Francesca Garaventa

Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms

Harmful benthic microalgae blooms are an emerging phenomenon causing health and economic concern, especially in tourist areas. This is the case of the Mediterranean Sea, where Ostreopsis ovata blooms occur in summer, with increasing regularity. Ostreopsis species produce palytoxin (PTX) and analogues, and a number of deaths directly associated with the ingestion of PTX contaminated seafood have been reported. PTX is considered one of the most toxic molecules occurring in nature and can provoke severe and sometimes lethal intoxications in humans. So far in temperate areas, O. ovata blooms were reported to cause intoxications of humans by inhalation and irritations by contact. In addition, invertebrate mass mortalities have been reported, possibly linked to O. ovata blooms, although other causes cannot be ruled out, such as oxygen depletion or high seawater temperature. In order to improve our knowledge about the direct toxicity of this species on invertebrate and vertebrate marine organisms, we performed an ecotoxicological screening to investigate the toxic effects of different concentrations of O. ovata (cultured in the laboratory and sampled in the field during blooms) on crustaceans and fish as model organisms. Artemia salina, Tigriopus fulvus, and Amphibalanus amphitrite larvae and juveniles of the sea bass Dicentrarchus labrax were used as model species. Toxic effects associated with cultured O. ovata cells were investigated using a crossed design: testing two different temperatures (20 and 25 °C), four different cell concentrations, and four treatments (untreated O. ovata culture, filtered and resuspended algal cells, growth medium devoid of algal cells, and sonicated algal cells). The results indicate that the toxicity of cultured O. ovata is related to the presence of living O. ovata cells, and that this effect is amplified by temperature. Furthermore, both tests with laboratory cultured algae and field sampled cells pointed out that A. salina is the most sensitive species even at concentrations below the Environmental Alarm Threshold set by the Italian Ministry of Health. Some possible explanations of such sensitivity are discussed, taking into account evidence of O. ovata cells ingestion and the activity of its toxins on the Na⁺/K⁺-ATPase.

Effect of silver nanoparticles on marine organisms belonging to different trophic levels.

Silver nanoparticles (Ag-NPs) are increasingly used in a wide range of consumer products and such an extensive use raises questions about their safety and environmental toxicity. We investigated the potential toxicity of Ag-NPs in the marine ecosystem by analyzing the effects on several organisms belonging to different trophic levels. Algae (Dunaliella tertiolecta, Skeletonema costatum), cnidaria (Aurelia aurita jellyfish), crustaceans (Amphibalanus amphitrite and Artemia salina) and echinoderms (Paracentrotus lividus) were exposed to Ag-NPs and different end-points were evaluated: algal growth, ephyra jellyfish immobilization and frequency of pulsations, crustaceans mortality and swimming behavior, and sea urchin sperm motility. Results showed that all the end-points were able to underline a dose-dependent effect. Jellyfish were the most sensitive species, followed by barnacles, sea urchins, green algae, diatoms and brine shrimps. In conclusion, Ag-NPs exposure can influence different trophic levels within the marine ecosystem.

Imposex in Hexaplex trunculus at some sites on the North Mediterranean Coast as a base-line for future evaluation of the effectiveness of the total ban on organotin based antifouling paints

Imposex — the superimposition of male sexual organs (penis and vas deferens) onto female Neogastropods such as Hexaplex trunculus (Linne , 1758) — is used world-wide as a biomarker of ecological impact of organotin based antifouling biocides (TBT and TPhT). To limit the impact of organotin pollution, since January 1, 2003, the International Maritime Organization (IMO) has enacted a global ban on the use of organotin compounds in antifouling systems. It is important to record imposex levels and organotin contamination before the implementation of the ban, in order to assess the current situation and be able, in the future, to verify the effects of the International Protocol. In this paper, recent imposex data measured in populations of Hexaplex trunculus from three different Mediterranean regions are compared: the Ligurian Sea (Italy), the Lagoon of Venice (Italy) and the western coast of Istria (Croatia). In the two former locations, a partial ban on TBT has been in force for vessels less than 25 m since 1982, while in the latter region no restrictions on organotin antifouling paints have been applied yet. Gastropod samples collected from the Venice lagoon were analysed with an acid extraction followed by Grignard derivatisation, clean up and GC-MS determination, in order to relate the levels of TBT, TPhT and their metabolites with the imposex degree detected. Biological data show that the levels of imposex were very high (VDS from 4.3 to 5) in all the sampling sites considered, particularly in the Croatian coast stations. The concentrations of organotin compounds — butyltins and phenyltins — measured in the samples from the lagoon of Venice were found to partition differently in the visceral coil and in the rest of the soft body of the analysed organisms.

