Paracentrotus Lividus Embryos Research Articles

Marine degradation and ecotoxicity of conventional, recycled and compostable plastic bags

Plastic bags are currently a major component of marine litter, causing aesthetical nuisance, and undesirable effects on marine fauna that ingest them or are entangled. Plastic litter also rises concern on the ecotoxicological effects due to the potential toxicity of the chemical additives leached in aquatic environments. Conventional plastic bags are made of polyethylene, either from first use or recycled, but regulations restricting single-use plastics and limiting lightweight carrier bags (<50 μm thickness) have fostered the replacement of thin PE bags by compostable materials advertised as safer for the environment. In this study, we assess the degradation of commercially available plastic bags in marine conditions at two scales: aquariums (60 days) and outdoors flow-through mesocosm (120 days). Strength at break point and other tensile strength parameters were used as ecologically relevant endpoints to track mechanical degradation. Ecotoxicity has been assessed along the incubation period using the sensitive Paracentrotus lividus embryo test. Whereas PE bags did not substantially lose their mechanical properties within the 60 d aquarium exposures, compostable bags showed remarkable weight loss and tensile strength decay, some of them fragmenting in the aquarium after 3–4 weeks. Sediment pore water inoculum promoted a more rapid degradation of compostable bags, while nutrient addition pattern did not affect the degradation rate. Longer-term mesocosms exposures supported these findings, as well as pointed out the influence of the microbial processes on the degradation efficiency of compostable/bioplastic bags. Compostable materials, in contrast toPE, showed moderate toxicity on sea-urchin larvae, partially associated to degradation of these materials, but the environmental implications of these findings remain to be assessed. These methods proved to be useful to classify plastic materials, according to their degradability in marine conditions, in a remarkably shorter time than current standard tests and promote new materials safer for the marine fauna.

Gene Expression Analysis of the Stress Response to Lithium, Nickel, and Zinc in Paracentrotus lividus Embryos.

Many anthropogenic pollutants such as metals are discharged into the marine environment through modern sources. Among these, lithium (Li), nickel (Ni), and zinc (Zn) can interfere with biological processes in many organisms when their concentration rises. These metals are toxic to sea urchin embryos, affecting their development. Indeed, animal/vegetal and dorso/ventral embryonic axes are differently perturbed: Li is a vegetalizing agent, Ni can disrupt dorso-ventral axis, Zn can be animalizing. To address the molecular response adopted by embryos to cope with these metals or involved in the gene networks regulating embryogenesis, and to detect new biomarkers for evaluating hazards in polluted environments in a well-known in vivo model, we applied a high-throughput screening approach to sea urchin embryos. After fertilization, Paracentrotus lividus embryos were exposed to Li, Ni, and Zn for 24/48 h. At both endpoints, RNAs were analyzed by NanoString nCounter technology. By in silico analyses, we selected a panel of 127 transcripts encoding for regulatory and structural proteins, ranked in categories: Apoptosis, Defense, Immune, Nervous, Development, and Biomineralization. The data analysis highlighted the dysregulation of many genes in a metal-dependent manner. A functional annotation analysis was performed by the KEEG Orthology database. This study provides a platform for research on metals biomarkers in sea urchins.

Vanadium Toxicity Monitored by Fertilization Outcomes and Metal Related Proteolytic Activities in Paracentrotus lividus Embryos.

Metal pharmaceutical residues often represent emerging toxic pollutants of the aquatic environment, as wastewater treatment plants do not sufficiently remove these compounds. Recently, vanadium (V) derivatives have been considered as potential therapeutic factors in several diseases, however, only limited information is available about their impact on aquatic environments. This study used sea urchin embryos (Paracentrotus lividus) to test V toxicity, as it is known they are sensitive to V doses from environmentally relevant to very cytotoxic levels (50 nM; 100 nM; 500 nM; 1 µM; 50 µM; 100 µM; 500 µM; and 1 mM). We used two approaches: The fertilization test (FT) and a protease detection assay after 36 h of exposure. V affected the fertilization percentage and increased morphological abnormalities of both egg and fertilization envelope, in a dose-dependent manner. Moreover, a total of nine gelatinases (with apparent molecular masses ranging from 309 to 22 kDa) were detected, and their proteolytic activity depended on the V concentration. Biochemical characterization shows that some of them could be aspartate proteases, whereas substrate specificity and the Ca2+/Zn2+ requirement suggest that others are similar to mammalian matrix metalloproteinases (MMPs).

