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This study investigates the combined impact of microplastics (MP) and Chlorpyriphos (CPF) on sea urchin larvae (Paracentrotus lividus) under the backdrop of ocean warming and acidification. While the individual toxic effects of these pollutants have been previously reported, their combined effects remain poorly understood. Two experiments were conducted using different concentrations of CPF (EC10 and EC50) based on previous studies from our group. MP were adsorbed in CPF to simulate realistic environmental conditions. Additionally, water acidification and warming protocols were implemented to mimic future ocean conditions. Sea urchin embryo toxicity tests were conducted to assess larval development under various treatment combinations of CPF, MP, ocean acidification (OA), and temperature (OW). Morphometric measurements and biochemical analyses were performed to evaluate the effects comprehensively. Results indicate that combined stressors lead to significant morphological alterations, such as increased larval width and reduced stomach volume. Furthermore, biochemical biomarkers like acetylcholinesterase (AChE), glutathione S-transferase (GST), and glutathione reductase (GRx) activities were affected, indicating oxidative stress and impaired detoxification capacity. Interestingly, while temperature increase was expected to enhance larval growth, it instead induced thermal stress, resulting in lower growth rates. This underscores the importance of considering multiple stressors in ecological assessments. Biochemical biomarkers provided early indications of stress responses, complementing traditional growth measurements. The study highlights the necessity of holistic approaches when assessing environmental impacts on marine ecosystems. Understanding interactions between pollutants and environmental stressors is crucial for effective conservation strategies. Future research should delve deeper into the impacts at lower biological levels and explore adaptive mechanisms in marine organisms facing multiple stressors. By doing so, we can better anticipate and mitigate the adverse effects of anthropogenic pollutants on marine biodiversity and ecosystem health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The goal of the present work is to study whether ocean- acidification (OA) and -warming (OW) could increase the toxicity of pollutants on P. lividus. We studied how model pollutants such as chlorpyrifos (CPF) and microplastics (MP), alone or in combination, impact the fertilisation process, and the development of larvae under conditions of OA (dissolved inorganic carbon increase of 126 × 10 -6  mol per kg of sea water) and OW (temperature increase of 4 °C) predicted by FAO (Food and Agriculture Organization) for the next 50 years. Fertilisation was determined by microscopic examination after 1 h. Growth, morphology, and alteration level were measured after 48 h of incubation. Results showed that CPF has a marked effect on the growth of larvae, but less on the fertilisation rate. When larvae are exposed to both MP and CPF, the effect on fertilisation and growth is higher than when CPF is added alone. Larvae exposed to CPF tend to adopt a rounded shape which is detrimental to their buoyancy and the combination with other stressors aggravate this situation. The variables most influenced by CPF or its mixtures are those related to body length, body width, and higher levels of body abnormalities, which is consistent with the degenerative effects caused by CPF on sea urchin larvae. The PCA analysis showed that temperature has more influence when embryos or larvae are exposed to a combination of stressors, demonstrating that global climate change drastically increase the impact of CPF on aquatic ecosystems. Overall, in this work we demonstrated that global climate change conditions increase the sensitivity of embryos to MP and CPF. Our findings support the idea that global change conditions could have a severe impact on marine life, increasing the negative effect of toxic agents commonly present in the sea and their mixtures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

One of the major consequences of increasing atmospheric CO 2 is a phenomenon known as ocean acidification. This alteration of water chemistry can modulate the impact on marine organisms of other stressors also present in the environment, such as microplastics (MP). The objective of this work was to determine the combined impact of microplastic pollution and ocean acidification on the early development of Paracentrotus lividus. To study these multi-stressor impacts on development P. lividus the sea urchin embryo test (SET) was used. Newly fertilised embryos of P. lividus were exposed to a control treatment (filtered natural seawater), MP (3000 particles/mL), acidified sea water (pH = 7.6), and a combination of MP and acidification (3000 particles/mL + pH = 7.6). After 48, 72, and 96 h measurements of growth and morphometric parameters were taken. Results showed that ocean acidification and MP cause alterations in growth and larval morphology both before and after the larvae start to feed exogenously. The exposure to MP under conditions of ocean acidification did not produce any additional effect on growth, but differences were observed at the morphological level related to a decrease in the width of larvae at 48 h. Overall, changes in larvae shape observed at three key points of their development could modify their buoyancy affecting their ability to obtain and ingest food. Therefore, ocean acidification and MP pollution might compromise the chances of P. lividus to survive in the environment under future scenarios of global climate change., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The effects of three relevant organic pollutants: chlorpyrifos (CPF), a widely used insecticide, triphenyl phosphate (TPHP), employed as flame retardant and as plastic additive, and bisphenol A (BPA), used primarily as plastic additive, on sea urchin (Paracentrotus lividus) larvae, were