained plastids Elphidium sp.

Hyaline

–

10

–

10

–

–

6.8 (3.1–13.7)

32.6

(0.8–1.2) (1.3–1.7) (4.5–6.1) (1.4–3.1)

(4.6–9.4) (2.8–3.7) (0.9–1.4) (1.3–2.5)

(6.6–13.9) (2.6–5.3) (1.7–2.2)

49.8 (37–67) 11.9 54 (36–87) 7.3 (5.3–10.1) 5.4 (4.9–6.1)

J.W. van Dam et al. / Marine Pollution Bulletin 65 (2012) 373–383

Test ultrastructure

Species

379

J.W. van Dam et al. / Marine Pollution Bulletin 65 (2012) 373–383

B)

Inhibition ΔF/F'm as % of control values

A)

C)

D)

0 20 40 60 H. depressa

80

Exposure Recovery

100

C. mayorii

A. radiata

Exposure Recovery

F)

E)

A. quoyi

Exposure Recovery

Exposure Recovery

G)

0

40 60 M. vertebralis (inshore) 100

Exposure Recovery 24

0

48

72

P. planatus

M. vertebralis (offshore)

Exposure Recovery

96 0

24

48

72

Exposure Recovery 24

96 0

48

72

72

96

Solvent control 0.3µg/L diuron 1 µg/L diuron 2 µg/L diuron 3 µg/L diuron 10 µg/L diuron 30 µg/L diuron 100µg/L diuron

20

80

48

24

0

96

Time (hours) Fig. 2. Diuron-induced inhibition of DF=F 0m of symbiotic microalgae of 7 species of foraminifera measured in hospite over time. Test specimens were incubated in diuron solutions for 48 h following washing and transfer to uncontaminated seawater. (A–D) Species hosting diatoms. (E and F) Species hosting dinoflagellates. (G) P. planatus hosts red algae. Data are means () ± SE, n = 9.

Inhibition Fv /F'm as % of control values

A)

C)

B)

D)

0 20 40 60 H. depressa

80

Exposure Recovery

C. mayorii

A. radiata

Exposure Recovery

A. quoyi

Exposure Recovery

Exposure Recovery

100 0

G)

F)

E)

0 20 40 60

M. vertebralis (inshore)

80 100

Exposure Recovery 0

24

48

72

96 0

P. planatus

M. vertebralis (offshore)

Exposure Recovery 24

48

72

Exposure Recovery

96 0

24

48

72

24

48

72

96

Solvent control 0.3µg/L diuron 1 µg/L diuron 2 µg/L diuron 3 µg/L diuron 10 µg/L diuron 30 µg/L diuron 100µg/L diuron

96

Time (hours) Fig. 3. Diuron-induced inhibition of Fv/Fm of symbiotic microalgae of 7 species of foraminifera measured in hospite over time. Test specimens were incubated in diuron solutions for 48 h following washing and transfer to uncontaminated seawater. (A–D) Species hosting diatoms. E and F) Species hosting dinoflagellates. (G) P. planatus hosts red algae. Data are means () ± SE, n = 9.

inhibition occurring at 10 lmol photons m2s1 PAR as opposed to 5 lmol photons m2s1 PAR (Fig. 6).

4. Discussion Herbicide residues are widespread contaminants of nearshore areas in the GBR, Australia (Lewis et al., 2009; Shaw et al., 2010). We investigated the toxic effects of the PSII herbicide diuron on the photochemistry of a wide range of symbiotic microalgae of benthic foraminifera in hospite. Photopigment profiles for species were in close accordance with reported profiles for associated symbiont types as summarized by Jeffrey et al. (1997) and confirmed our expectations on the specificity of symbiotic partnerships evaluated here. Given their specific pigment composition, species could be readily grouped by symbiont taxa: species hosting diatoms were characterized by high fucoxanthin levels, species hosting dinoflagellates by peridinin and species hosting green algae by violaxanthin and chlorophyll b. The profile of the kleptoplast Elphidium sp. proved very similar to the diatom bearing species,

consistent with previous findings by Lopez (1979), who speculated Elphidium to obtain its chloroplasts from ingested diatoms. The main light-harvesting pigments in red algae (water-soluble phycobilisomes) were not measured (Bryant and Sidler, 2004), but nonetheless the presence of red algae could be verified by the presence of zeaxanthin. Thus, pigment based analysis could confirm the main symbionts as described in earlier morphological and culture studies (e.g. Lee and Anderson, 1991; Leutenegger, 1983). Habitat and occurrence of symbiotic foraminifera are determined by different ecological parameters, including temperature, water turbulence and irradiance intensity (Hohenegger et al., 1999). Examination of light adaptation status showed species were adapted to different light conditions. Many foraminiferal families (mainly of the order Miliolida – including the Soritidae to which Marginopora species belong), are known to build opaque test walls (so-called porcelaneous or imperforate foraminifera), reducing light penetration and water flow and restricting most species to the upper layers of the photic zone (Hohenegger, 1994). In contrast, all diatom hosting foraminifera examined here (with the