PAHs and PCBs Affect Functionally Intercorrelated Genes in the Sea Urchin Paracentrotus lividus Embryos.

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) represent the most common pollutants in the marine sediments. Previous investigations demonstrated short-term sublethal effects of sediments polluted with both contaminants on the sea urchin Paracentrotus lividus after 2 months of exposure in mesocosms. In particular, morphological malformations observed in P. lividus embryos deriving from adults exposed to PAHs and PCBs were explained at molecular levels by de novo transcriptome assembly and real-time qPCR, leading to the identification of several differentially expressed genes involved in key physiological processes. Here, we extensively explored the genes involved in the response of the sea urchin P. lividus to PAHs and PCBs. Firstly, 25 new genes were identified and interactomic analysis revealed that they were functionally connected among them and to several genes previously defined as molecular targets of response to the two pollutants under analysis. The expression levels of these 25 genes were followed by Real Time qPCR, showing that almost all genes analyzed were affected by PAHs and PCBs. These findings represent an important further step in defining the impacts of slight concentrations of such contaminants on sea urchins and, more in general, on marine biota, increasing our knowledge of molecular targets involved in responses to environmental stressors.

Towards standard methods for the classification of aquatic toxicity for biologically active household chemicals (BAHC) present in plastics, pharmaceuticals, and cosmetic products

A standard method to test the aquatic toxicity of biologically active household chemicals (BAHC), including those with very low water solubility, is proposed. The method uses the common marine models Paracentrotus lividus embryos and Acartia clausi larvae, in order to advance towards derivation of water quality criteria for these emerging pollutants that currently lack environmental standards. Depending on the water solubility and octanol–water partition coefficient (Kow) of the substance, the protocol consists of testing the toxicity of the substances by serial dilutions of water stocks, dimethyl-sulfoxide stocks, or 100 mg/L lixiviates in seawater. When this method is applied to eleven model BAHC, the pharmaceutical fluoxetine, the antioxidant butylated hydroxytoluene, and the UV filters broadly present in cosmetics octocrylene and 4-methylbenzylidene camphor, are classified as very toxic to aquatic life, since their EC50 values are < 1 mg/L. In general, both biological models, P. lividus and A. clausi, yield the same classification of the substances tested, but variations in the classification of aquatic toxicity depending on methodological aspects are discussed. The use of A. clausi nauplii provides more protecting value to the toxicity parameters obtained by using this protocol.Graphic abstract

Elutriate preparation affects embryo development test with Paracentrotus lividus: An in-depth study on the differences between two protocols and three different sediment/water mixing times

Coastal areas are under continuous and increasing pressure from different human activities. A mixture of contaminants (e.g. hydrocarbons, pesticides, persistent organic pollutants (POPs), emerging contaminants, and others), originating mainly from populated, industrialised and agricultural areas, can reach the marine environment through different means such as wastewater discharge, soil runoffs, leaching from agriculture, and volatilisation/deposition. In this context, marine sediments have increasingly been considered repositories for a variety of pollutants that can accumulate and be stored for long periods, acting as a secondary source of contaminants during subsequent dredging operation or vessel manoeuvring. Chemical and ecotoxicological analyses of sediments are routinely conducted to evaluate the potential hazard/risk to the environment, either on bulk sediment or elutriate. In general, sediment elutriates are commonly prepared according to ASTM Guide even if alternative protocols are proposed by USACE for the various condition that they have to represent. The goal of the present study was to determine if the toxicological properties of ASTMprepared elutriates are comparable to those obtained from the USACE protocol. Sediment coming from 3 harbours (Olbia, Cagliari, and Toulon), as part of the “Se.D.Ri.Port” Interreg Project, were processed to obtain elutriates according to ASTM Guide and USACE Dredging Elutriate protocol and tested with the sea urchin Paracentrotus lividus embryo development test. Moreover, the significance of different stirring times of water/sediment mixture (1 h, 3 h, and 24 h) was tested with both the ASTM and USACE protocol. In addition to the biological analysis, for each sediment sample, heavy metals concentration, granulometry, and organic matter were determined. Even if for the ports of Toulon and Cagliari, the ASTM and USACE elutriates showed comparable results with P. lividus bioassay, for the port of Olbia the two protocols showed different criticalities. Preliminary results show that for the site Olbia elutriates prepared with the USACE protocol resulted in higher toxicity than elutriates obtained with ASTM (p < 0.001). In conclusion, differences in preparation protocols appear to be significant and can lead to different results in biological testing. To overcome this problem and to obtain more reliable evaluations of risk to the environment, standardisation and regulation must be the next goals in sediment management procedure.

