Multiple stressors and benthic harmful algal blooms (BHABs): Potential effects of temperature rise and nutrient enrichment

Marine Pollution Bulletin 131 (2018) 552–564

Contents lists available at ScienceDirect

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

Multiple stressors and benthic harmful algal blooms (BHABs): Potential effects of temperature rise and nutrient enrichment

T

⁎

A. Frickea,b,c, , A. Peya, F. Giannia,b, R. Leméeb, L. Mangialajoa,b a

Université Côte d'Azur, CNRS, ECOMERS, Parc Valrose 28, Nice 06108, France Sorbonne Université, CNRS, Laboratoire d'Océanographie de Villefranche, LOV, F-06230 Villefranche sur mer, France c Instituto Argentino de Oceanografía (IADO), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Florida 4750, Bahía Blanca, B8000FWB, n/a, Argentina b

A R T I C LE I N FO

A B S T R A C T

Keywords: Ostreopsis cf. ovata Actinocyclus sp. Prorocentrum lima Microphytobenthos Environmental change Mediterranean Sea

Blooms of Ostreopsis cf. ovata, causing health incidence and mass human intoxications in the Mediterranean, gained special attention over the past decades. To study the potential effects of temperature and nutrient enrichment on this benthic dinoflagellate and other associated microalgae in situ, a multifactorial experiment was set up along a temperature gradient of a heat pump system in Monaco. Microalgae were quantified in experimental units, in the natural biofilm and in the water column. No significant interaction was observed between temperature and nutrients. A species- and bloom phase-dependent effect of the increased temperature was recorded, while the nutrient enrichment had a significant effect only at the end of the experiment (when cell abundances were low). Temperature effects were also visible in the biofilm and the surrounding water. The observed assemblages were mainly driven by changes in abundances of Ostreopsis cf. ovata and Actinocyclus sp., affected in different ways.

1. Introduction To date, there is hardly any marine system unaffected by human impacts, reaching levels unprecedented in history (Halpern et al., 2008). While certain environmental changes are associated with global processes, e.g. rising sea water temperature and ocean acidification (Boyd et al., 2016), others result from local human activities, e.g. pollution, overfishing, eutrophication (Grall and Chauvaud, 2002). The marine ecosystems have been increasingly affected by the simultaneous impact of such multiple stressors, which can create hotspots of particular vulnerability, leading to changes in ecosystem health and functioning (Gruber, 2011; Halpern et al., 2015). Due to human impact at both the local and global scale, the frequency and intensity of harmful algal blooms (HABs) and microalgal community shifts toward toxic species have increased worldwide (Heisler et al., 2008; Hallegraeff, 2010; Fu et al., 2012). In order to better understand the future public health, ecological and economic risks linked to HABs, this phenomenon has to be approached in the context of climate change that could potentially affect their distribution and frequency, as much as that of related health impact consequences (Miraglia et al., 2009). However, the predictions of the impact of global change on algal blooms are fraught with uncertainties (Hallegraeff,

⁎

2010; Wells et al., 2015; Davidson et al., 2016). Several studies have been conducted to investigate the global change impact on algal blooms, but the interactions between different drivers are rarely considered (but see O'Neil et al., 2012; Vidyarathna and Graneli, 2013; Rigosi et al., 2014; Tawong et al., 2015; Thomas et al., 2017). An emergent phenomenon in the framework of HABs is represented by benthic algal blooms (Valiela et al., 1997; Teichberg et al., 2010; Fricke et al., 2016) and in particular those of several Ostreopsis species (e.g. Fukuyo and Ishimaru, 1986; Mangialajo et al., 2008; Shears and Ross, 2010). Blooms of Ostreopsis spp. can affect marine invertebrates (see review in Faimali et al., 2012), but also humans, mostly through respiratory and cutaneous symptoms (Vila et al., 2016), with potentially relevant economic consequences (Lemee et al., 2012). Blooms are regularly reported in tropical to subtropical areas (Grzebyk et al., 1994; Morton et al., 1997; Parsons and Preskitt, 2007; Nascimento et al., 2012), as well as in temperate areas such as New-Zealand (Chang et al., 2000; Shears and Ross, 2009, 2010; Rhodes, 2011), Tasmania/South Australia (Rhodes, 2011), Japan (Taniyama et al., 2003), Eastern Russia (Selina et al., 2014) and the Mediterranean Sea that is particularly concerned by this phenomenon. Since the first record in 1972 on the French Riviera (Taylor, 1979), proliferations of Ostreopsis cf. ovata have been increasing in magnitude and frequency (Mangialajo et al., 2008;

Corresponding author at: Leibniz Centre for Tropical Marine Research (ZMT), Fahrenheitstrasse 6, 28359 Bremen, Germany. E-mail address: [email protected] (A. Fricke).

https://doi.org/10.1016/j.marpolbul.2018.04.012 Received 7 December 2017; Received in revised form 7 March 2018; Accepted 7 April 2018 0025-326X/ © 2018 Published by Elsevier Ltd.

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

temperature (> 25 °C) and phosphate concentrations (N/P around 25) as thresholds for cyst germination and the onset of Ostreopsis blooms in the Northern Adriatic Sea. A laboratory experiment crossing the two drivers (Vidyarathna and Graneli, 2013) proved a positive effect of temperature and a negative effect of N-limitation. However, field data on the impact of simultaneous stressors remain very scant and the evidence for an interaction between temperature and nutrients and its potential direct and indirect effects is uncertain. The aim of the present study, using an innovative in situ approach for the first time a sea-water heat-pump as proxy of global warming, is to investigate the potential interactive effects of sea temperature rise and nutrient enrichment on benthic harmful algae blooms, in particular of Ostreopsis cf. ovata in the Mediterranean Sea. The heat pump system provides a unique opportunity to study the role of time-wise temperature variation due to pulsing heated water outflow directly in situ. In an orthogonal approach, artificial nutrient diffusers (based on Littler et al., 2010) were used in order to simulate localised eutrophication and investigate the potential single and combined effects of such stressors on the benthic microalgae community.

Totti et al., 2010; Cohu et al., 2011), and public awareness on potentially harmful benthic microalgae has increased. Given their health, ecological and economic importance, better knowledge regarding the potential drivers of Ostreopsis blooms is crucial as a basis for predicting and taking into account for future scenarios. Climate change-driven temperature rise is known to foster the expansion of marine organisms, shifting their distribution ranges and enabling them to occupy new habitats. This is a common phenomenon in the Mediterranean Sea (Lejeusne et al., 2010; Smith et al., 2016), considered to be under a process of tropicalization (Coll et al., 2010; Vergés et al., 2014; Hyndes et al., 2016). This process potentially affects the distribution of microalgae (Gómez, 2011; Glibert et al., 2014; Lakkis and Sabour, 2014) and might consequently foster their blooms. Land clearing, production and applications of fertilizer, discharge of human waste, animal production and combustion of fossil fuels have highly enriched coastal waters that generally have higher concentrations of nitrogen (N) and phosphate (P) compared even to recent decades (Cloern, 2001). Nutrient enrichment is a global phenomenon often associated with undesirable changes in ecosystems (Rabalais et al., 2009), such as HABs (Heisler et al., 2008). Coastal eutrophication can be climatically driven, either through alterations of terrestrial runoff (Hama and Handa, 1994) or atmospheric deposition (Paerl et al., 1990), and is expected to increase over the next decades (Coll et al., 2010). Nutrients and temperature commonly interact and potentially impact algal growth and community composition in different ways (Peñuelas et al., 2013), affecting the chemical and physical properties of aquatic systems (Sterner and Grover, 1998; Xia et al., 2016) and driving physiological processes (Raven and Geider, 1988; Berges et al., 2002). Temperature rises may lead to an increased uptake of nutrients in microalgae (Lomas and Glibert, 1999), whereas their growth might be limited by species-specific temperature limits (Ras et al., 2013). Several studies reported interactive effects of nutrients and temperatures (mostly synergetic, but sometimes null), on a wide array of organisms, macroalgae (Endo et al., 2013; Strain et al., 2014; Gao et al., 2016), as well as microalgae (Lomas and Glibert, 1999; Roleda et al., 2013). The effect of temperature and nutrients on Ostreopsis species have been recently reviewed by several authors (Pistocchi et al., 2011; Carnicer et al., 2015; Accoroni and Totti, 2016). The role of temperature seems to be determinant, and several threshold values have been proposed for the different strains. The correlation at small temporal and spatial scales is not obvious, according to results of laboratory experiments, and some authors suggest that the temperature may not affect intensity of blooms, while their toxicity may be enhanced (Carnicer et al., 2016). Concerning the nutrients, O. cf. ovata might be favoured by its dorso-ventrally flattened cell shape, which not only allows it to easily move in the interstitial environment (Hoppenrath et al., 2014), but provide a high surface: volume ratio, which seems to increase its nutrient uptake rates (Fraga et al., 2012). So far, different laboratory studies report a strong direct impact of nutrients on the chemistry and growth of O. cf. ovata (e.g. Pistocchi et al., 2011; Pezzolesi et al., 2016), which is not only able to use up different dissolved inorganic (NH4+ and NO3−) and organic (N-urea) N sources, but also showed clear preferences for NH4+ (Jauzein et al., 2017). Consequently a potential link between anthropogenic driven land-use change and Ostreopsis blooms appearances might be probable. Interestingly, the findings of field observations are less clear. Ostreopsis cf. ovata can form dense blooms in nutrient enriched environments (e.g. Parsons and Preskitt, 2007; Skinner et al., 2013) but is also found in rather pristine oligotrophic environments (Shears and Ross, 2009; Pistocchi et al., 2011). This suggest (1) the importance of other potentially complementary environmental components and/or (2) the presence of various strains of O. cf. ovata in the Mediterranean that may produce more or less secondary metabolites, as shown by Sato et al. (2011), and that may also respond differently to nutrient inputs. Accoroni et al. (2014, 2015) defined the combination of water

