Over the last few decades, the incidence of toxic phytoplankton blooms has apparently been increasing in coastal waters worldwide (GEOHAB 2012). Toxic blooms cause negative impacts and economic losses in many parts of the world and their consequences and mechanisms depend on the species involved. Broadly, there are four categories of deleterious effects: risks to human health; loss of natural or cultured seafood resources; impairment of tourism and recreational activities; and damage to non-commercial marine resources and wildlife (GEOHAB 2001, Garcés and Camp 2011, Anderson et al. 2012). Several taxonomic groups of microalgae are known as harmful marine species: dinoflagellates, diatoms, raphidophyceans, cyanophytes and haptophytes (Moestrup 1994).

Among the genera responsible for harmful algal blooms (HABs), the genus Alexandrium is one of the most important, in terms of severity, diversity and distribution of bloom impacts (Anderson et al. 2012). Alexandrium minutum Halim is well known for the production of paralytic shellfish poisoning toxins and its presence can affect copepods, shellfish, and other marine organisms (Zhenxing et al. 2006). A. minutum was first described in the Mediterranean Sea forming a red water discoloration in Alexandria Harbour, Egypt (Halim 1960); it has since then been reported widely on the Mediterranean and Atlantic Spanish coast (Delgado et al. 1990, Franco et al. 1994, Frangópulos et al. 2000, Figueroa et al. 2007, Penna et al. 2008 and ICES 2011).

The haptophyte alga Prymnesium parvum N. Carter has been responsible for toxic events, with severe ecological impacts, in many parts of the world (Moestrup 1994). This organism produces a set of highly potent exotoxins, commonly called prymensins, that have been shown to have several biological effects, including ichthyotoxic, neurotoxic, cytotoxic, hepatotoxic and hemolytic activity towards a range of marine organisms (Igarashi et al. 1996, Manning and La Claire 2010). P. parvum was described for the first time in England (Carter 1937) and has been responsible for massive fish mortalities around the world (Granéli and Turner 2006, Johnsen et al. 2010).

Because of these deleterious effects, several methods for determining the toxicity of marine microalgae have been used to date. The most common is the mouse bioassay developed by the Japanese Ministry of Health and Welfare (Yasumoto et al. 1978). This method is characterized by being accurate and of short-term response, but the controversy in the use of mammals for bioassays has led to the search for and practice of new alternative bioassays (FAO 2005). In this research field, phytoplankton toxicity has been tested in marine organisms, including fish and invertebrates; the tests include mortality tests with crustaceans of the genus Artemia (Houdan et al. 2004, Zhenxing et al. 2006, Faimali et al. 2012). Moreover, toxicity of extracts from different diatoms has been evaluated in sea urchin embryos (Buttino et al. 1999, Hansen et al. 2003). It should be noted that the toxic capacity of some toxic microalgae is still unknown and that toxicity induction depending on the environmental conditions, mainly nutrient availability, has led to some controversy (Granéli and Johanson 2003, Remmel et al. 2011).

Due to the high toxicity reported for A. minutum and other dinoflagellates, its toxicity level should be fully evaluated to prevent the risk of bloom episodes in coastal areas. In addition, the involvement of the genus Prymnesium in fish mortalities makes its toxicity evaluation important in order to ensure the good quality of areas with fish aquaculture and shellfish exploitation.

Toxicity may be variable between strains of potentially toxic species (e.g. Ichimi et al. 2002, Montoya et al. 2010, Varela et al. 2012). It is therefore necessary to evaluate the toxicity of local strains of known potentially toxic species, such as Alexandrium minutum and Prymnesium parvum.

In the present study, bioassays with three different marine invertebrates (with different levels of biological organization) were applied as a detection tool to assess the toxic capacity of these marine microalgae species. A set of ecotoxicological tests was carried out to investigate the effects of A. minutum and P. parvum using different treatments on larvae of the sea urchin Paracentrotus lividus (Lamarck, 1816), the branchiopod Artemia franciscana (Leach, 1819) and adults of the amphipod Corophium multisetosum (Stock, 1952).

The branchiopod A. franciscana is used routinely as a test organism for screening in ecotoxicological studies (Sorgeloos et al. 1978). Its life cycle begins with the hatching of dormant cysts that, once rehydrated in salt water, start the development as nauplii larvae, which are the material for the bioassays. These crustaceans are characterized by their adaptability to a wide range of salinity and temperature conditions: their short life cycle; their high adaptability to adverse environmental conditions; their high fecundity; their bisexual/parthenogenetic reproduction strategy (with nauplii or cysts production); their small body size; and their adaptability to variable food resources. They are also non-selective filter feeders sensitive to toxic substances (Sarabia 2002, Faimali et al. 2012).