A preliminary gene expression analysis on Paracentrotus lividus embryos exposed to UVB, Cadmium and their combination

Paracentrotus lividus is a Mediterranean and Eastern Atlantic sea urchin species, very sensitive to chemical and physical environmental changes and widely used in eco-toxicological studies. Here, we applied a high throughput screening approach on P. lividus embryos exposed to UVB radiation (UV), Cadmium Chloride (Cd) and their combination (Cd/UV), to deeply characterize the molecular responses adopted by embryos to cope with these stressors. in vitro eco-toxicological assays were performed by exposing embryos to Cd (10−4 M) soon after fertilization, to UV (200 and 400J/m2) at early stage of development, while in co-exposure experiments, Cd-exposed embryos were irradiated with UV at 200 J/m2. By NanoString nCounter technology, custom-made probes were developed and hybridized on total RNA extracted from exposed embryos at 51h after fertilization. By in silico analyses, we selected and retrieved at the NCBI nucleotide database a panel of P. lividus transcripts encoding for many regulatory and structural proteins that we ranked in categories, i.e., Apoptosis, Biomineralization, Defense, Development, Immunity, Signaling and Transcription Factors. The analysis of 127 transcripts highlighted the dysregulation of many genes, some specifically activated to cope with stress agents, others involved in the complex molecular network of genes that regulate embryo development. We revealed the downregulation of Biomineralization and Development genes and the upregulation of Defensive genes in Cd and Cd/UV embryos. Our approach, using sea urchin embryo as an in vivomodel, contributes to advance our knowledge about cellular responses to UV, Cd and their combination.

Diatom-Derived Polyunsaturated Aldehydes Activate Similar Cell Death Genes in Two Different Systems: Sea Urchin Embryos and Human Cells

Programmed cell death, such as apoptosis and autophagy, are key processes that are activated early on during development, leading to remodelling in embryos and homeostasis in adult organisms. Genomic conservation of death factors has been largely investigated in the animal and plant kingdoms. In this study, we analysed, for the first time, the expression profile of 11 genes involved in apoptosis (extrinsic and intrinsic pathways) and autophagy in sea urchin Paracentrotus lividus embryos exposed to antiproliferative polyunsaturated aldehydes (PUAs), and we compared these results with those obtained on the human cell line A549 treated with the same molecules. We found that sea urchins and human cells activated, at the gene level, a similar cell death response to these compounds. Despite the evolutionary distance between sea urchins and humans, we observed that the activation of apoptotic and autophagic genes in response to cytotoxic compounds is a conserved process. These results give first insight on death mechanisms of P. lividus death mechanisms, also providing additional information for the use of this marine organism as a useful in vitro model for the study of cell death signalling pathways activated in response to chemical compounds.

Cadmium stress effects indicating marine pollution in different species of sea urchin employed as environmental bioindicators.

In recent years, researches about the defense strategies induced by cadmium stress have greatly increased, invading several fields of scientific research. Mechanisms of cadmium-induced toxicity continue to be of interest for researchers given its ubiquitous nature and environmental distribution, where it often plays the role of pollutant for numerous organisms. The presence in the environment of this heavy metal has been constantly increasing because of its large employment in several industrial and agricultural activities. Cadmium does not have any biological role and, since it cannot be degraded by living organisms, it is irreversibly accumulated into cells, interacting with cellular components and molecular targets. Cadmium is one of the most studied heavy metal inductors of stress and a potent modulator of several processes such as apoptosis, autophagy, reactive oxygen species, protein kinase and phosphatase, mitochondrial function, metallothioneins, and heat-shock proteins. Sea urchins (adults, gametes, embryos, and larvae) offer an optimal opportunity to investigate the possible adaptive response of cells exposed to cadmium, since these cells are known to accumulate contaminants. In this review, we will examine several responses to stress induced by cadmium in different sea urchin species, with a focus on Paracentrotus lividus embryos. The sea urchin embryo represents a suitable system, as it is not subjected to legislation on animal welfare and can be easily used for toxicological studies and as a bioindicator of environmental pollution. Recently, it has been included into the guidelines for the use and interpretation of assays to monitor autophagy.