2. Material and methods 2.1. Experimental site and sampling stations The environmental conditions of the populated Monegasque coastline provide ideal conditions for the development of microalgae, as Ostreopsis blooms were frequently reported and monitored over the past seven years over summer time, when water temperature reached values around 22 to 25 °C (Cohu et al., 2011). The study was carried out during summer 2014 on an artificial dyke, close to the outflow of the sea-water heat-pump (SWHP) serving the various activities within the adjacent casino of the Monte Carlo Sporting Club, Monaco (43°44′47″N; 7°26′44″E) (Fig. 1). The heat pump system has an average flow rate of 1551 m3 h−1 and discharges at the surface sea water collected from the surface, close to the reject. For the analyses, two factors were considered: i) DISTANCE from the heat pump outlet, related to the temperature (3 levels: 2, 10 and 30 m) and ii) DIRECTION referring to the two considered spatial gradients from the point of outlet (2 levels: East and West). A total of six sampling stations were designated along the East-ward and West-ward transects from the outlet, at 2, 10 and 30 m distance (30W, 10W, 2W, 2E, 10E, 30E). 2.2. Experimental timing Three consecutive experimental runs were performed during the Ostreopsis cf. ovata bloom period to assess the consistency of results over time, (see below). As it was not possible to regularly follow the bloom in the studied site, in order to choose the dates of the experimental runs, the known temporal trends of Ostreopsis blooms (ex. Mangialajo et al., 2011) were followed. Unluckily, for logistical reasons, a run in the first half of July growing phase of the bloom was not possible and the experiment therefore started during the stationary phase of the bloom, from 23rd of July 2014 (T1). The declining phase was followed from 5th of August 2014 (T2) and the end of the bloom (T3) from 4th of September 2014 (at the end of the bloom). T1 and T2 were run for a period of three days, as bad weather conditions were expected and the risk of losing the units was high. In T3 the exposure time was doubled. 2.3. Experimental set-up and sampling To investigate the potential responses of Ostreopsis cf. ovata and other associated microalgae to the altered temperature regime and test for a potential interactive effect of nutrient enrichment, a field experiment (hereafter “EXPT”) was designed along the two temperature gradients resulting from the heat pump. Following the experimental approach of Littler et al. (2010), 36 porous terracotta flower pots were 553

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

corresponding 1 l bottles were carefully placed and fixed over the SUs, containing cells and surrounding water inside (Fig. 2B–C). One EXPT sample out of 36 was lost at each experimental run (T1: 30WNUTb, T2: 10ECa, T3: 10WNUTa). SUs were detached from their support by cutting the cable ties and closed sampling bottles were transferred to the surface. At the surface, they were vigorously shaken prior to opening, and subsequently gently cleaned with a toothbrush (Al-Handal et al., 2016), in order to separate as much as possible the attached cells from the SUs. To investigate the microalgal cell abundances at the study site along the temperature gradients, microalgal surveys were conducted at the end of each experimental run. For this purpose the classical Ostreopsis cf. ovata cell quantification, coupling the sampling of the cells in the biofilm (BIOFILM, expressed as cells/g of collected macroalgae) and in the surrounding water (WATER) was applied at 50 cm depth (Mangialajo et al., 2011). Firstly the WATER sample was collected, in order to avoid resuspension of Ostreopsis cf. ovata cells, about 20 cm above the macroalgal communities, using 250 ml plastic flasks. Then, 5 to 10 g of algae (Laurencia Lamouroux 1813 complex, Corallina caespitosa R.H. Walker, J. Brodie & L.M. Irvine in Walker et al. 2009 and Dictyota Lamouroux 1809 spp.) were gently collected in a 250 ml plastic flask together with the surrounding water, to limit the loss of microalgal cells. Three samples of BIOFILM and WATER were collected at each station and at each experimental run. Immediately after collection, samples for EXPT, BIOFILM and WATER were fixed by adding acidic Lugol at 1% (vol./vol.). A total of 213 samples (105 EXPT, 54 BIOFILM, and 54 WATER) were collected.

2.4. Microalgal quantification In the laboratory all samples were quantified, using an inverted light microscope (10 times magnification, Axio Observer D1, Zeiss, Germany). For BIOFILM samples, microalgae were separated from their hosts by vigorously shaking the bottle (Mangialajo et al., 2008a). Abundances of Ostreopsis cf. ovata and the associated species were quantified, using 1 ml volume Sedgwick Rafter © counting cells, and expressed as cell numbers per gram fresh weight (cells g FW−1). Surrounding WATER microalgal abundances were assessed following the Utermöhl method, by sedimentation of 50 ml of sea water. Concentrations of Ostreopsis cf. ovata and associated species in the surrounding water were recorded as the number of cells per liter (cells l−1). For EXPT, 1 ml volume Sedgwick Rafter © counting cells were used for samples taken in T1 and T2, whereas due to the lower cell abundances 50 ml volume Utermöhl chambers (Utermöhl, 1931) were used in T3. Cell abundances were related to the volume of individual SU samples (cells SU−1). In addition we calculated the attached number of cells per cm2 by relating average treatment values of the EXPT, after substraction of corresponding Plankton values, to a approached SU surface area of 163 cm2, calculated by the formula: (R + r) ∗ m ∗ π + π ∗ R2 + (G2 − G1) + 6 ∗ h ∗ ls, whereas R and r = radius of bottom and top area of flower pot, respectively, m = surface line of flower pot, G2 = area of lid, G1 = base area of flower pot, h = height of screws and ls = lengths of screws.

Fig. 1. Study site. (A) Location of the sea-water heat-pump (SWHP) of the Monte Carlo Sporting Club in the study area (Principality of Monaco). (B) The six considered stations along the two gradients (Westward and Eastward) from the outflow (black arrow; 30W, 10W, 2W, 2E, 10E, 30E).

used to construct Settlement Units (SUs), that were exposed at 0.5 m depth (6 at each station). Each SU was composed of a ceramic flower pot (7 cm in diameter), which was screwed individually inside the lids of 1 L plastic bottles on top of a supporting squared PVC plate (thickness = 3 mm, 13 × 13 cm), fixed by cable ties on two screws drilled into the rocks (Fig. 2A–D). A SU was transformed into a nutrient disperser (NU) by “enriching” it with 30 g of the granulate fertilizer ®Osmocote NPK 17-9-11 (17% Total nitrogen (N), consisting of 7.7% nitric nitrogen (NO3) and 9.3% ammoniacal nitrogen (NH4), 9% water-soluble phosphorus pentoxide (P2O5) and 11% water-soluble potassium oxide (K2O)), a fertilizer commonly used in various marine enrichment studies (Teichberg et al., 2013; Amundrud et al., 2015). To test the effectiveness and spatial range of nutrient enrichment, prior to the experimental run, a NU was exposed at the study site (in July 2014). This preliminary study revealed that ammonium, used as enrichment indicator, showed increased levels only in the boundary layer of the NU, whereas already at 0.2 m distance no pronounced enrichment effect was observed (Supplemental Fig. 1). On the base of this finding, the minimum distance among replicates was set at 0.5 m during the experimental runs. At the beginning of each experimental run, 18 NUs (n = 3 per sampling station) and other 18 “unenriched” SUs, used as control (C; n = 3 per sampling station), were exposed at the study site. For sampling, at the end of each experimental run, the

2.5. Environmental conditions 2.5.1. Water temperature To measure the temperature increase due to the heat pump outflow along the two chosen gradients, temperature loggers (Tidbit v2 Water Temperature Data Logger - UTBI-001, Hobo) tied close to the flower pots (Fig. 2D), were setup at each station. The water temperature was measured hourly, during the 3 experimental runs. Unfortunately, the logger set up in the warmest station of the West gradient (2W) was lost in T2. 554

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Fig. 2. Experimental set-up. (A) plan of individual settlement units (SU), (B–C) transformation of SU to sampling bottle by super-imposing the 1 l bottle to the lid and cutting the cable ties, (D) individual SU with attached temperature logger (TEMP) and clod card (CLOD).

room temperature for 4 h in the dark. The detection limit was 0.002 μM l−1. Milli Q water served as blanc and base for the calibration curve. Dissolved inorganic Nitrogen (DIN) was calculated by summing measured values of NO2−, NO3− and NH4+ , allowing the calculations of nitrogen to phosphorus (N/P) ratio, identified as important marker for Ostreopsis bloom development by Accoroni et al. (2014, 2015).