The sea urchin Paracentrotus lividus is also used widely for testing particular contaminants (Fernández 2002) and determining phytotoxicity (Buttino et al. 1999, Hansen et al. 2003, Privitera et al. 2012). P. lividus has a wide geographic distribution, is abundant, and is easily harvested and maintained in the laboratory (Garmendia et al. 2009). Moreover, obtaining gametes and in vitro fecundation do not involve difficulties, while the embryonic development is short, thus allowing viable larvae to be obtained in a short time (Garmendia et al. 2009).

Marine and estuarine amphipods are regarded as one of the most suitable model species for integrating the effects of multiple toxic compounds (USEPA 2001, OSPAR Commision 2005). However, C. multisetosum has not been used previously for determining microalgae toxicity.

The bioassays were applied with the aim of (i) evaluating the sensitivity of the abovementioned species to the toxicity of A. minutum and P. parvum in the presence and absence of cells; and (ii) comparing the sensitivity of the three organisms to the toxicity of the mentioned phytoplankton species.

The abiotic parameters in all microalgae culture flasks (oxygen, salinity and temperature) are reported in Table 2. The LC₁₀ and LC₅₀ values for SDS in P. lividus and the LC₅₀ value for NH₄⁺ in Corophium are reported in Table 3.

The LC₅₀-SDS for P. lividus in the initial test was 4.05 mg L^–1; it was 2.65 mg L^–1 in the definitive test. Furthermore, NOEC and LOEC values for the initial test were 2.1 and 3.15 mg L^–1, respectively. LOEC for the definitive test was 2.03 mg L^–1 (Table 3). The LC₅₀- NH₄⁺ for C. multisetosum was 25.47 mg L^–1. Moreover, NOEC and LOEC values were 8 and 16 mg L^–1, respectively.

Lethal concentrations (mg L^–1) for SDS in P. lividus and for NH₄⁺ in C. multisetosum, as calculated in this study, lie within the range of those obtained previously by Menchaca (2010) and Pérez (2006), respectively (Table 3). Our results have therefore confirmed the good quality of the model organisms (differences in the quality of gonad material were observed in the two different periods where sea urchins were collected, as the LC₅₀ value for SDS in P. lividus obtained in May (the initial test) was higher than the value obtained in June (the definitive test). This observation is in accordance with Garmendia et al. (2010), who also detected variability in the quality of the gonad material throughout the year. Therefore, this assumption must be considered when interpreting the differences found in results of the two sets of trials performed with P. lividus exposed to A. minutum.

The dinoflagellate Alexandrium minutum was toxic for A. franciscana nauplii at 5000 cell mL^–1, while the LC₅₀ value increased with exposure time. The 48-hour exposure proved to be much more harmful, indicating that both concentration and exposure time contributed to the toxic response in the nauplii population. The toxic effect of A. minutum on A. franciscana nauplii survival has rarely been examined. Our results agree with those of Zhenxing et al. (2006), who found that four strains of Alexandrium spp. were lethal for A. salina at a density of 2000 cell mL^–1. Regarding the filtrate of A. minutum, the maximum concentration (100000 cell mL^–1) showed no toxic effect in the A. franciscana nauplii, which is explained by the fact that their toxicity is linked to the intact cell, as has been reported for Ostreopsis cf. ovata (Faimali et al. 2012)

In contrast, A. franciscana nauplii survival was not affected by the presence of P. parvum culture or filtrate at the maximum exposure concentration (250000 cell mL^–1). These results contrast with those of Houdan et al. (2004), who found an LC₅₀ value of a P. parvum culture (in the stationary phase) lower than 100000 cell mL^–1 for A. salina. Some authors have suggested that this sensitivity disagreement could be related to differences in microalgae culture conditions (Granéli and Johanson 2003). According to the aforementioned authors, excretion of toxic metabolites might be a general mechanism among toxic haptophytes when exposed to stress events, e.g. nutrient-deficient conditions. However, recent studies hypothesize that the majority of Prymnesium toxicity is cellular-based rather than related to exotoxins production (Remmel et al. 2011). Further, reduction of A. franciscana movement with exposure time, as shown in the present study at the maximum exposure concentration of P. parvum, is in accordance with the results of Remmel and Hambright (2012). These investigators observed that Prymensium cells were attached to most exterior surfaces, to fish gills and to feeding and respiratory appendages in zooplankton, which contributed to the movement depletion of individuals. Moreover, they found that the number of attached cells increased with exposure time and density of Prymnesium.