First molecular evidence of the toxicogenetic effects of copper on sea urchin Paracentrotus lividus embryo development

Bioassays with sea urchin embryos are widely used to define the environmental quality of marine waters. Anomalies during embryogenesis are generally considered as end-points, whereas a toxigenomic approach, despite it is wide use in other species, is yet in its infancy. In the present study we evaluated toxigenic effects induced by copper on the sea urchin Paracentrotus lividus embryo, combining morphological observations with gene expression analysis. Many anthropogenic activities release copper in the marine environment, with harmful effects on aquatic organisms. In the present study P. lidivus embryos were exposed to different concentrations of copper (24, 36, 48 μg/L) and the activation of fifty specific marker genes, involved in different biological processes (stress, skeletogenesis, development/differentiation, detoxification) was investigated at early blastula, late gastrula and pluteus stage. At blastula stage no morphological anomalies were found, with early down-regulation of genes involved in development/differentiation and a moderate up-regulation of some detoxification genes. At gastrula stage a slight increase in developmental anomalies (up to 19% of malformed embryos) was followed by an increased number of targeted genes belonging to the same two classes, relative to the blastula stage. At pluteus stage morphological anomalies increased in a dose dependent manner. All the analyzed genes were strongly up-regulated, stress and skeletogenic genes showing a “late response” and almost all genes were targeted by copper at all the concentrations tested. The present study represents the first molecular report on the potential negative effect of copper on P. lividus embryos in the environment. Gene expression analysis should be considered as a promising tool for future environmental biomonitoring programs.

Molecular and Morphological Toxicity of Diatom-Derived Hydroxyacid Mixtures to Sea Urchin Paracentrotus lividus Embryos.

Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.

Leachates of micronized plastic toys provoke embryotoxic effects upon sea urchin Paracentrotus lividus

Microplastics are defined as plastic fragments <5 mm, and they are found in the ocean where they can impact on the ecosystem. Once released in seawater, microplastics can be internalized by organisms due to their small size, moreover they can also leach out several additives used in plastic manufacturing, such as plasticizers, flame retardants, etc., resulting toxic for biota. The aim of this study was to test the toxicity of micronized PVC products with three different colors, upon Paracentrotus lividus embryos. In particular, we assessed the effects of micronized plastics and microplastic leachates. Results showed a decrease of larval length in plutei exposed to low concentrations of micronized plastics, and a block of larval development in sea urchin embryos exposed to the highest dose. Virgin PVC polymer did not result toxic on P. lividus embryos, while an evident toxic effect due to leached substances in the medium was observed. In particular, the exposure to leachates induced a development arrest immediately after fertilization or morphological alterations in plutei. Finally, PVC products with different colors showed different toxicity, probably due to a different content and/or combination of heavy metals present in coloring agents.

Toxicity of diatom-derived polyunsaturated aldehyde mixtures on sea urchin Paracentrotus lividus development

Diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal, derive from the oxidation of fatty acids and have cytotoxic and anticancer effects. PUAs, tested separately, induce malformations in sea urchin Paracentrotus lividus embryos. Decadienal induces the worst malformations and lowest survival rates. Interestingly, decadienal, heptadienal and octadienal place in motion several genes to counteract their negative effects. To date, no studies are available reporting on the effects of PUA mixtures on marine invertebrates. Here we test binary and ternary mixtures on embryonic development of P. lividus. Our findings demonstrate that mixtures of PUAs act (i) at morphological level in synergistic way, being much more severe compared to individual PUAs; (ii) at molecular level also reveal an additive effect, affecting almost all fifty genes, previously tested using individual PUAs. This study is relevant from an ecological point of view since diatoms are a major food source for both pelagic and benthic organisms. This work opens new perspectives for understanding the molecular mechanisms that marine organisms use in reacting to environmental natural toxin mixtures such as diatom PUAs.