2.5.2. Nutrient levels In order to assess the ambient nutrient levels and the enrichment effect, 36 samples were collected at each experimental run. In order to limit resuspension of cells, nutrient samples were taken from the water surrounding SU during the sampling, before shaking (see above), using 50 ml sterile plastic bottles. Unfortunately nutrient samples for the enriched T1 treatments get lost. To exclude the presence of additional nutrient variations in the ambient water along the considered temperature gradients, supplementary samples were taken for NO3−, NO2−, PO43− and Si(OH)4 in duplicates along the East-ward and Westward gradients at 2, 10, 30 and 50 m distances at the beginning of the second experimental run (5th of August 2014). Samples intended for nutrient analysis were divided into two parts: i) for NO3−, NO2−, PO43− and Si(OH)4 samples were placed in 60 ml plastic bottles, and ii) for NH4+ samples were placed in 15 ml centrifuge tubes, and stored at −20 °C until analyses. Concentrations of NO3−, NO2−, PO43− and Si(OH)4 were measured using a Seal Analytical continuous flow Autoanalyser III (AA3) according to the protocols of Raimbault et al. (1999) and Aminot and Kérouel (2007). Limits of detection were: NO3 = 8.74 nM, NO2− = 2.53 nM, PO43− = 5.02 nM and Si(OH)4 = 15.82 nM. Concentrations of Ammonium were determined following the protocol of Oriol et al. (2014). From each sample 10 ml were filled in 50 ml polycarbonate tubes (OAK Ridge, Nalgene), diluted with 1250 μl reactive agent and incubated at

2.5.3. Wave exposure In order to exclude a potential bias due to a different exposure of SUs to hydrodynamic conditions, 36 clod cards were prepared following a modified protocol of Doty (1971). For this purpose 36 plaster cylinders, mounted in 200 ml plastic cups with a central plastic ring allowing fixation in the field, where exposed for 24 h during the third experimental run (Fig. 2D). The PERMANOVA performed on the dissolution rate did not show any significant difference in relation to the DISTANCE from the heat pump outlet, the DIRECTION (E or W) or the TREATMENT (enriched or unenriched) (Supplemental Table S1). 2.6. Statistical analyses Potential differences in seawater temperature, nutrient concentrations (NO2−, NO3, NH4+, PO43, Si(OH)4) and microalgal communities composition, were assessed by different 2- and 3-ways permutational 555

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

3.2. Ambient nutrient concentrations

multi-variate analyses of variance (PERMANOVA), testing the differences between the six different sampling stations (30W, 10W, 2W, 2E, 10E and 30E), related to DISTANCE (2, 10 and 30 m) from the heat pump outlet, DIRECTION (W or E) of the transect and the nutrient enrichment conditions (TREATMENT: C: control, N: enriched). For nutrients, distance matrices were calculated applying Euclidean distances on normalized data. For the microalgal community abundances, Bray-Curtis similarity indices were used for the calculation of similarity matrices, prior to root transformation in order to scale-down the importance of dominant taxa. The corresponding Principal Coordinate Analyses (PCOs) were produced for the microalgal communities, superimposed by environmental variables applying Pearson correlations. Similarity percentages (SIMPER) were used to determine the relative contribution of single parameters to similarity/dissimilarity, measured in average squared distances (av. sq. dis.). Different 2-ways and 3-ways analyses of variance (ANOVAs) were performed to test the differences between the separate species cell abundances in the experimental units (cells SU−l), in the biofilm (cells g FW−1) and in the surrounding water (cells l−1), following the same experimental design (without the factor Treatment for the Biofilm and surrounding water analyses) (Evans, 1996). Data were log transformed prior analysis. Homogeneity of variances was tested using Cochran's test. Heteroscedastic data were analysed with the nonparametric Kruskal–Wallis test. Tukey's test of Honest Significant Difference (HSD) or Kruskal-Wallis multiple comparisons were used for post-hoc tests. Data were processed using STATISTICA software (StatSoft), version 7.1. As the temporal trend of the bloom was not relevant for the hypotheses, separate analyses were performed for both PERMANOVAs and ANOVAs (or Kruskall-Wallis tests), for the three experimental runs (T1-T3). When a main factor or an interaction was identified as significant (p < 0.05), post-hoc pair-wise tests were conducted to detect differences within levels of each fixed relevant factor.

The measured mean ambient nutrient values ranged from 0.58 μM (30E at T1) to 9.98 μM (10W at T2) for dissolved inorganic nitrogen (DIN; including 0.02 to 1.04 μM for NH4+, 0.01 to 0.57 μM for NO2− and 0.68 to 9.02 μM for NO3−), from 0.05 μM (30E at T1) to 1.20 μM (2E at T2) for dissolved inorganic phosphate (DIP; PO43−) and from 0.24 μM (10W at T3) to 5.38 μM (10E at T2) for Si(OH)4 (Supplemental Fig. 2). A temporal shift in the nutrient concentrations was observed, characterized by an increase in nearly all measured concentrations at T2 (Supplemental Fig. 2). Concerning the nutrient ratios calculated for the ambient (control) treatments, N/P ratios were close to the threshold of 25, defined by Accoroni et al. (2014, 2015) for Ostreopsis bloom onset, at the start of the experiment (T1, maximum values: 23.5). The N/P ratios decreased over time, with maximum values of T2:15.1 and T3:22.0 (Supplemental Fig. 3), due to a temporally increase in PO43− (at T2) and later increase in NH4+ (at T3) (Supplemental Fig. 2). 3.3. Experimental nutrient enrichment

3. Results

The effectiveness of the enriched SUs was proved by significantly higher nutrient concentrations, as showed in the PERMANOVAs in both T2 and T3 (Factor: treatment, T2: Pseudo-F = 8.48, p = 0.001; T3: Pseudo-F = 4.62, p = 0.019; Supplemental Table S3). The enriched units showed variable, but consistently higher concentrations for NO2−, NO3−, NH4+, PO43−, whereas no differences were observed for Si (OH)4 (shown for T2 and T3 in Supplemental Fig. 4). The high values (e.g. NH4 > 1000 μM) recorded in the enriched treatments at T3 are potentially due to the longer exposure time and the sampling strategy that did not allow to sample the water before closing the sample. Except higher nutrient concentrations in controls at the W sites at T1 (Factor: Direction, Pseudo-F = 7.8, p = 0.01; Supplemental Table S3), no other main factors or interactions were significant, showing the homogeneity of nutrient concentrations (both controls and enriched) in the experimental setup.

3.1. Temperature regime

3.4. Microalgal composition and dynamics

Average water temperatures varied over the experimental period, driven mostly by the activities of the heat pump, but also by daily variation and weather-driven changes (Fig. 3A). The temperature gradient related to the distance from the heat pump was clear since the temperature ranged between stations 2 m and 30 m from 24 °C to 26 °C with variations from 0.05 °C to 1.2 °C at T1, from 25 °C to 27 °C with variations from 0.08 °C to 1.1 °C at T2, and from 24 °C to 26 °C with variations of 0.3 °C to 1.5 °C at T3. Over the whole experiment, highest mean values of seawater temperature were generally measured at 2W (Supplemental Table S2, Fig. 3B), whereas the lowest mean values were found in the 30E and 30W stations. Consistently with their position along the transect, the temperature values in 10E and 10W were generally intermediate values, whereas 10E did not significantly differ from 2E at T1 and showed some higher temperature values at T2. Temperature values in 10W showed significantly lower values than in 10E at T2 and T3. Overall, a gradient of 30W = 30E < 10W ≤ 10E ≤ 2E < 2W was observed over the experimental time, and at small scale the hydrological regime seemed to influence the seawater temperature along the gradients. The highest mean temperature difference between the 2 m and the 30 m stations was +0.8 °C at T3. The highest daily alterations were observed during September (T3), peaking at 10 W with 2 °C amplitude (min: 23.9 °C, max: 25.9 °C). Comparing the temperature regimes over time, a seasonal shift was observed in the 30 m treatments, from 24.4 °C ± 0.05 °C (July, T1), 25.6 °C ± 0.01 °C (August, T2), 24.4 °C ± 0.03 °C (September, T3).

Ostreopsis cf. ovata was very abundant in the observed assemblages. For the EXPT it reached a maximum of 489,850 cells SU−1 in the enriched (10E) and a calculated maximum average of 1940 cells cm−2 in the non-enriched (10W) treatments (Fig. 4); and was found with a maximum of 1,040,000 cells g FW−1 (on a Laurencia) in the biofilm (Fig. 5), and of 21,480 cells l−1 in the surrounding water (Fig. 6), at T1. In the following sampling times, the Ostreopsis cf. ovata bloom decreased, showing maximum values of 217,000 cells SU−1 (corresponding to an average of 811 cells cm−2) in the non-enriched 30W at T2, and of 34,036 cells SU−1 (corresponding to an average of 115 cells cm−2) in the non- enriched 30E treatments, at T3. In correspondence, the maximum values of the biofilm decreased from 488,699 to 9223 cells mg FW−1 (both on Dictyota) and of the surrounding water from 3800 cells l−1 to 240 l−1. Apart Ostreopsis cf. ovata, the other dinoflagellates composing the community were Coolia monotis Meunier 1919, Prorocentrum lima (Ehrenberg) F. Stein 1878 and P. micans Ehrenberg 1834 (Figs. 5, 6, 7), but in very low abundances, compared to Ostreopsis cf. ovata. Their generally low abundance and patchy distribution, may not always have allowed a correct interpretation of the observed potential responses to temperature and nutrient enrichment. On the contrary, the diatom Actinocyclus Ehrenberg 1837 sp. showed high abundances, generally lower than Ostreopsis cf. ovata ones throughout the experiment in the natural biofilms, but reached temporally higher cell abundances in T2 in the non-enriched EXPT (controls) and the surrounding water, with maximum values of 4658 cells cm−2 (Fig. 4) and 3329 cells l−1 (Fig. 6), respectively at T2. 556

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Fig. 3. Temperature gradients. Water temperatures measured at different distances from the heat-pump outlet (2, 10 and 30 m) along the West-ward and East-ward (W and E) transects. (A) raw data and (B) mean values ( ± S.E.) of the six different sampling stations (30W, 10W, 2W, 2E, 10E, 30E), calculated from one (for T1 and T2) and three entire days (T3) during the experimental runs. Small letters indicate post hoc results of PERMANOVAs reported in Supplemental Table 4.* missing data for 2W in T2.

As showed in the PERMANOVA and the corresponding SIMPER results (Supplemental Table 4), the observed differences between the microalgal assemblages were mainly due to changes in the abundances of Ostreopsis cf. ovata and the diatom Actinocyclus sp.

temperature seems to be the variable mostly driving the dispersion of points. At T3 the segregation of stations is less evident, potentially due to the effect of nutrients in a longer experiment and a generally lower abundance of cells (end of the bloom for the most abundant species).