J.W. van Dam et al. / Marine Pollution Bulletin 65 (2012) 373–383

Inhibition ΔF/F m’ as % of control values

380

0

A

20

B

C

40 H. depressa A. radiata C. mayorii A. quoyi

60 80 100 0 0.1

M. vertebralis inshore M. vertebralis offshore

1

100 0.1 0

10

1

10

P. planatus

100 0.1 0

1

10

100

10

100

Inhibition Fv/Fm as % of control values

0

D

20

E

F

40 60

H. depressa A. radiata C. mayorii A. quoyi

80 100 0.1 0

1

M. vertebralis inshore M. vertebralis offshore

100 0.1 0

10

1

10

P. planatus

100 0.1 0

1

Diuron concentration (µg L-1) Fig. 4. Inhibition of quantum PSII yield of symbiotic microalgae relative to control values, measured in hospite in 7 species of foraminifera exposed to elevated diuron concentrations in the range 0.3–100 lg L1 after 24 h exposure. Incubations performed at 5 lmol quanta m2s1 PAR. (A–C) Dose–response of inhibition of effective quantum yield DF/Fm0 . (D–F) Dose–response of inhibition of potential quantum yield Fv/Fm, measured after 30 min dark adaptation. (A and D) Species bearing diatoms. (B and E) Species bearing dinoflagellates. (C and F) P. planatus hosts red algae. Data are means () ± SE (error bars were often smaller than symbols), n = 9.

Inhibition Fv/Fm as % of control values

0

A

20

B

C

40 60 80 100 0.1 0

H. depressa - 10 µE A. radiata - 10 µE C. mayorii - 10 µE A. quoyi - 10 µE Elphidium sp.* - 10 µE H. depressa (microspheric)* - 10 µE O. ammonoides - 10 µE

1

10

M. vertebralis inshore - 20 µE M. vertebralis offshore - 20 µE S. orbiculus* - 20 µE

100 0.1 0

1

10

100 0.1 0

P. planatus - 10 µE P. antillarum* - 10 µE P. marginalis* - 10 µE

1

10

100

Diuron concentration (µg L-1)

IC25 at 5 µmol photons m-2s-1 PAR (µg L-1 diuron)

Fig. 5. Inhibition of quantum PSII yield of symbiotic microalgae measured in hospite in 13 species of foraminifera exposed to elevated diuron concentrations in the range 0.3– 30 lg L1. Dose–response of inhibition of potential quantum yield (Fv/Fm) relative to control values, measured after 30 min dark adaptation (t = 24 h). Incubations performed at 20 (dinoflagellate hosting) or 10 (other species) lmol quanta m2s1 PAR (lE). (A) Species bearing diatoms and the kleptoplastic Elphidium sp. (B) Species bearing dinoflagellates. (C) Species bearing red or green algae. Data are means () ± SE, n = 15. ⁄n = 6–9.

60 M. vertebralis (offshore)

50

A. quoyi C. mayorii M. vertebralis (inshore)

40

30 A. radiata

20 H. depressa

Diatoms 10

y = 4.5361x R² = 0.9441

Dinoflagellates regression (diatoms)

0 0

5

10

15

IC25 at 10/20 µmol photons m-2s-1 PAR (µg L-1 diuron) Fig. 6. Twenty-four hours IC25 Fv/Fm of symbiotic diatoms and dinoflagellates of 6 species of foraminifera in hospite at 5 lmol photons m2s1 PAR in plotted against 24 h IC25 Fv/Fm at 10 (diatoms) or 20 (dinoflagellates) lmol photons m2s1 PAR. The slope of the regression line (as shown for diatom hosting foraminifera) illustrates the approximate four-fold increase in diuron toxicity at the higher light intensity.