Response to metals treatment of Fra1, a member of the AP-1 transcription factor family, in P. lividus sea urchin embryos

Lithium (Li), Nickel (Ni), and Zinc (Zn) are metals normally present in the seawater, although they can have adverse effects on the marine ecosystem at high concentrations by interfering with many biological processes. These metals are toxic for sea urchin embryos, affecting their morphology and developmental pathways. In particular, they perturb differently the correct organization of the embryonic axes (animal-vegetal, dorso-ventral): Li is a vegetalizing agent and Ni disrupts the dorso-ventral axis, while Zn has an animalizing effect. To deeply address the response of Paracentrotus lividus embryos to these metals, we studied the expression profiling of Pl-Fra transcription factor (TF), relating it to Pl-jun, a potential partner for AP-1 complex formation, and to Pl-MT, known to be an AP-1 target and to have a protective role against heavy metals. The AP-1 TFs are found throughout the animal kingdom and are involved in many cellular events, i.e. cell proliferation and differentiation, immune and stress responses, cancer growth. Here, we isolated the complete Pl-Fra cDNA and showed that Pl-Fra transcript, already present in the unfertilized eggs, was newly synthesized from the blastula stage, while its spatial distribution was mainly observed in skeletogenic cells, similarly to Pl-jun. Interestingly, Pl-Fra expression was induced by the different metals and the induction kinetics revealed its persistent expression during treatments. Moreover, its temporal and spatial behavior in response to the three metals was comparable to that of Pl-jun and Pl-MT. The understanding of AP-1 functions in invertebrates may provide new knowledge about the mechanisms of response to metal injuries, as well as it might lead to acknowledge the TFs as new type of biomarkers for the evaluation of hazards in polluted environment.

In situ embryo toxicity test with sea urchin: Development of exposure chamber for test execution

In situ toxicity tests represent a technique rarely performed owing to the lack of standard methodologies or to technical or economic problems. Nevertheless, its application would allow for a more realistic interpretation of pollution effects than those obtained by standard laboratory tests. The goal of this study is to develop and validate a specific exposure chamber for in situ exposition of Paracentrotus lividus embryos to obtain a defined methodology to perform the sea urchin embryo toxicity test in field conditions. After a first part of the study to verify the feasibility of the test chamber and the methodology, this approach was used as a tool to investigate whether the cruise ship “Costa Concordia” shipwrecked on Giglio Island (Tuscany, Italy), could have acted as a source of pollution. The results obtained for in situ tests showed, on average, percentages of normal embryos lower than those obtained in laboratory conditions and a greater sensitivity than for those obtained in the laboratory owing to the time-integration of results. Thus the exposure chamber and the in situ methodology so far developed would appear to be suitable tools for future application in the environmental quality evaluation of marine waters.

A sea urchin invivo model to evaluate Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity ofthe sea urchin embryo to anti-EMT drugs and pinpoint the sea urchin embryo as a valuable invivo model system for studying EMT and the screening of anti-EMT candidates.

Amino-modified polystyrene nanoparticles affect signalling pathways of the sea urchin (Paracentrotus lividus) embryos

Polystyrene nanoparticles have been shown to pose serious risk to marine organisms including sea urchin embryos based on their surface properties and consequently behaviour in natural sea water. The aim of this study is to investigate the toxicity pathways of amino polystyrene nanoparticles (PS-NH2, 50 nm) in Paracentrotus lividus embryos in terms of development and signalling at both protein and gene levels. Two sub-lethal concentrations of 3 and 4 μg/mL of PS-NH2 were used to expose sea urchin embryos in natural sea water (PS-NH2 as aggregates of 143 ± 5 nm). At 24 and 48 h post-fertilisation (hpf) embryonic development was monitored and variations in the levels of key proteins involved in stress response and development (Hsp70, Hsp60, MnSOD, Phospho-p38 Mapk) as well as the modulation of target genes (Pl-Hsp70, Pl-Hsp60, Pl-Cytochrome b, Pl-p38 Mapk, Pl-Caspase 8, Pl-Univin) were measured. At 48 hpf various striking teratogenic effects were observed such as the occurrence of cells/masses randomly distributed, severe skeletal defects and delayed development. At 24 hpf a significant up-regulation of Pl-Hsp70, Pl-p38 Mapk, Pl-Univin and Pl-Cas8 genes was found, while at 48 hpf only for Pl-Univin was observed. Protein profile showed different patterns as a significant increase of Hsp70 and Hsp60 only after 48 hpf compared to controls. Conversely, P-p38 Mapk protein significantly increased at 24 hpf and decreased at 48 hpf. Our findings highlight that PS-NH2 are able to disrupt sea urchin embryos development by modulating protein and gene profile providing new understandings into the signalling pathways involved.