3.5. Responses of the microalgal assemblages to the experimental temperature and nutrient regimes (EXPT)

3.5.1. Microalgal responses to temperature In T1 (stationary phase of the bloom), Ostreopsis cf. ovata responded significantly to the temperature gradient in both, the BIOFILM and the EXPT (Supplemental Table 5, Figs. 5, 6), but this response was not detectable in the WATER abundances (Supplemental Table 5, Fig. 7). The temperature had a positive effect on the cell abundances, resulting in a positive linear correlation for the BIOFILM (Supplemental Table 6). Also in T2 a significant response of Ostreopsis cf. ovata to the temperature was observed along the gradients (Supplemental Table 5, Figs. 5, 6), that was not detectable in the WATER. At this time the temperature had a negative effect on cell abundances, with a significant negative linear correlation for both BIOFILM and EXPT (Supplemental Table 6). At the end of the bloom (T3), the patterns were less clear, a significant response was detected from the PERMANOVA at the scale of the stations along the gradients (Supplemental Table 5, Figs. 5, 6, 7), but not in agreement with the temperature. Corresponding correlations were not significant (Supplemental Table 6). Actinocyclus sp. responded significantly to the temperature at T1, during the growing phase of its bloom, in the EXPT, the BIOFILM and the WATER samplings (Supplemental Table 5, Figs. 5, 6, 7). Positive

The two drivers “Temperature” and “Nutrient enrichment” affected the microalgal communities in different ways during the three experimental runs (Supplemental Table 4), but never in combination: no significant interaction involving the factor Treatment and Distance was observed. The effect of temperature, expressed as the distance from the outflow, was highlighted by the PCOs (Fig. 7) and PERMANOVAs on the communities at the three experimental runs (Supplemental Table 4). At T1 and T2, differences in agreement with the studied gradients were observed in the two directions from the outflow, while at T3, only the western gradient showed significant differences in the community structure and composition. No effect of the nutrient enrichment on microalgal communities was detected at T1 and T2, while a significant effect was detected at T3. The PCOs (Fig. 7) showed that the stations at 30 m from the outlet are quite separate from the other points, especially at T1 and T2. Superimposition of variables by linear correlation shows that the 557

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Fig. 4. Ostreopsis cf. ovata, Actinocyclus sp., Coolia monotis, Prorocentrum lima and P. micans abundances in the EXPT (mean values ± S.E). Black bars indicate enriched conditions, while grey bars indicate controls. Percentage values at y-axes indicate scaling differences within each taxon. Small letters indicate post hoc results of PERMANOVAs reported in Supplemental Table 5.

Coolia monotis and Prorocentrum lima showed a significant peak of abundance, during the growing phase of their blooms, in the warmest station (2W) in the EXPT at T1 (Fig. 5). Besides this observation, no significant patterns consistent with the gradients were recorded, potentially due to the low abundances of cells which did not allow precise estimation.

correlations with the temperature were observed for the EXPT and the BIOFILM sampling (Supplemental Table 6). In T2, during the stationary phase of its bloom (Figs. 5, 6), Actinocyclus sp. responded to the temperature gradient in both the EXPT and the WATER samplings (Supplemental Table 5, Figs. 5, 7), showing positive correlations with the corresponding temperatures (Supplemental Table 6). In T3, during in the decreasing phase of the bloom, no clear patterns or correlations were detected for this species. 558

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Fig. 5. Ostreopsis cf. ovata, Actinocyclus sp., Coolia monotis, Prorocentrum lima and P. micans abundances in the BIOFILM (mean values ± S.E). Percentage values at yaxes indicate scaling differences within each taxon. Small letters indicate post hoc results of PERMANOVAs reported in Supplemental Table 5.

temperature and nutrient enrichment has been detected, either at the community level, or for the dominant species, in contrast to what was observed in other studies on planktonic microalgal communities (Deng et al., 2014; Rigosi et al., 2014). As the named studies mainly concern free floating planktonic assemblages, it might be possible that the less motile benthic microalgal communities are more tolerant toward environmental variations than we think, or that uncontrolled grazer activity mediate potential effects, as it was observed for benthic microalgae grown in a seagrass mesocosm experiment by Alsterberg et al. (2013). In the present study, temperature seemed to play an important role for the benthic microalgal communities, significantly affecting them in the 3 experimental runs. In contrast the nutrient enrichment had a significant effect on the microalgal community only in T3. The two most abundant species were the dinoflagellate Ostreopsis cf. ovata and the diatom Actinocyclus sp., both providing species- and bloom phasespecific responses to the two drivers considered. As shown in previous studies, the response of O. cf. ovata to water temperature may depend on species identity as well as on the population's origin (reviewed by

3.5.2. Microalgal responses to elevated nutrient levels (EXPT) A significant negative response to nutrient enrichment was observed in the microalgal communities at T3, mainly driven by a general decrease in cell numbers of Actinocyclus and Ostreopsis (Supplemental Table 4). Interestingly, Ostreopsis cf. ovata did not respond to the nutrient enrichment in the three considered runs of the experiment (Supplemental Table 5, Fig. 4). On the contrary, Actinocyclus seemed to respond in both T2 and T3 (Supplemental Table 5, Fig. 4) in a negative way, with abundances being higher in the controls than in the treatments. 4. Discussion The innovative experimental approach of the in situ combination of temperature increase and nutrient enrichment proposed in this study was conceived to comply with the priority needs in environmental change research of i) studying the combined effects of global and local stressors, and ii) enhanced realism in research through field experiments (Wernberg et al., 2012). Interestingly, no interaction of 559

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Fig. 6. Ostreopsis cf. ovata, Actinocyclus sp., Coolia monotis, Prorocentrum lima and P. micans abundances in the surrounding WATER (mean values ± S.E). Percentage values at y-axes indicate scaling differences within each taxon. Small letters indicate post hoc results of PERMANOVAs reported in Supplemental Table 5.

decreasing phase of the bloom can be negatively correlated with the temperature. Ostreopsis cf. ovata abundances were significantly higher at the warmer stations in the first run of the experiment, corresponding to the stationary phase of the bloom, in one of the two gradients in the nutrient enrichment experiment and in both gradients when considering the biofilm of natural macroalgal communities. Interestingly, Ostreopsis abundances showed an opposite pattern in T2, the declining phase of the bloom, in both the enrichment experiment and in the biofilm of macroalgal communities. No particular pattern was observed in T3, at the end of the bloom. The recorded different responses of Ostreopsis cf. ovata to the increased temperature in the different phases of the bloom

Carnicer et al., 2016), reflecting the likelihood that populations from diverse environments may require different optimal temperatures for growth. For the Mediterranean Sea, Ostreopsis spp. proliferation is reported between 18 and 30 °C (Scalco et al., 2012), blooms are typical summer events (Mangialajo et al., 2011) and temperature is considered as one of the most determinant factors (Pistocchi et al., 2011). Temperatures around 25–26 °C have been suggested for triggering bloom onset in the Ligurian Sea (Mangialajo et al., 2008a; Pistocchi et al., 2011; Cohu et al., 2013), in agreement with the findings of the present study. Once initiated, Ostreopsis cf. ovata bloom can develop independently of declining water temperatures (Cohu et al., 2011; Accoroni et al., 2015), and the results of the present study show that the 560

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Fig. 7. Principal Coordinate analyses (PCOs) of experimental (EXPT) microalgal communities. The twelve combinations of Stations (30W, 10W, 2W, 2E, 10E, 30E) and Treatments (NUT = enriched, C = control) are reported with different symbols per each experimental run (T1–T3). Analyses performed on Bray Curtis similarities matrices, prior root transformation of data. Overlaid variables (Pearson correlation) indicate direction of water temperature (encircled with dashed line) and + 3− , Si(OH)4, N/P). different nutrient levels (e.g. NO2−, NO3− , NH4 , PO4

negatively to the nutrient enrichment in both T2 and T3 (respectively the stationary and the decreasing phases of the bloom). Some species of the Coscinodiscales are known to have very high nutrient uptake rates that disadvantage them compared to other species in competing for nutrients (Nishikawa and Yamaguchi, 2008). It is not clear how this could have led to a negative effect of enrichment on Actinocyclus abundances in the present study, but a competitive effect with other small autotrophic organisms not included in the study cannot be excluded. Ostreopsis cf. ovata abundances were not affected by the nutrient enrichment during the whole experiment. Unluckily, the growing phase of the bloom was not included in the experiment and we cannot exclude different results in this phase where nutrients ratios seem to play a role (Accoroni et al., 2014, 2015). On the other way, an absence of effect of nutrients may be in agreement with the contrasting results recorded in the literature (reviewed by Pistocchi et al., 2011; Carnicer et al., 2015; Accoroni and Totti, 2016). Although Ostreopsis cf. ovata is able to effectively use different inorganic and organic nitrogen sources, observable already after 24 h (Jauzein et al., 2017), Pezzolesi et al. (2016) showed that under laboratory conditions an effect of nutrient enrichment on Ostreopsis cf. ovata in the growth pattern becomes visible between 3 and 6 days. Consequently our results may also have been affected by a potentially too short exposure time. In conclusion, based on our observations, further enrichment studies during the early growth phase, and with longer exposition (in agreement with the weather conditions), may provide further interesting insights on to the nutrient effects on the onset of Ostreopsis cf. ovata bloom and the potential

may explain some inconsistency observed in past studies: while most studies (in the field and in laboratory) found a positive correlation with temperature (e.g. Accoroni et al., 2015; Graneli et al., 2011; Yamaguchi et al., 2012), some negative responses were also observed (Cohu et al., 2011; Scalco et al., 2012; Carnicer et al., 2016). It can be hypothesized that the temperature is not only affecting the absolute abundances, but potentially shifting the blooms in time, causing an early bloom in the warmest stations, and a consequent early decline. Such a potential shift in the season due to global warming, may have unexpected indirect consequences on the marine ecosystem by potentially affecting seasonal phases of the life cycle (i.e. the reproduction or the recruitment) of particular species, which might not be affected at the actual seasonal bloom dynamic (Brierley and Kingsford, 2009; Barton et al., 2016; Poloczanska et al., 2016) The diatom Actinocyclus sp. shows a positive response to temperature in the first two runs of the enrichment experiment (T1, growing phase of the bloom and T2, stationary phase of the bloom), while this trend was visible uniquely in the first run when considering the biofilm of natural macroalgal communities and the surrounding water. This is in agreement with findings on some other species of the Coscinodiscales, which growth seem to increase with temperature (Montagnes and Franklin, 2001; Nishikawa and Yamaguchi, 2008). In the present experiment, nutrients seem to be a less important driver in fostering benthic microalgal blooms: an enrichment effect was observed on the microalgal assemblages only in the third experimental run (T3), when the abundances of microalgae were lower. This effect was unexpectedly mostly driven by Actinocyclus sp. that responded