The toxicity of A. minutum and P. parvum had not been evaluated previously using sea urchin embryo bioassays. However, a dose-dependent toxic effect of several marine microalgae on sea urchin cell division has been recorded in several studies (Paul and Fenical 1984, Lemée et al. 1993, Pedrotti et al. 1996, Buttino et al. 1999). In the present study, A. minutum was toxic to sea urchin embryos at 5000 cell mL^–1 (LOEC value). These results are in accordance with those of Privitera et al. (2012), who demonstrated the toxic effect in competent sea urchin larvae when exposed to the dinoflagellate Ostreopsis cf. ovata. On the other hand, filtrate of the dinoflagellate showed no toxic effect on the development of the sea urchin. This scenario is also in agreement with Faimali et al. (2012), who demonstrated that dinoflagellates do not show significant toxicity with growth medium devoid of cells.

By contrast, the culture and filtrate of P. parvum significantly inhibited embryo development at 200000 and 300000 cell mL^–1. These effective concentrations are within the range of those calculated by Hansen et al. (2003), who reported a cell division reduction of newly-fertilizing eggs of the sea urchin Sphaerechinus granularis exposed to a haptophyte. However, Remmel and Hambright (2012) suggested that the presence of toxins in the filtrate could be an artefact caused by the vacuum pressure used in the filtration process, which can cause cell disruption and the subsequent release of intracellular toxins. The result of the filtrate reported in the present study should therefore be considered carefully.

In the present study, adult amphipods were not sensitive to the toxicity of A. minutum and P. parvum at the concentrations tested. This finding contrasts with those of other authors who have routinely used adult zooplankton organisms (Euterpina acutifrons and Acartia grani) to evaluate the toxicity of A. minutum (Frangópulos et al. 2000, Costa and Fernández 2002). However, Romano et al. (2003) observed that secondary metabolites, such as unsaturated aldehydes produced by some algae during stress events, such as depredation (Pohnert 2000), were not toxic in adult copepods, while they inhibited the development of oocytes and embryos. Romano et al. (2003) hypothesized that this behaviour could be a defence mechanism of microalgae, designed to regulate the populations of herbivorous zooplankton.

Assuming similar test conditions (photoperiod, temperature, salinity and oxygen) for all the bioassays performed, among the tested organisms A. franciscana nauplii showed the highest sensitivity for Alexandrium minutum according to LC₅₀-48h. These results are in agreement with those of Faimali et al. (2012) and Privitera et al. (2012), who suggested that the development stages could affect the sensitivity of the organisms, increasing the mortality with the ingestion rate. Moreover, Zhenxing et al. (2006) confirmed a decrease in the survival of Artemia salina nauplii with the feeding rates of Alexandrium spp. However, in the present study, owing to logistic difficulties, the presence of cell content inside the alimentary canal of A. franciscana could not be identified. As a result, it cannot be concluded that the higher sensitivity of A. franciscana nauplii is related to the ingestion of the microalgae. In the case of P. parvum, difficulties in the LC₅₀ calculation did not permit comparison of the sensitivity between the tested organisms.

The presence of toxic microalgae on the coasts of the Basque Country is monitored every year, within the context of the EU Water Framework Directive (WFD) (e.g. Borja et al. 2009). In 2008 the cell density of Alexandrium minutum was lower than 100 cell L^–1, much less than the value considered as a limit for toxicity risk found in the present study. Prymnesium parvum has not been reported specifically in the WFD reports. The inverted light-microscope technique based upon fixed samples used in this monitoring programme for phytoplankton identification and counting does not usually allow species or even genus-level identification, of small naked flagellates such us Prymnesium spp. The species level identification of prymnesiophyceans requires the analysis of the body scales through electron microscopy (Seoane et al. 2009) or molecular methods.

Regarding toxicity of Alexandrium minutum, it must be considered that the long culture maintenance of the dinoflagellate in the present work may have led to the decrease in toxin production. Martins et al. (2004) demonstrated that saxitoxin production is not a constitutive, stable characteristic of Alexandrium, but can be lost during routine culture maintenance over a prolonged interval.

Only larvae of Paracentrotus lividus and Artemia franciscana have been demonstrated to be sensitive to the toxicity of the two marine microalgae Alexandrium minutum and Prymnesium parvum. The algae strains tested were isolated much in advance compared with the performed test. Therefore, the minimum toxic concentration values established in the present study for the model organisms cannot be considered as a real toxic value. We suggest performing the bioassays of the present study with the model organisms, using recent isolated A. minutum or P. parvum during toxic blooms and combining different parameters (nutrients, light and temperature) to study the potential toxicity in different conditions. Nevertheless, our results place P. lividus and A. franciscana bioassays as a viable alternative for the detection of A. minutum and P. parvum toxicity in the context of routine monitoring. Other standardized toxicological assays using more distant taxonomic groups are also available and have been applied extensively (Ribo and Kaiser 1987, Zon and Peterson 2005). Therefore, besides working on the use of the bioassays described in the present work, we also suggest the use of alternative methodologies in order to explore a wider range of tests for the detection of toxic events.