Antisettlement activity of synthetic analogues of polymeric 3-alkylpyridinium salts isolated from the sponge Reniera sarai

Based on the high, non-toxic and reversible antifouling activity of the polymeric 3-alkylpyridinium salts isolated from the sponge Reniera sarai, the anti-settlement activity and toxicity of a series of synthetic analogues has been studied. All the test compounds were less efficient than the natural polymers, suggesting that the high and reversible anti-macrofouling activity of the natural polymers could derive from their detergent-like properties. The values obtained for EC50sett. of inhibition of cyprid settlement and EC50imm. as naupliar toxicity for the synthetic compounds indicate that the presence of single or multiple charges in the structure is not relevant for the antifouling activity which, conversely, is favoured by increasing the length of the alkyl chain, or by the presence of uncharged pyridine units. The compound 1,8-di(3-pyridyl)octane was the most efficient (EC50sett.= 0.44 μg ml–1), although with a higher toxicity on naupliar stage of B. amphitrite than the natural polymers.

New implications in the use of imposex as a suitable tool for tributyltin contamination: experimental induction in Hexaplex trunculus (Gastropoda, Muricidae) with different stressors

Imposex, i.e. the development of additional male sex organs (penis and/or vas deferens), in females of gonochorist marine and freshwater gastropods, is known to be caused by tributyltin (TBT), and it has been widely used as a biomonitoring tool in environmental surveys for TBT pollution assessment. In this study, we experimentally tested the potential to induce imposex by another endocrine disruptor (polychlorinated biphenyls [PCBs] mixture—Aroclor 1260). Adults of Hexaplex trunculus with low imposex level, coming from an Italian Marine Protected Area, were injected separately with different doses of tributyltin chloride (TBTCl) and Aroclor 1260. The compounds were dissolved in ethanol and the organisms were narcotised by immersion in MgCl2 solution before injection. Before and after the experiment, butyltin compounds (BuTs) and PCB tissue concentrations were determined. A significant increase in imposex with respect to non-treated organisms was observed in all treatments, including artefact controls. No clear correlation was observed between BuTs and PCB tissue concentrations and indices of imposex incidence. Based on these results, no assumption can be formulated about PCB effect on imposex development. Nevertheless, they suggest that the imposex level increase, at least in H. trunculus, in laboratory conditions might not be caused by TBT only, but it would rather be a non-specific response to different stress stimuli.

Standardization of laboratory bioassays with Balanus amphitrite larvae for preliminary oil dispersants toxicological characterization

The Italian National regulations on oil-dispersants use (D.D. 23 December 2002) require for these products to pass several laboratory screenings before they can be applied in oil-spill clean-up. Although legislation recommend the use of the American mysid shrimp Americamysis bahia, for laboratory toxicity testing, there is growing interest in employing local marine crustacean species more representative than A. bahia, in quantifying the risk of significant harm to Mediterranean ecosystems. The aim of this study (in the framework of the National Project ‘Taxa Project’, supported by the Italian Ministry for the Environment and Territory) is to improve new specific bioassays for assessing acute or sublethal responses to oil dispersants using the larval stages of the sessile crustacean Balanus amphitrite. The bioassays were standardized using sodium dodecyl sulphate (SDS) as toxic reference compound. Results of acute toxicity (48 h LC50, 7.49 mg l−1) and behavioural tests (7 d EC50, 7.79 mg l−1) with barnacle larvae showed that their susceptibility to SDS could be comparable with that of A. bahia (96 h LC50; 6.6 mg l−1). Therefore, a B. amphitrite bioassay could be proposed to replace the A. bahia bioassay in a standardized toxicological screening of new products for oil-pollution remediation technologies in the Mediterranean Sea.