561

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Acknowledgments

interactions with temperature. A shift in abundances of cells was observed between the two dominant microalgae (Ostreopsis cf. ovata and Actinocyclus sp.) in the enrichment experiment, highlighting how the peak of Actinocyclus sp. corresponded to the decreasing phase of Ostreopsis cf. ovata. A similar pattern has been observed at a beach in Croatia by Pfannkuchen et al. (2012), where a bloom of Actinocyclus sp. replaced Ostreopsis cf. ovata after its decay. The common conjoined occurrence of Ostreopsis and Coscinodiscales blooms, observed at different sites in the Mediterranean (Vila et al., 2001; Pfannkuchen et al., 2012; Carnicer et al., 2015) suggests a potential regulation of Ostreopsis cf. ovata blooms based on interspecific competition. Indeed, a recent work revealed an inhibitory effect of polyunsaturated aldehydes (PUAs), produced and released by Diatoms, on the growth of Ostreopsis cf. ovata (Pichierri et al., 2016, 2017). Vice versa, the recorded decrease of benthic microalgae during Ostreopsis blooms (Accoroni et al., 2016), might be related to the production of dinoflagellate toxins, suppressing sympatric species (Pfannkuchen et al., 2012). In addition, also close related taxa, like Coolia or Prorocentrum, seem to interact with Ostreopsis blooms, as they were commonly observed within the mucus (Pfannkuchen et al., 2012; Carnicer et al., 2015). In our study we observed Coolia monotis and Prorocentrum lima within the Ostreopsis bloom, showing a peak of abundance together with Ostreopsis at one of the warmest stations (2W) in their growing phase in the experiment. However, this trend was not confirmed in the biofilm of natural macroalgal communities, neither in the surrounding water, probably due to their low abundances and the consequent high variability that did not allow robust quantification of cells. Overall, allelopathic interactions seem to play a crucial role in triggering the bloom activity and possibly affecting also its toxicity (e.g. Pichierri et al., 2016, 2017). As species identity plays an important role, we recommend future studies to concern also potential genetic shifts, as well as including the accompanying taxa, and investigate the concentration and composition of toxic secondary metabolites (e.g. PUAs, palytoxin and derivates). One of the key factors limiting algal bloom predictions is the understanding of interacting effects of environmental stressors, such as temperature, salinity, nutrient availability or irradiance (Scalco et al., 2012; Glibert et al., 2014; Tawong et al., 2015; Gao et al., 2016). Moreover predictions routinely focus on the abiotic drivers, yet biotic interactions, such as competition (for example between Ostreopsis cf. ovata and Actinocyclus sp.), predation, parasitism, associated bacteria composition and virus regulation, can strongly influence how climate change affects these organisms (O'Neil et al., 2012). Studies focused on multiple stressors provide robust evidence that both biotic and abiotic drivers commonly interact synergistically in marine ecosystems (Crain et al., 2008; Strain et al., 2014). The present study showed that this is not always the case, but failure to incorporate these interactions limits the ability to predict responses of species to global change (Gilman et al., 2010). The results presented corroborate that temperature plays a crucial role in regulating benthic microalgal blooms (see also Graneli et al., 2011), suggesting that ocean warming is a potential driver of the increase in magnitude and frequency of blooms over the last decades (Shears and Ross, 2009; Parsons et al., 2012; Accoroni and Totti, 2016). In contrast the roles of increased nutrients seem to be more dependent on the species composition and the characteristics of the local environment. In the light of future climate scenarios predicting a warming trend and an increase in the frequency of exceptional events such as storms, cyclones and atmospheric stability, further studies on how the future changes in climate will potentially influence the magnitude and frequency of benthic HABs should represent a research priority. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2018.04.012.

The present research has been produced with the financial assistance of the European Union under the ENPI CBC Mediterranean Sea Basin Programme, within the project M3-HABs (Project Reference Number: IIB/2.1/0096) and of the national French Program OPTIMA PAC (FUI-AAP11, co-funded by the Région PACA, the Conseil Général des Alpes Maritimes and the Environment Directorate of the Principality of Monaco). The authors wish to warmly thank the personnel of the Direction of the Environment of the Principauté de Monaco, for their logistical support (and for their patience in waiting for good weather conditions), and for productive scientific and technical discussions, together with the other partners of the 2 projects (M3HABs and OPTIMA PAC). We thank the Hustedt Diatom Study Centre (AWI Bremerhaven, Germany) for its support. Aurélie Dufour and Cécile Jauzein helped with nutrient analyses. References Accoroni, S., Totti, C., 2016. The toxic benthic dinoflagellates of the genus Ostreopsis in temperate areas: a review. Adv. Oceanogr. Limnol. 7. http://dx.doi.org/10.4081/ aiol.2016.5591. Accoroni, S., Romagnoli, T., Pichierri, S., Totti, C., 2014. New insights on the life cycle stages of the toxic benthic dinoflagellate Ostreopsis cf. ovata. Harmful Algae 34, 7–16. http://dx.doi.org/10.1016/j.hal.2014.02.003. Accoroni, S., Glibert, P.M., Pichierri, S., Romagnoli, T., Marini, M., Totti, C., 2015. A conceptual model of annual Ostreopsis cf. ovata blooms in the northern Adriatic Sea based on the synergic effects of hydrodynamics, temperature, and the N:P ratio of water column nutrients. Harmful Algae 45, 14–25. http://dx.doi.org/10.1016/j.hal. 2015.04.002. Accoroni, S., Romagnoli, T., Pichierri, S., Totti, C., 2016. Effects of the bloom of harmful benthic dinoflagellate Ostreopsis cf. ovata on the microphytobenthos community in the northern Adriatic Sea. Harmful Algae 55, 179–190. http://dx.doi.org/10.1016/j. hal.2016.03.003. Al-Handal, A.Y., Fricke, A., Wulff, A., 2016. Observations on diatoms inhabiting natural and artificial substrates in Kongsfjorden, Svalbard, the Arctic. Polar Biol. 1–20. http://dx.doi.org/10.1007/s00300-016-1998-7. Alsterberg, C., Eklöf, J.S., Gamfeldt, L., Havenhand, J.N., Sundbäck, K., 2013. Consumers mediate the effects of experimental ocean acidification and warming on primary producers. Proc. Natl. Acad. Sci. U. S. A. 110, 8603–8608. http://dx.doi.org/10. 1073/pnas.1303797110. Aminot, A., Kérouel, R., 2007. Dosage automatique des nutriments dans les eaux marines: méthodes en flux continu. Editions Quae. Amundrud, S.L., Srivastava, D.S., O'Connor, M.I., 2015. Indirect effects of predators control herbivore richness and abundance in a benthic eelgrass (Zostera marina) mesograzer community. J. Anim. Ecol. 84, 1092–1102. Barton, A.D., Irwin, A.J., Finkel, Z.V., Stock, C.A., 2016. Anthropogenic climate change drives shift and shuffle in North Atlantic phytoplankton communities. Proc. Natl. Acad. Sci. 113, 2964–2969. http://dx.doi.org/10.1073/pnas.1519080113. Berges, J., Varela, D., Harrison, P., 2002. Effects of temperature on growth rate, cell composition and nitrogen metabolism in the marine diatom Thalassiosira pseudonana (Bacillariophyceae). Mar. Ecol. Prog. Ser. 225, 139–146. http://dx.doi.org/10.3354/ meps225139. Boyd, P.W., Cornwall, C.E., Davison, A., Doney, S.C., Fourquez, M., Hurd, C.L., Lima, I.D., McMinn, A., 2016. Biological responses to environmental heterogeneity under future ocean conditions. Glob. Chang. Biol. 22, 2633–2650. http://dx.doi.org/10.1111/gcb. 13287. Brierley, A.S., Kingsford, M.J., 2009. Impacts of climate change on marine organisms and ecosystems. Curr. Biol. 19, R602–614. http://dx.doi.org/10.1016/j.cub.2009.05.046. Carnicer, O., Guallar, C., Andree, K.B., Diogène, J., Fernández-Tejedor, M., 2015. Ostreopsis cf. ovata dynamics in the NW Mediterranean Sea in relation to biotic and abiotic factors. Environ. Res. http://dx.doi.org/10.1016/j.envres.2015.08.023. Carnicer, O., García-Altares, M., Andree, K.B., Tartaglione, L., Dell'Aversano, C., Ciminiello, P., de la Iglesia, P., Diogène, J., Fernández-Tejedor, M., 2016. Ostreopsis cf. ovata from western Mediterranean Sea: physiological responses under different temperature and salinity conditions. Harmful Algae 57, 98–108. http://dx.doi.org/ 10.1016/j.hal.2016.06.002. Chang, F.H., Shimizu, Y., Hay, B., Stewart, R., Mackay, G., Tasker, R., 2000. Three recently recorded Ostreopsis spp. (Dinophyceae) in New Zealand: temporal and regional distribution in the upper North Island from 1995 to 1997. N. Z. J. Mar. Freshw. Res. 34, 29–39. Cloern, J., 2001. Our evolving conceptual model of the coastal eutrophication problem. Mar. Ecol. Prog. Ser. 210, 223–253. http://dx.doi.org/10.3354/meps210223. Cohu, S., Thibaut, T., Mangialajo, L., Labat, J.-P., Passafiume, O., Blanfune, A., Simon, N., Cottalorda, J.-M., Lemee, R., 2011. Occurrence of the toxic dinoflagellate Ostreopsis cf. ovata in relation with environmental factors in Monaco (NW Mediterranean). Mar. Pollut. Bull. 62, 2681–2691. http://dx.doi.org/10.1016/j.marpolbul.2011.09.022. Cohu, S., Mangialajo, L., Thibaut, T., Blanfune, A., Marro, S., Lemee, R., 2013. Proliferation of the toxic dinoflagellate Ostreopsis cf. ovata in relation to depth, biotic substrate and environmental factors in the North West Mediterranean Sea. Harmful