exception of A. quoyi) are hyaline (perforate) species that construct tests adjusted for light penetration, allowing for a broader depthrange to be inhabited (Hohenegger, 1994). Consistent with our RLC results, dinoflagellate bearing M. vertebralis was able to effectively utilize a much broader range of light intensities than diatom hosting species. Correspondingly, M. vertebralis is often found on shallower reef flats or sandy patches where abundant light filters through, in contrast to the diatom hosting species that often inhabit deeper, shaded or cryptic habitats. Of all species tested here, diatom- and chlorophyte hosting foraminifera were most sensitive to diuron, reflected in both ICand LOEC-values (Table 2). A. quoyi proved an exception with 24 h IC10 and IC25 values approximately three-fold higher (thus less sensitive) than other diatom bearing foraminifera. As A. quoyi and other diatom-hosting species tested here share similar habitat, the reason for its insensitivity remains unknown but may be attributed to differences in test ultrastructure, influencing uptake and transport of diuron to its target sites (test ultrastructure is listed in Table 2). As mentioned, A. quoyi carries diatoms but belongs to the porcelaneous miliolida (Loeblich and Tappen, 1964), Therefore, in addition to reduced light penetration, we propose that herbicide penetration may also be reduced in porcelaneous species. This hypothesis is further supported by the fact that we also observed

J.W. van Dam et al. / Marine Pollution Bulletin 65 (2012) 373–383

slower uptake, reduced sensitivity and lengthy recovery in porcelaneous species as compared to hyaline. The dinoflagellate hosting species tested here also build porcelaneous tests and were affected similar to A. quoyi. The relatively small S. orbiculus proved somewhat more sensitive than the ‘robust’ M. vertebralis, even though these species are found side by side in shallow waters, indicating size may also influence herbicide kinetics. M. vertebralis collected offshore showed a slightly higher vulnerability to low diuron concentrations than its inshore counterpart, but it is uncertain whether reduced susceptibility in inshore areas is related to adaptation or acclimatization to chronic pollution exposure. Foraminifera have been shown to host a variety of clades of dinoflagellates (compiled by Stat et al., 2006) and similar to corals (van Oppen et al., 2001), foraminifera may favor certain dinoflagellate clades which are better suited to tolerate local conditions. Red algae symbionts proved most resilient to diuron of all species tested here, with only the higher light/high diuron treatment causing any significant damage to PSII (reductions in Fv/Fm). Red algae and cyanobacteria contain a photosystem structurally unlike that of other algae or high plants, primarily evolved to optimize light utilization. State transitions in the photosystem can balance the excitation energy distribution between PSI and PSII (Fork and Satoh, 1986), potentially decreasing energy flowing through PSII and boosting PSI. In this way, a blocked PSII can be partially by-passed, restricting herbicide effects and oxidative stress. Similarly, studies on the effects of PSII herbicides on crustose coralline red algae reported a lower sensitivity to diuron (Harrington et al., 2005; Negri et al., 2011). PSII herbicides have been reported as rapid inhibitors of photosynthetic yield. The relatively long time required for diuron to reach maximum effect on PSII in foraminiferal symbionts (24– 48 h) and the time required for recovery (Figs. 2 and 3) is unusual compared to both free-living microalgae and corals. Studies on herbicide kinetics in free-living algae indicated uptake and transport rates to vary substantially between species; however maximum inhibition was always reached within relatively short time-spans (within 20 min for diuron) (Bengtson-Nash et al., 2005; Magnusson et al., 2010; Schreiber et al., 2007). Tests with scleractinian corals indicated > 50% inhibition of DF=F 0m of symbionts in hospite to occur within 20 min and maximum effect was reached within 90 min, even though the herbicide has to cross multiple membrane layers (of both host-animal and algal origin) to reach its target sites (Jones and Kerswell, 2003). Analogous to herbicide uptake kinetics, studies on herbicide elimination observed rapid recovery in corals (Jones and Kerswell, 2003; Jones et al., 2003). The delayed response and recovery of symbionts as observed within foraminifera may be due to differences in penetration and elimination kinetics, targetsite delivery or structural dissimilarities in the D1 protein. The D1 protein however, is highly conservative and most likely, diuron uptake through the test and transport through the host membranes is the main factor influencing herbicide biokinetics. In both corals and foraminifera, symbionts have been shown to reside within host cells (intracellular) (Lee and Anderson, 1991; Muscatine and Porter, 1977) and experiments with symbionts isolated from foraminifera are required to shed further light on transport and host influence. Although slow herbicide kinetics may suggest a greater resilience to the negative effects of chemical pollution, alternatively it may also influence elimination processes and recovery potential following longer exposures. Direct comparisons of our experimental findings with other studies on the phytotoxicity of diuron in marine ecosystems indicate that symbiotic foraminifera react in a different way to environmental stress when compared with other marine autotrophs. For example, 10 h IC50 values for a reduction in DF=F 0m for the scleractinian corals Acropora formosa, Seriatopora hystrix, Montipora digitata, Porites cylindrica and isolated symbiotic dinoflagellates