Old model organisms and new behavioral end-points: Swimming alteration as an ecotoxicological response

Behavioral responses of aquatic organisms have received much less attention than developmental or reproductive ones due to the scarce presence of user-friendly tools for their acquisition. The technological development of data acquisition systems for quantifying behavior in the aquatic environment and the increase of studies on the understanding the relationship between the behavior of aquatic organisms and the physiological/ecological activities have generated renewed interest in using behavioral responses also in marine ecotoxicology. Recent reviews on freshwater environment show that behavioral end-points are comparatively fast and sensitive, and warrant further attention as tools for assessing the toxicological effects of environmental contaminants. In this mini-review, we perform a systematic analysis of the most recent works that have used marine invertebrate swimming alteration as behavioral end-point in ecotoxicological studies by assessing the differences between behavioral and acute responses in a wide range of species, in order to compare their sensitivity.

Antifouling Activity of Synthetic Alkylpyridinium Polymers Using the Barnacle Model

Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.

Effects of polystyrene microbeads in marine planktonic crustaceans

Plastic debris accumulates in the marine environment, fragmenting into microplastics (MP), causing concern about their potential toxic effects when ingested by marine organisms. The aim of this study was to verify whether 0.1µm polystyrene beads are likely to trigger lethal and sub-lethal responses in marine planktonic crustaceans. MP build-up, mortality, swimming speed alteration and enzyme activity (cholinesterases, catalase) were investigated in the larval stages of Amphibalanus amphitrite barnacle and of Artemia franciscana brine shrimp exposed to a wide range of MP concentrations (from 0.001 to 10mgL-1) for 24 and 48h. The results show that MP were accumulated in crustaceans, without affecting mortality. Swimming activity was significantly altered in crustaceans exposed to high MP concentrations (> 1mgL-1) after 48h. Enzyme activities were significantly affected in all organisms exposed to all the above MP concentrations, indicating that neurotoxic effects and oxidative stress were induced after MP treatment. These findings provide new insight into sub-lethal MP effects on marine crustaceans.

Ecotoxicological effects of sediments from Mar Piccolo, South Italy: toxicity testing with organisms from different trophic levels

The Mar Piccolo of Taranto (Ionian Sea, Southern Italy) is a semi-enclosed and strongly polluted basin. For decades, it has been subjected to different anthropogenic impacts. These stressors caused severe sediments contamination with high concentration of different pollutants (PAHs, PCB, heavy metals). In order to assess the current status of sediments contamination, an ecotoxicological investigation combined with chemical analysis (heavy metals, PAH, and PCB) has been performed. In order to derive ecologically relevant conclusions, a multiorganisms and multiend-points approach has been applied, exposing organisms from different trophic levels to elutriate and whole sediment. The battery of bioassays consists of a microalgal growth inhibition test (Dunaliella tertiolecta), acute and sublethal assays (end-points: mortality, immobilization and swimming speed alteration) on crustaceans larvae and juveniles, and rotifers (Amphibalanus amphitrite, Artemia salina, Corophium insidiosum and Brachionus plicatilis), and embryotoxicity test on echinoderms (Paracentrotus lividus). Considering the high levels of sediment contamination highlighted from chemical analysis, an unexpected very low toxic effect was observed, even considering the sublethal end-point (larval swimming speed alteration). The results of this study suggest a very complex contaminants dynamic in the Mar Piccolo sediments that, despite a strong level of contamination, seems to not affect in a proportional manner the biological compartment.