562

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

Heisler, J., Glibert, P.M., Burkholder, J.M., Anderson, D.M., Cochlan, W., Dennison, W.C., Dortch, Q., Gobler, C.J., Heil, C.A., Humphries, E., Lewitus, A., Magnien, R., Marshall, H.G., Sellner, K., Stockwell, D.A., Stoecker, D.K., Suddleson, M., 2008. Eutrophication and harmful algal blooms: a scientific consensus. Harmful Algae 8, 3–13. http://dx.doi.org/10.1016/j.hal.2008.08.006. Hoppenrath, M., Murray, S.A., Chomérat, N., Horiguchi, T., 2014. Marine benthic dinoflagellates - unveiling their worldwide biodiversity. Kleine Senckenberg-Reihe, Band 54, Schweizerbart, Stuttgart, Germany, pp. 276. Hyndes, G.A., Heck, K.L., Vergés, A., Harvey, E.S., Kendrick, G.A., Lavery, P.S., McMahon, K., Orth, R.J., Pearce, A., Vanderklift, M., Wernberg, T., Whiting, S., Wilson, S., 2016. Accelerating tropicalization and the transformation of temperate seagrass meadows. Bioscience 66, 938–948. http://dx.doi.org/10.1093/biosci/biw111. Jauzein, C., Couet, D., Blasco, T., Lemée, R., 2017. Uptake of dissolved inorganic and organic nitrogen by the benthic toxic dinoflagellate Ostreopsis cf. ovata. Harmful Algae 65, 9–18. http://dx.doi.org/10.1016/j.hal.2017.04.005. Lakkis, S., Sabour, W., 2014. “Tropicalization” of the East Mediterranean enhancing invasion of tropical species in Syro-Lebanese seawaters. Mar. J. 1. Lejeusne, C., Chevaldonné, P., Pergent-Martini, C., Boudouresque, C.F., Pérez, T., 2010. Climate change effects on a miniature ocean: the highly diverse, highly impacted Mediterranean Sea. Trends Ecol. Evol. 25, 250–260. http://dx.doi.org/10.1016/j. tree.2009.10.009. Lemee, R., Mangialajo, L., Cohu, S., Amzil, Z., Blanfune, A., Chomerat, N., Ganzin, N., Gasparini, S., Grossel, H., Guidi-Guivard, L., Hoareau, L., le Duff, F., Marro, S., Simon, N., Nezan, E., Pedrotti, M.-L., Sechet, V., Soliveres, O., Thibaut, T., 2012. Interactions between scientists, managers and policy makers in the framework of the French MediOs project on Ostreopsis (2008–2010). Cryptogam. Algol. 33, 137–142. Littler, M.M., Littler, D.S., Brooks, B.L., 2010. The effects of nitrogen and phosphorus enrichment on algal community development: artificial mini-reefs on the Belize Barrier Reef sedimentary lagoon. Harmful Algae 9, 255–263. http://dx.doi.org/10. 1016/j.hal.2009.11.002. Lomas, M.W., Glibert, P.M., 1999. Interactions between NH+4 and NO−3 uptake and assimilation: comparison of diatoms and dinoflagellates at several growth temperatures. Mar. Biol. 133, 541–551. http://dx.doi.org/10.1007/s002270050494. Mangialajo, L., Bertolotto, R., Cattaneo-Vietti, R., Chiantore, M., Grillo, C., Lemee, R., Melchiorre, N., Moretto, P., Povero, P., Ruggieri, N., 2008. The toxic benthic dinoflagellate Ostreopsis ovata: quantification of proliferation along the coastline of Genoa, Italy. Mar. Pollut. Bull. 56, 1209–1214. http://dx.doi.org/10.1016/j. marpolbul.2008.02.028. Mangialajo, L., Ganzin, N., Accoroni, S., Asnaghi, V., Blanfuné, A., Cabrini, M., CattaneoVietti, R., Chavanon, F., Chiantore, M., Cohu, S., Costa, E., Fornasaro, D., Grossel, H., Marco-Miralles, F., Masó, M., Reñé, A., Rossi, A.M., Sala, M.M., Thibaut, T., Totti, C., Vila, M., Lemée, R., 2011. Trends in Ostreopsis proliferation along the northern Mediterranean coasts. Toxicon Off. J. Int. Soc. Toxinology 57, 408–420. http://dx. doi.org/10.1016/j.toxicon.2010.11.019. Miraglia, M., Marvin, H.J.P., Kleter, G.A., Battilani, P., Brera, C., Coni, E., Cubadda, F., Croci, L., De Santis, B., Dekkers, S., Filippi, L., Hutjes, R.W.A., Noordam, M.Y., Pisante, M., Piva, G., Prandini, A., Toti, L., van den Born, G.J., Vespermann, A., 2009. Climate change and food safety: an emerging issue with special focus on Europe. Food Chem. Toxicol. 47, 1009–1021. http://dx.doi.org/10.1016/j.fct.2009.02.005. Montagnes, D.J.S., Franklin, M., 2001. Effect of temperature on diatom volume, growth rate, and carbon and nitrogen content: reconsidering some paradigms. Limnol. Oceanogr. 46, 2008–2018. http://dx.doi.org/10.4319/lo.2001.46.8.2008. Morton, S.L., Sexton, J.P., Andersen, R.A., 1997. Harmful Algal Cultures Available at the Provasoli-Guillard National Center for Culture of Marine Phytoplankton. Instituto Espanol de Oceanografia, Centro Oceanografico de Vigo, Vigo (Espana). Nascimento, S.M., Franca, J.V., Goncalves, J.E., Ferreira, C.E., 2012. Ostreopsis cf. ovata (Dinophyta) bloom in an equatorial island of the Atlantic Ocean. Mar. Pollut. Bull. 64, 1074–1078. http://dx.doi.org/10.1016/j.marpolbul.2012.03.015. Nishikawa, T., Yamaguchi, M., 2008. Effect of temperature on light-limited growth of the harmful diatom Coscinodiscus wailesii, a causative organism in the bleaching of aquacultured Porphyra thalli. Harmful Algae 7, 561–566. http://dx.doi.org/10.1016/ j.hal.2007.12.021. O'Neil, J.M., Davis, T.W., Burford, M.A., Gobler, C.J., 2012. The rise of harmful cyanobacteria blooms: the potential roles of eutrophication and climate change. Harmful Algae 14, 313–334. http://dx.doi.org/10.1016/j.hal.2011.10.027. Oriol, L., Garcia, N., Cariou, T., 2014. Dosage de l'azote ammoniacal en milieu marin par fluorimétrie. Paerl, H.W., Rudek, J., Mallin, M.A., 1990. Stimulation of phytoplankton production in coastal waters by natural rainfall inputs: nutritional and trophic implications. Mar. Biol. 107, 247–254. http://dx.doi.org/10.1007/BF01319823. Parsons, M.L., Preskitt, L.B., 2007. A survey of epiphytic dinoflagellates from the coastal waters of the island of Hawaii. Harmful Algae 6, 658–669. http://dx.doi.org/10. 1016/j.hal.2007.01.001. Parsons, M.L., Aligizaki, K., Bottein, M.-Y.D., Fraga, S., Morton, S.L., Penna, A., Rhodes, L., 2012. Gambierdiscus and Ostreopsis: Reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology. Harmful Algae 14, 107–129. http://dx.doi.org/10.1016/j.hal.2011.10.017. (Harmful Algae–The requirement for species-specific information). Peñuelas, J., Sardans, J., Estiarte, M., Ogaya, R., Carnicer, J., Coll, M., Barbeta, A., RivasUbach, A., Llusià, J., Garbulsky, M., Filella, I., Jump, A.S., 2013. Evidence of current impact of climate change on life: a walk from genes to the biosphere. Glob. Chang. Biol. 19, 2303–2338. http://dx.doi.org/10.1111/gcb.12143. Pezzolesi, L., Vanucci, S., Dell'Aversano, C., Dello Iacovo, E., Tartaglione, L., Pistocchi, R., 2016. Effects of N and P availability on carbon allocation in the toxic dinoflagellate Ostreopsis cf. ovata. Harmful Algae 55, 202–212. http://dx.doi.org/10.1016/j.hal. 2016.02.011.