381

from Stylophora pistillata ranged from 2.3 to 6 lg L1 diuron (Jones and Kerswell, 2003; Jones et al., 2003). For the free living marine algae Phaeodactylum tricornutum (diatom, 1 h), Nephroselmis pyriformis (green alga, 15 min) and Navicula sp. (diatom, 3 d), a 50% reduction in DF=F 0m was observed at 2.1–5.9 lg L1 diuron (Bengtson-Nash et al., 2005; Magnusson et al., 2010; Schreiber et al., 2002). Chesworth et al. (2004) found a 50% reduction in DF=F 0m in the seagrass Zostera marina at 3.1 lg L1 diuron (10 d). In contrast, the present study found 50% inhibition of DF=F 0m to be reached only for 2 out of 7 species after 24 h, with the most sensitive species tested being H. depressa, hosting diatoms (IC50 = 11.3 lg L1 diuron) (Fig. 4). The apparent resilience of foraminiferal symbionts as compared to other taxa may reflect light conditions as used in our experiments. Since most foraminifera studied here are low-light adapted species (Table 2), very low experimental light intensities (5–20 lmol photons m2s1 PAR) were used in comparison with studies on e.g. corals (20–300 lmol photons m2s1 PAR). At higher light intensities, effects of blocked photosynthetic electron flow may be intensified (Jones and HoeghGuldberg, 2001), as excess excitation energy that cannot be transferred through the electron transport chain is instead passed over to form highly reactive oxygen radicals (Bowyer et al., 1991), capable of damaging PSII while causing further photogeneration of reactive oxygen (Rutherford and Krieger-Liskay, 2001). This process of photoinhibition may cause a considerably greater impact than the loss of photosynthate; it follows that higher experimental light intensities may induce further reductions in PSII yield. This corresponds with our results that show increased toxicity of diuron to symbionts of foraminifera in hospite at higher light intensities (Fig 6). Despite the low light intensities used here, effect concentrations for a reduction of potential quantum PSII yield (Fv/Fm) (Table 2) of symbiotic algae in foraminifera were directly comparable with those for many corals. Fv/Fm is observed after dark adaptation when all PSII reaction centers are open (even those that have herbicide bound to the D1 protein), and as such should not be influenced by any bound diuron but will only exhibit a reduction if previous damage to PSII reaction centers has occurred, e.g. through photoinhibition during light periods (Jones, 2004). Consequently, Fv/Fm is a measure for damage to PSII. Negri et al. (2005) reported 24 h LOECvalues of 1 lg L1 diuron for reduced Fv/Fm in adult branches of the hard corals Pocillopora damicornis and Acropora millepora. However, Fv/Fm quickly recovered after transfer to uncontaminated medium (Negri et al., 2005), possibly due to a combination of photosystem repair mechanisms and host-mediated expulsion of damaged symbionts (bleaching) (Jones and Hoegh-Guldberg, 1999). Most corals are sessile organisms and adapted to high-light conditions (125– 250 lmol photons m2s1 PAR), often being exposed to excessive levels of solar irradiance. As a consequence, considerable photodamage may occur during days of intense illumination. Corals have adapted to their ecological niche and can actively limit the amount of light energy converted and the availability of reactive oxygen through several pathways, including photoprotective (quenching) pigments, biochemical antioxidants and enzymatic repair cycles (Lesser, 2006). Thus, under normal circumstances, photodamage is readily repaired. In contrast to corals, many foraminifera are low-light adapted species (<60 lmol photons m2s1 PAR, Table 2), existing primarily in cryptic habitats or shaded areas of the reef. Furthermore, foraminifera can actively move to avoid localized high light conditions. The low resilience of most symbiotic foraminifera to photoinhibition (reduction Fv/Fm) as was observed in this study, even under very low light, suggests photoprotective mechanisms may be considerably less established in foraminifera than in corals. Jones (2004) observed rapid tissue bleaching in corals after a sustained diuron-induced decrease of Fv/Fm. This correlation between dissociation of symbiosis and decreased Fv/Fm in corals has