investigated. Experiments consisted of exposing sea urchin fertilized eggs throughout their development to the 4-arm pluteus larval stage. The antioxidant enzymes glutathione reductase (GR) and catalase (CAT), the phase II detoxification enzyme glutathione S-transferase (GST), and the neurotransmitter catabolism enzyme acetylcholinesterase (AChE) were assessed in combination with responses at the individual level (larval growth). CPF was the most toxic compound with 10 and 50% effective concentrations (EC 10 and EC 50 ) values of 60 and 279 μg/l (0.17 and 0.80 μM), followed by TPHP with EC 10 and EC 50 values of 224 and 1213 μg/l (0.68 and 3.7 μM), and by BPA with EC 10 and EC 50 values of 885 and 1549 μg/l (3.9 and 6.8 μM). The toxicity of the three compounds was attributed to oxidative stress, to the modulation of the AChE response, and/or to the reduction of the detoxification efficacy. Increasing trends in CAT activity were observed for BPA and, to a lower extent, for CPF. GR activity showed a bell-shaped response in larvae exposed to CPF, whereas BPA caused an increasing trend in GR. GST also displayed a bell-shaped response to CPF exposure and a decreasing trend was observed for TPHP. An inhibition pattern in AChE activity was observed at increasing BPA concentrations. A potential role of the GST in the metabolism of CPF was proposed, but not for TPHP or BPA, and a significant increase of AChE activity associated with oxidative stress was observed in TPHP-exposed larvae. Among the biochemical responses, the GR activity was found to be a reliable biomarker of exposure for sea urchin early-life stages, providing a first sign of damage. These results show that the integration of responses at the biochemical level with fitness-related responses (e.g., growth) may help to improve knowledge about the impact of toxic substances on marine ecosystems., (© 2022. The Author(s).)

The aim of this work was to estimate the potential risk of the combined effect of global change factors (acidification, temperature increase) and microplastic (MP) pollution on the growth and development of the sea urchin P. lividus. Embryo-larval bioassays were conducted to determine growth and morphology after 48 h of incubation with MP (1000 and 3000 particles/mL); with filtered sea water at pH = 7.6; and with their combinations. A second experiment was conducted to study the effect of pH and MP in combination with a temperature increase of 4 °C compared to control (20 °C). We found that the inhibition of growth in embryos reared at pH = 7.6 was around 75%. Larvae incubated at 3000 MP particles/mL showed a 20% decrease in growth compared to controls. The exposure to MP also induced an increase in the postoral arm separation or rounded vertices. The combined exposure to a pH 7.6 and MP caused a significant decrease of larval growth compared to control, to MP and to pH 7.6 treatments. Morphological alterations were observed in these treatments, including the development of only two arms. Increasing the temperature resulted in an increased growth in control, in pH 7.6 and pH 7.6 + MP3000 treatments, but the relative stomach volume decreased. However, when growth parameters were expressed per Degree-Days the lower growth provoked by the thermal stress was evidenced in all treatments. In this work we demonstrated that MP could aggravate the effect of a decreased pH and that an increase in water temperature generated an additional stress on P. lividus larvae, manifested in a lower growth and an altered development. Therefore, the combined stress caused by ocean warming, ocean acidification, and microplastic pollution, could threaten sea urchin populations leading to a potential impact on coastal ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have established for first time an ecotoxicological bioassay using cryopreserved sea urchin embryos (Paracentotus lividus) and provided a comparison to the already standardized sea urchin embryo-larval bioassay, using selected (organic and inorganic) pollutants and sediment elutriates from 4 different locations from Ria de Vigo harbour (Galicia, NW Iberian Peninsula). A cryopreservation protocol was designed in order to enable the successful cryopreservation and cryobanking of gametes and embryos to be used for marine quality assessment and ensure the accessibility to high quality reproductive material all year round, as an option to conditioning adults for out of season reproduction. The calculated EC50 using the cryopreserved blastula was 53.7 μg L(-1) for copper, 81.0 μg L(-1) for lead, 300.6 μg L(-1) for BP-3 and 300.6 μg L(-1) for 4-MBC. The sensitivity of the classic sea urchin embryo-larval bioassay was compared with the bioassay conducted with cryopreserved blastula. The results showed that the use of cryopreserved blastula bioassay allows detecting lower concentrations of pollutants in comparison with the classic bioassay., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The cryopreservation of embryos can be a powerful biotechnological tool to supply all year-round biological material for sea urchin aquaculture production. This study investigates different methodological and biological factors that may affect the result of the cryopreservation process of sea urchin (Paracentrotus lividus) embryos. Our data indicate that neither embryo density nor the use of different cryopreservation containers presented effect on the cryopreservation outcome. Contrary to other marine invertebrates, for sea urchin embryo cryopreservation ultrapure water cannot be used as CPA solvent, yielding zero survival. After studying the reproductive parameters along the reproductive season, we found a positive correlation between both male and female Condition Index (C.I.), and between the oocyte weight and C.I. Both the histology study of female gonads and the C.I. variation, suggest that the sea urchin natural spawning period in the Ría de Vigo occurs between June and July. We found no correlation between any of the reproductive parameters monitored and the cryopreservation outcome.