Algae 24, 32–44. http://dx.doi.org/10.1016/j.hal.2013.01.002. Coll, M., Piroddi, C., Steenbeek, J., Kaschner, K., Ben Rais Lasram, F., Aguzzi, J., Ballesteros, E., Bianchi, C.N., Corbera, J., Dailianis, T., Danovaro, R., Estrada, M., Froglia, C., Galil, B.S., Gasol, J.M., Gertwagen, R., Gil, J., Guilhaumon, F., KesnerReyes, K., Kitsos, M.-S., Koukouras, A., Lampadariou, N., Laxamana, E., López-Fé de la Cuadra, C.M., Lotze, H.K., Martin, D., Mouillot, D., Oro, D., Raicevich, S., RiusBarile, J., Saiz-Salinas, J.I., San Vicente, C., Somot, S., Templado, J., Turon, X., Vafidis, D., Villanueva, R., Voultsiadou, E., 2010. The biodiversity of the Mediterranean Sea: estimates, patterns, and threats. PLoS One 5, e11842. http://dx. doi.org/10.1371/journal.pone.0011842. Crain, C.M., Kroeker, K., Halpern, B.S., 2008. Interactive and cumulative effects of multiple human stressors in marine systems. Ecol. Lett. 11, 1304–1315. http://dx.doi. org/10.1111/j.1461-0248.2008.01253.x. Davidson, K., Anderson, D.M., Mateus, M., Reguera, B., Silke, J., Sourisseau, M., Maguire, J., 2016. Forecasting the risk of harmful algal blooms. Harmful Algae 53, 1–7. http:// dx.doi.org/10.1016/j.hal.2015.11.005. Deng, J., Qin, B., Paerl, H.W., Zhang, Y., Wu, P., Ma, J., Chen, Y., 2014. Effects of nutrients, temperature and their interactions on spring phytoplankton community succession in lake Taihu, China. PLoS One 9, e113960. http://dx.doi.org/10.1371/ journal.pone.0113960. Doty, M.S., 1971. Measurement of water movement in reference to benthic algal growth. Bot. Mar. 14. http://dx.doi.org/10.1515/botm.1971.14.1.32. Endo, H., Suehiro, K., Kinoshita, J., Gao, X., Agatsuma, Y., 2013. Combined effects of temperature and nutrient availability on growth and phlorotannin concentration of the brown alga Sargassum patens (Fucales; Phaeophyceae). Am. J. Plant Sci. 04, 14. http://dx.doi.org/10.4236/ajps.2013.412A2002. Evans, J.D., 1996. Straightforward Statistics for the Behavioral Sciences. Brooks/Cole Publishing Company. Faimali, M., Giussani, V., Piazza, V., Garaventa, F., Corrà, C., Asnaghi, V., Privitera, D., Gallus, L., Cattaneo-Vietti, R., Mangialajo, L., Chiantore, M., 2012. Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms. Emerg. Persistent Impacts Mar. Org. Detect. Methods Action Mech. 76, 97–107. http://dx.doi.org/10.1016/j.marenvres.2011.09.010. Fraga, S., Rodríguez, F., Bravo, I., Zapata, M., Marañón, E., 2012. Review of the Main Ecological Features Affecting Benthic Dinoflagellate Blooms. Cryptogam. Algol. 33, 171–179. http://dx.doi.org/10.7872/crya.v33.iss2.2011.171. Fricke, A., Kopprio, G.A., Alemany, D., Gastaldi, M., Narvarte, M., Parodi, E.R., Lara, R.J., Hidalgo, F., Martínez, A., Sar, E.A., Iribarne, O., Martinetto, P., 2016. Changes in coastal benthic algae succession trajectories and assemblages under contrasting nutrient and grazer loads. Estuar. Coasts 39, 462–477. http://dx.doi.org/10.1007/ s12237-015-9999-2. Fu, F., Tatters, A., Hutchins, D., 2012. Global change and the future of harmful algal blooms in the ocean. Mar. Ecol. Prog. Ser. 470, 207–233. http://dx.doi.org/10.3354/ meps10047. Fukuyo, Y., Ishimaru, T., 1986. In: Maclean, J.L., Dizon, L.B., Hosillos, L.V. (Eds.), Toxic Dinoflagellates in Japan. Gao, G., Clare, A.S., Rose, C., Caldwell, G.S., 2016. Eutrophication and warming-driven green tides (Ulva rigida) are predicted to increase under future climate change scenarios. Mar. Pollut. Bull. http://dx.doi.org/10.1016/j.marpolbul.2016.10.003. Gilman, S.E., Urban, M.C., Tewksbury, J., Gilchrist, G.W., Holt, R.D., 2010. A framework for community interactions under climate change. Trends Ecol. Evol. 25, 325–331. http://dx.doi.org/10.1016/j.tree.2010.03.002. Glibert, P.M., Icarus Allen, J., Artioli, Y., Beusen, A., Bouwman, L., Harle, J., Holmes, R., Holt, J., 2014. Vulnerability of coastal ecosystems to changes in harmful algal bloom distribution in response to climate change: projections based on model analysis. Glob. Chang. Biol. 20, 3845–3858. http://dx.doi.org/10.1111/gcb.12662. Gómez, F., 2011. Diversity and distribution of the dinoflagellates Brachidinium, Asterodinium and Microceratium (Brachidiniales, Dinophyceae) in the open Mediterranean Sea. Acta Bot. Croat. 70, 209–214. http://dx.doi.org/10.2478/ v10184-010-0019-0. Grall, J., Chauvaud, L., 2002. Marine eutrophication and benthos: the need for new approaches and concepts. Glob. Chang. Biol. 8, 813–830. http://dx.doi.org/10.1046/j. 1365-2486.2002.00519.x. Graneli, E., Vidyarathna, N.K., Funari, E., Cumaranatunga, P.R.T., Scenati, R., 2011. Can increases in temperature stimulate blooms of the toxic benthic dinoflagellate Ostreopsis ovata? Harmful Algae 10, 165–172. http://dx.doi.org/10.1016/j.hal.2010. 09.002. Gruber, N., 2011. Warming up, turning sour, losing breath: ocean biogeochemistry under global change. Philos. Trans. R. Soc. Lond. Math. Phys. Eng. Sci. 369, 1980–1996. http://dx.doi.org/10.1098/rsta.2011.0003. Grzebyk, D., Berland, B., Thomassin, B.A., Bosi, C., Arnoux, A., 1994. Ecology of ciguateric dinoflagellates in the coral reef complex of Mayotte Island (S.W. Indian Ocean). J. Exp. Mar. Biol. Ecol. 178, 51–66. Hallegraeff, G.M., 2010. Ocean climate change, phytoplankton community responses, and harmful algal blooms: a formidable predictive challenge. J. Phycol. 46, 220–235. http://dx.doi.org/10.1111/j.1529-8817.2010.00815.x. Halpern, B.S., McLeod, K.L., Rosenberg, A.A., Crowder, L.B., 2008. Managing for cumulative impacts in ecosystem-based management through ocean zoning. Ocean Coast. Manag. 51, 203–211. http://dx.doi.org/10.1016/j.ocecoaman.2007.08.002. Halpern, B.S., Frazier, M., Potapenko, J., Casey, K.S., Koenig, K., Longo, C., Lowndes, J.S., Rockwood, R.C., Selig, E.R., Selkoe, K.A., Walbridge, S., 2015. Spatial and temporal changes in cumulative human impacts on the world's ocean. Nat. Commun. 6, 7615. http://dx.doi.org/10.1038/ncomms8615. Hama, J., Handa, N., 1994. Variability of the biomass, chemical composition and productivity of phytoplankton in Kinu-ura Bay, Japan during the rainy season. Estuar. Coast. Shelf Sci. 39, 497–509. http://dx.doi.org/10.1006/ecss.1994.1078.