382

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more often been reported as a consequence of various environmental stressors, including temperature stress (Fitt and Warner, 1995; Saxby et al., 2003), high irradiance intensities (Jones and Hoegh-Guldberg, 2001) and reduced salinity (Kerswell and Jones, 2003). Even though bleaching was not measured in the present study, significant decreases in Fv/Fm after short-term exposure to low diuron concentrations (1–10 lg L1) under low light conditions (5–20 lmol photons m2s1 PAR) suggests considerable photodamage has occurred and that cryptic, low-light adapted species may be just as vulnerable to herbicide-induced photostress as high-light species, possibly due to a lack of necessary means to deal with photoinhibition. The present study indicated that the photophysiological response of symbiotic foraminifera to herbicide exposure is distinct to that of other tropical symbiotic organisms like for example corals. Where the dinoflagellate symbionts in corals are more sensitive to reversible reductions in photosynthetic efficiency than foraminifera, active photoprotection and enzymatic repair limit the damage done to PSII. In contrast, even low light can cause considerable photodamage to symbiotic microalgae of foraminifera in hospite in the presence of low concentrations of diuron. Furthermore, the toxicity of diuron in foraminifera is significantly enhanced by increasing light intensities. These characteristics make symbiotic foraminifera good proxies to test for phytotoxicity on the GBR. Test structure and host size may influence uptake and transport kinetics of herbicides, while symbiont phylum and clade determine sensitivity of the algal photosystem. It has been argued that distribution and abundance of symbiotic foraminifera in the GBR is linked to water quality factors such as sedimentation and nutrient concentrations (Uthicke and Altenrath, 2010; Uthicke and Nobes, 2008). As shown here, PSII herbicides are capable of impacting photosynthesis at low concentrations. Reduced photosynthetic output limits energy transfer to the host animals, potentially reducing fitness and resilience to other stressors. Environmental herbicide concentrations measured in the GBR are typically below levels at which direct adverse effects may occur on photosynthesis (e.g. diuron < 20 ng L1) (Kennedy et al., 2010), yet during major flooding events concentrations have been determined near inshore reefs that are sufficiently high for effects to be detectable (diuron 0.1–1 lg L1) and that may last for weeks (Lewis et al., 2009). Furthermore, flood plumes often transport mixtures of pollutants and as multiple factors potentially interfere with the same physiological mechanism (e.g. PSII electron flow), additive or even synergistic toxic effects may occur (Bengtson-Nash et al., 2005). A recent study has confirmed that the phytotoxicity of PSII herbicides commonly detected in GBR waters towards tropical microalgae is additive (Magnusson et al., 2010) but little is known of how other water quality parameters may interact with herbicides to affect free and symbiotic microalgae. During periods of flooding, the margin of safety between observed herbicide concentrations and measurable effects is relatively small (van Dam et al., 2011) and photosynthesis can be persistently inhibited. Studies on chronic effects of herbicides on free-living benthic microalgae (10 days; diuron 6 23.3 lg L1) revealed a linear relationship between PSII yield and growth rate (Magnusson et al., 2008), while chronic exposure of corals to diuron (2–3 months; diuron 6 10 lg L1) caused flow-on effects to the host, including reduced reproductive output due to a decrease in energy translocation (Cantin et al., 2007). These effects may also be relevant to foraminifera and future studies should focus on chronic exposures and impacts on the host animal, as well as on interactions between simultaneously occurring chemical and physical stressors. Evidence is emerging that pollution may decrease resilience of reef biota to further stressors as e.g. global climate change (Wooldridge and Done, 2009). Summer monsoonal rainfall and subsequent flood events often occur as seawater temperatures approach thermal tolerance levels