wWeathering of petroleum spilled in the marine environment may not only change its physical and chemical properties but also its effects on the marine ecosystem. The objective of this study was to evaluate the toxicity of the water-accommodated fraction (WAF) obtained from a standard fuel oil following an environmentally realistic simulated weathering process for a period of 80 d. Experimental flasks with 40 g L(-1) of fuel oil were incubated at 18°C with a 14 h light:10 h dark photoperiod and a photosynthetically active radiation (PAR) intensity of 70 μE m(-2) s(-1). Samples were taken at four weathering periods: 24 h, 7, 21 and 80 d. WAF toxicity was tested using the sea urchin (Paracentrotus lividus) and mussel (Mytilus galloprovincialis) embryo-larval bioassays and the aromatic hydrocarbons levels (AH) in the WAF were measured by gas chromatography/mass spectrometry. In contrast with the classic assumption of toxicity decrease with oil weathering, the present study shows a progressive increase in WAF toxicity with weathering, being the EC(50) after 80d eightfold lower than the EC(50) at day 1, whereas AH concentration slightly decreased. In the long term, inoculation of WAF with bacteria from a hydrocarbon chronically-polluted harbor slightly reduced toxicity. The differences in toxicity between fresh and weathered fuels could not be explained on the basis of the total AH content and the formation of oxidized derivatives is suggested to explain this toxicity increase., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The toxicity of the alternative antifouling compound Sea-Nine to the early developmental stages of the sea urchin Paracentrotus lividus was investigated. The inhibition of the fertilization rate and the induction of transmissible damages to the offspring, measured as inhibition of embryonic development and larval growth, were studied by preexposure of gametes to a range of Sea-Nine concentrations. Sperm and egg exposures resulted in a significant decrease of the fertilization rate and induced a transmissible damage to the offspring. The effects of Sea-Nine throughout the embryonic development were also studied by a 48 h exposure of fertilized eggs. The larval growth was the most sensitive response tested, with toxic effects detected at 8.6 nM=2.4 microg/L (EC(10)). The inhibition of P. lividus embryonic development and larval growth was also used to study the loss of toxicity in Sea-Nine solutions exposed for 8h to direct sunlight and maintained for 28 h in dark conditions. The results showed that the toxicity of Sea-Nine solutions did not decrease but a slight increase in toxicity was observed in comparison with control solutions. The risk of Sea-Nine maximum concentrations measured in marinas around Europe to P. lividus early developmental stages was calculated and the obtained risk quotient was 5.5, indicating that adverse ecological effects of this compound are likely to occur.

The effects of the new antifouling compound zinc pyrithione (Zpt) on the embryonic development of sea urchin (Paracentrotus lividus) and mussel (Mytilus edulis) were investigated in laboratory toxicity tests. The median effective concentrations (EC50) were 7.7 nM for sea urchin embryos and 8 nM for mussel embryos. Toxic effects of Zpt on the larval growth of the sea urchin were detected at 0.5 nM. Predicted environmental concentrations of Zpt in pleasure craft harbours are higher than the predicted no effect concentrations for sea urchin and mussel embryos, indicating that Zpt may pose a threat to those species from exposure in the field.