563

Marine Pollution Bulletin 131 (2018) 552–564

A. Fricke et al.

examination of nitrogen, temperature, light, and other nutrients. Water Res. 32, 3539–3548. http://dx.doi.org/10.1016/S0043-1354(98)00165-1. Strain, E.M.A., Thomson, R.J., Micheli, F., Mancuso, F.P., Airoldi, L., 2014. Identifying the interacting roles of stressors in driving the global loss of canopy-forming to matforming algae in marine ecosystems. Glob. Chang. Biol. 20, 3300–3312. http://dx. doi.org/10.1111/gcb.12619. Taniyama, S., Arakawa, O., Terada, M., Nishio, S., Takatani, T., Mahmud, Y., Noguchi, T., 2003. Ostreopsis sp. a possible origin of palytoxin (PTX) in parrotfish Scarus ovifrons. Toxicon 42, 29–33. https://doi.org/10.1016/S0041-0101(03)00097-7. Tawong, W., Yoshimatsu, T., Yamaguchi, H., Adachi, M., 2015. Effects of temperature, salinity and their interaction on growth of benthic dinoflagellates Ostreopsis spp. from Thailand. Harmful Algae 44, 37–45. http://dx.doi.org/10.1016/j.hal.2015.02.011. Taylor, F.J.R., 1979. Description of the benthic dinoflagellate [algae] associated with maitotoxin and ciguatoxin, including observations on Hawaiian material. In: Toxic Dinoflagellate Blooms. Elsevier, Amsterdam (Netherlands), pp. 71–76. Teichberg, M., Fox, S.E., Olsen, Y.S., Valiela, I., Martinetto, P., Iribarne, O., Muto, E.Y., Petti, M.a.V., Corbisier, T.N., Soto-Jiménez, M., Páez-Osuna, F., Castro, P., Freitas, H., Zitelli, A., Cardinaletti, M., Tagliapietra, D., 2010. Eutrophication and macroalgal blooms in temperate and tropical coastal waters: nutrient enrichment experiments with Ulva spp. Glob. Chang. Biol. 16, 2624–2637. http://dx.doi.org/10.1111/j.13652486.2009.02108.x. Teichberg, M., Fricke, A., Bischof, K., 2013. Increased physiological performance of the calcifying green macroalga Halimeda opuntia in response to experimental nutrient enrichment on a Caribbean coral reef. Aquat. Bot. 104, 25–33. http://dx.doi.org/10. 1016/j.aquabot.2012.09.010. Thomas, M.K., Aranguren-Gassis, M., Kremer, C.T., Gould, M.R., Anderson, K., Klausmeier, C.A., Litchman, E., 2017. Temperature-nutrient interactions exacerbate sensitivity to warming in phytoplankton. Glob. Chang. Biol. 23, 3269–3280. http:// dx.doi.org/10.1111/gcb.13641. Totti, C., Accoroni, S., Cerino, F., Cucchiari, E., Romagnoli, T., 2010. Ostreopsis ovata bloom along the Conero Riviera (northern Adriatic Sea): relationships with environmental conditions and substrata. Harmful Algae 9, 233–239. http://dx.doi.org/ 10.1016/j.hal.2009.10.006. Utermöhl, H., 1931. Neue Wege in der Quantitativen Erfassung des Planktons. (Mit besondere Beriicksichtigung des Ultraplanktons). Verhandlungen Int. Ver. Limnol. 5, 567–596. Valiela, I., McClelland, J., Hauxwell, J., Behr, P.J., Hersh, D., Foreman, K., 1997. Macroalgal blooms in shallow estuaries: controls and ecophysiological and ecosystem consequences. Limnol. Oceanogr. 42, 1105–1118. http://dx.doi.org/10.4319/lo. 1997.42.5_part_2.1105. Vergés, A., Steinberg, P.D., Hay, M.E., Poore, A.G.B., Campbell, A.H., Ballesteros, E., Heck, K.L., Booth, D.J., Coleman, M.A., Feary, D.A., Figueira, W., Langlois, T., Marzinelli, E.M., Mizerek, T., Mumby, P.J., Nakamura, Y., Roughan, M., van Sebille, E., Gupta, A.S., Smale, D.A., Tomas, F., Wernberg, T., Wilson, S.K., 2014. The tropicalization of temperate marine ecosystems: climate-mediated changes in herbivory and community phase shifts. Proc. R. Soc. B 281, 20140846. http://dx.doi.org/10. 1098/rspb.2014.0846. Vidyarathna, N.K., Graneli, E., 2013. Physiological responses of Ostreopsis ovata to changes in N and P availability and temperature increase. Harmful Algae 21-22, 54–63. http://dx.doi.org/10.1016/j.hal.2012.11.006. Vila, M., Camp, J., Garcés, E., Masó, M., Delgado, M., 2001. High resolution spatiotemporal detection of potentially harmful dinoflagellates in confined waters of the NW Mediterranean. J. Plankton Res. 23, 497–514. http://dx.doi.org/10.1093/ plankt/23.5.497. Vila, M., Abós-Herràndiz, R., Isern-Fontanet, J., Àlvarez, J., Berdalet, E., 2016. Establishing the link between blooms and human health impacts using ecology and epidemiology. Sci. Mar. 80, 107–115. http://dx.doi.org/10.3989/scimar.04395.08A. Wells, M.L., Trainer, V.L., Smayda, T.J., Karlson, B.S.O., Trick, C.G., Kudela, R.M., Ishikawa, A., Bernard, S., Wulff, A., Anderson, D.M., Cochlan, W.P., 2015. Harmful algal blooms and climate change: learning from the past and present to forecast the future. Harmful Algae 49, 68–93. http://dx.doi.org/10.1016/j.hal.2015.07.009. Wernberg, T., Smale, D.A., Thomsen, M.S., 2012. A decade of climate change experiments on marine organisms: procedures, patterns and problems. Glob. Chang. Biol. 18, 1491–1498. http://dx.doi.org/10.1111/j.1365-2486.2012.02656.x. Xia, R., Zhang, Y., Critto, A., Wu, J., Fan, J., Zheng, Z., Zhang, Y., 2016. The potential impacts of climate change factors on freshwater eutrophication: implications for research and countermeasures of water management in China. Sustainability 8, 229. http://dx.doi.org/10.3390/su8030229. Yamaguchi, H., Tanimoto, Y., Yoshimatsu, T., Sato, S., Nishimura, T., Uehara, K., Adachi, M., 2012. Culture method and growth characteristics of marine benthic dinoflagellate Ostreopsis spp. isolated from Japanese coastal waters. Fish. Sci. 78, 993–1000. http:// dx.doi.org/10.1007/s12562-012-0530-4.

Pfannkuchen, M., Godrijan, J., Pfannkuchen, D.M., Ivesa, L., Kruzic, P., Ciminiello, P., Dell'Aversano, C., Dello Iacovo, E., Fattorusso, E., Forino, M., Tartaglione, L., Godrijan, M., 2012. Toxin-producing Ostreopsis cf. ovata are likely to bloom undetected along coastal areas. Environ. Sci. Technol. 46, 5574–5582. http://dx.doi. org/10.1021/es300189h. Pichierri, S., Pezzolesi, L., Vanucci, S., Totti, C., Pistocchi, R., 2016. Inhibitory effect of polyunsaturated aldehydes (PUAs) on the growth of the toxic benthic dinoflagellate Ostreopsis cf. ovata. Aquat. Toxicol. 179, 125–133. http://dx.doi.org/10.1016/j. aquatox.2016.08.018. Pichierri, S., Accoroni, S., Pezzolesi, L., Guerrini, F., Romagnoli, T., Pistocchi, R., Totti, C., 2017. Allelopathic effects of diatom filtrates on the toxic benthic dinoflagellate Ostreopsis cf. ovata. Mar. Environ. Res. 131, 116–122. http://dx.doi.org/10.1016/j. marenvres.2017.09.016. Pistocchi, R., Pezzolesi, L., Guerrini, F., Vanucci, S., Dell'Aversano, C., Fattorusso, E., 2011. A review on the effects of environmental conditions on growth and toxin production of Ostreopsis ovata. Toxicon 57, 421–428. http://dx.doi.org/10.1016/j. toxicon.2010.09.013. Poloczanska, E.S., Burrows, M.T., Brown, C.J., García Molinos, J., Halpern, B.S., HoeghGuldberg, O., Kappel, C.V., Moore, P.J., Richardson, A.J., Schoeman, D.S., Sydeman, W.J., 2016. Responses of marine organisms to climate change across oceans. Front. Mar. Sci. 3. http://dx.doi.org/10.3389/fmars.2016.00062. Rabalais, N.N., Turner, R.E., Díaz, R.J., Justić, D., 2009. Global change and eutrophication of coastal waters. ICES J. Mar. Sci. J. Cons. 66, 1528–1537. http://dx.doi.org/10. 1093/icesjms/fsp047. Raimbault, P., Pouvesle, W., Diaz, F., Garcia, N., Sempéré, R., 1999. Wet-oxidation and automated colorimetry for simultaneous determination of organic carbon, nitrogen and phosphorus dissolved in seawater. Mar. Chem. 66, 161–169. http://dx.doi.org/ 10.1016/S0304-4203(99)00038-9. Ras, M., Steyer, J.-P., Bernard, O., 2013. Temperature effect on microalgae: a crucial factor for outdoor production. Rev. Environ. Sci. Biotechnol. 12, 153–164. http://dx. doi.org/10.1007/s11157-013-9310-6. Raven, J.A., Geider, R.J., 1988. Temperature and Algal Growth - RAVEN - 1988 - New Phytologist - Wiley Online Library [WWW Document]. http://onlinelibrary.wiley. com/doi/10.1111/j.1469-8137.1988.tb00282.x/abstract (accessed 10.18.16). Rhodes, L., 2011. World-wide occurrence of the toxic dinoflagellate genus Ostreopsis Schmidt. Toxicon 57, 400–407. http://dx.doi.org/10.1016/j.toxicon.2010.05.010. Rigosi, A., Carey, C.C., Ibelings, B.W., Brookes, J.D., 2014. The interaction between climate warming and eutrophication to promote cyanobacteria is dependent on trophic state and varies among taxa. Limnol. Oceanogr. 59, 99–114. http://dx.doi.org/10. 4319/lo.2014.59.1.0099. Roleda, M.Y., Slocombe, S.P., Leakey, R.J.G., Day, J.G., Bell, E.M., Stanley, M.S., 2013. Effects of temperature and nutrient regimes on biomass and lipid production by six oleaginous microalgae in batch culture employing a two-phase cultivation strategy. Bioresour. Technol. 129, 439–449. http://dx.doi.org/10.1016/j.biortech.2012.11. 043. Sato, S., Nishimura, T., Uehara, K., Sakanari, H., Tawong, W., Hariganeya, N., Smith, K., Rhodes, L., Yasumoto, T., Taira, Y., Suda, S., Yamaguchi, H., Adachi, M., 2011. Phylogeography of Ostreopsis along West Pacific Coast, with Special Reference to a Novel Clade from Japan. PLoS One 6, e27983. http://dx.doi.org/10.1371/journal. pone.0027983. Scalco, E., Brunet, C., Marino, F., Rossi, R., Soprano, V., Zingone, A., Montresor, M., 2012. Growth and toxicity responses of Mediterranean Ostreopsis cf. ovata to seasonal irradiance and temperature conditions. Harmful Algae 17, 25–34. http://dx.doi.org/ 10.1016/j.hal.2012.02.008. Selina, M.S., Morozova, T.V., Vyshkvartsev, D.I., Orlova, T.Y., 2014. Seasonal dynamics and spatial distribution of epiphytic dinoflagellates in Peter the Great Bay (Sea of Japan) with special emphasis on Ostreopsis species. Harmful Algae 32, 1–10. http:// dx.doi.org/10.1016/j.hal.2013.11.005. Shears, N.T., Ross, P.M., 2009. Blooms of benthic dinoflagellates of the genus Ostreopsis; an increasing and ecologically important phenomenon on temperate reefs in New Zealand and worldwide. Harmful Algae 8, 916–925. http://dx.doi.org/10.1016/j.hal. 2009.05.003. Shears, N.T., Ross, P.M., 2010. Toxic cascades: multiple anthropogenic stressors have complex and unanticipated interactive effects on temperate reefs. Ecol. Lett. 13, 1149–1159. http://dx.doi.org/10.1111/j.1461-0248.2010.01512.x. Skinner, M.P., Lewis, R.J., Morton, S., 2013. Ecology of the ciguatera causing dinoflagellates from the Northern Great Barrier Reef: changes in community distribution and coastal eutrophication. Mar. Pollut. Bull. 77, 210–219. http://dx.doi.org/10. 1016/j.marpolbul.2013.10.003. Smith, S.M., Fox, R.J., Donelson, J.M., Head, M.L., Booth, D.J., 2016. Predicting rangeshift success potential for tropical marine fishes using external morphology. Biol. Lett. 12. http://dx.doi.org/10.1098/rsbl.2016.0505. Sterner, R.W., Grover, J.P., 1998. Algal growth in warm temperate reservoirs: kinetic

564