for many species which are therefore exposed to combinations of low salinity, high turbidity, nutrients and herbicides during episodes of thermal stress. Stressor interactions are a key challenge in environmental risk assessment and benthic foraminifera can provide a valuable alternative suite of host-symbiont partnerships available for use as ecological biomarker organisms and interaction-toxicity testing. Acknowledgements We thank the crew of RV Cape Ferguson, Florita Flores and Till Röthig for assistance in field collections and laboratory experiments. This research was supported by the Australian Government’s Marine and Tropical Sciences Research Facility (MTSRF). JvD received financial support from The University of Queensland. Entox is a partnership between Queensland Health and The University of Queensland. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.marpolbul.2011.08.008. References Alve, E., 1995. Benthic foraminiferal responses to estuarine pollution – A review. J. Foraminiferal Res. 25, 190–203. Bengtson-Nash, S.M., McMahon, K., Eaglesham, G., Müller, J.F., 2005. Application of a novel phytotoxicity assay for the detection of herbicides in Hervey Bay and the Great Sandy Straits. Mar. Pollut. Bull. 51, 351–360. Bowyer, J.R., Camilleri, P., Vermaas, W.F.J., 1991. In: Baker, N.R., Percival, M.P. (Eds.), Herbicides, Topics in Photosynthesis. Elsevier, Amsterdam, pp. 27–85. Bryant, D.A., Sidler, W.A., 2004. Phycobilisome and phycobiliprotein structures. In: Govindjee (Ed.), The Molecular Biology of Cyanobacteria. Springer, Netherlands, pp. 139–216. Cantin, N., van Oppen, M., Willis, B., Mieog, J., Negri, A., 2009. Juvenile corals can acquire more carbon from high-performance algal symbionts. Coral Reefs 28, 405–414. Cantin, N.E., Negri, A.P., Willis, B.L., 2007. Photoinhibition from chronic herbicide exposure reduces reproductive output of reef-building corals. Mar. Ecol.-Prog. Ser. 344, 81–93. Carnahan, E.A., Hoare, A.A., Hallock, P., Lidz, B.H., Reich, C.D., 2008. Distribution of heavy metals and foraminiferal assemblages in sediments of Biscayne Bay, Florida, USA. J. Coastal Res. 24, 159–169. Chesworth, J.C., Donkin, M.E., Brown, M.T., 2004. The interactive effects of the antifouling herbicides Irgarol 1051 and diuron on the seagrass Zostera marina (L). Aquat. Toxicol. 66, 293–305. Fabricius, K.E., 2005. Effects of terrestrial runoff on the ecology of corals and coral reefs: review and synthesis. Mar. Pollut. Bull. 50, 125–146. Fitt, W.K., Warner, M.E., 1995. Bleaching patterns of four species of Caribbean reef corals. Biol. Bull. 189, 298–307. Fork, D.C., Satoh, K., 1986. The control by state transitions of the distribution of excitation energy in photosynthesis. Ann. Rev. Plant Physiol. 37, 335–361. Gattuso, J.-P., Allemand, D., Frankignoulle, M., 1999. Photosynthesis and calcification at cellular, organismal and community levels in Coral Reefs: a review on interactions and control by carbonate chemistry. Am. Zool. 39, 160– 183. Genty, B., Briantais, J.M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron-transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990, 87–92. Hallock, P., 1981a. Algal symbiosis: a mathematical analysis. Mar. Biol. 62, 249–255. Hallock, P., 1981b. Production of carbonate sediments by selected large benthic foraminifera on two Pacific coral reefs. J. Sediment. Petrol. 51, 467–474. Hallock, P., 2000. Symbiont-bearing foraminifera: harbingers of global change? Micropaleontology 46, 95–104. Hallock, P., Lidz, B.H., Cockey-Burkhard, E.M., Donnelly, K.B., 2003. Foraminifera as bioindicators in coral reef assessment and monitoring: the FORAM index. Environ. Monit. Assess. 81, 221–238. Harrington, L., Fabricius, K., Eaglesham, G., Negri, A., 2005. Synergistic effects of diuron and sedimentation on photosynthesis and survival of crustose coralline algae. Mar. Pollut. Bull. 51, 415–427. Hohenegger, J., 1994. Distribution of living larger foraminifera NW of Sesoko-Jima, Okinawa, Japan. Mar. Ecol. 15, 291–334. Hohenegger, J., Yordanova, E., Nakano, Y., Tatzreiter, F., 1999. Habitats of larger foraminifera on the upper reef slope of Sesoko Island, Okinawa, Japan. Mar. Micropaleontol. 36, 109–168. Hughes, T.P., Baird, A.H., Bellwood, D.R., Card, M., Connolly, S.R., Folke, C., Grosberg, R., Hoegh-Guldberg, O., Jackson, J.B.C., Kleypas, J., Lough, J.M., Marshall, P.,

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