Autophagy is required for sea urchin oogenesis and early development

Autophagy is a major intracellular pathway for the degradation and recycling of cytosolic components. Emerging evidence has demonstrated its crucial role during the embryo development of invertebrates and vertebrates. We recently demonstrated a massive activation of autophagy in Paracentrotus lividus embryos under cadmium stress conditions, and the existence of a temporal relationship between induced autophagy and apoptosis. Although there have been numerous studies on the role of autophagy in the development of different organisms, information on the autophagic process during oogenesis or at the start of development in marine invertebrates is very limited. Here we report our recent data on the occurrence of autophagy at these key phases of development. In order to investigate autophagy trends we performed in vivo assays to detect autophagolysomes, as well as in situ analysis with anti-LC3 antibody to detect autophagosomes before the fusion with lysosomes. From data generated through confocal laser scanning microscopy and quantification of autophagic signals we have drawn several unequivocal conclusions. The results showed a copious and rising number of autophagic organelles that had specific localization. Interestingly the increase in autophagy that occurred just after fertilization has been proved to be crucial for correct initiation of the developmental programme: irreversible developmental delays and morphologic anomalies were induced by short autophagic inhibition. This work focused on the sea urchin model system and corroborates evidence on the need for self-digestion during development, enriching the knowledge on autophagy, a biological mechanism belonging to evolutionarily different organisms.

Spermiotoxicity and embryotoxicity of permethrin in the sea urchin Paracentrotus lividus.

The toxicity of permethrin on the fertilization and early development of sea urchin Paracentrotus lividus embryos were studied. Spermiotoxicity was evaluated on the basis of fertilization rate. Embryotoxicity was determined by comparing the frequency of normal development and malformations in embryos exposed to permethrin throughout their development. Permethrin inhibited fertilization success, and yielded IC25 and IC50 values of 0.58 (CL = 0.44-0.77) and 0.94 (CL = 0.92-0.95) µg/L, respectively. The embryotoxicity of permethrin was concentration dependent indicating a decreased percentage of normally developed plutei with increasing permethrin concentrations: IC25 = 0.195 µg/L (CL = 0.15-0.26) and IC50 = 0.346 µg/L (CF = 0.29-0.41). Associated with the decrease in normal pluteus frequency was an increase in larval malformations as skeleton deformities. The results suggest that permethrin is more highly toxic to embryos than to sperm, and that this insecticide may present a potential risk for the sea urchin in contaminated marine environments.

Sea urchin (Paracentrotus lividus) larval rearing — Culture from cryopreserved embryos

This is the first attempt to cryopreserve Paracentrotus lividus embryos for the aquaculture industry. The cryopreservation protocol used for sea urchin embryos has been proven not only to be able to produce viable outcome, but also embryos that can grow into competent larvae, metamorphose and settle. Cryopreserved blastulas yielded an initial survival of 50% at day 3 and 29% by the end of larval rearing (day 20), which represents the 71% survival of control, non-cryopreserved blastulas. Cryopreserved blastulas suffered a delay in growth and development, achieving the prism or 4-arm pluteus stage at 96 h after fertilization. Larval development showed differences with the control blastulas after 10 days of the larval rearing (staying at 4-arm pluteus until day 8, early 6-arm pluteus by day 10). By day 13 cryopreserved blastulas achieved the 8-arm pluteus stage and, from that point on, developed the rudiment and larvae achieved competence to metamorphose almost at the same time than fresh controls. The settlement percentage of cryopreserved larvae was 7%, which represents the 25% of control larvae settlement.