Developmental and biochemical markers of the impact of pollutant mixtures under the effect of Global Climate Change.

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.

Impact of microplastics and ocean acidification on critical stages of sea urchin (Paracentrotus lividus) early development.

One of the major consequences of increasing atmospheric CO2 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.

Combined effect of microplastics and global warming factors on early growth and development of the sea urchin (Paracentrotus lividus).

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.

Assessing the cytotoxic effect of hexabromocyclododecane (HBCD) on liver tissue cultures from fathead minnow (Pimephales promelas).

Hexabromocyclododecane (HBCD) is a ubiquitous environmental contaminant of current concern despite its global ban in 2013 due to its characteristics as a persistent organic pollutant. While the toxicity of HBDC in vertebrates has been extensively studied, the specific molecular mechanisms underlying its toxicity in fish are not fully understood to date. Therefore, the aim of this work was to determine the in vitro cytotoxicity of HBCD in the fathead minnow (Pimephales promelas) using liver explants, and to investigate the molecular mechanisms underlying these effects. Explants were incubated with nine different concentrations of HBCD (0.00032, 0.0016, 0.008, 0.04, 0.2, 1, 5, 25 and 125 mg HBCD/L) for 6 and 24 h, and cytotoxicity was tested by using the Lactate Dehydrogenase (LDH) assay. The expression of genes with a key role in the regulation of apoptosis, oxidative stress, cryoprotective responses to reactive oxygen species (ROS), and xenobiotic metabolism was also measured in liver explants after exposure to 0.00032, 0.0016, 0.008, 0.2, and 25 mg HBCD/L. After 6 h, a concentration-dependent significant increase in cytotoxicity was found between 0.008 and 1 mg/L HBCD, followed by a decrease between 1 and 25 mg/L. Cytotoxicity reached 100 % at a concentration of 125 mg/L HBCD. After 24 h, HBCD showed a biphasic response with a concentration-dependent decrease in cytotoxicity between 0.0016 and 1 mg/L that returned to baseline levels at 5 mg/L. Then, cytotoxicity increased at concentrations greater than 5 mg/L to reach a maximum value at 125 mg/L. Changes in the expression of genes related to apoptosis (apoEn, apoIn, caspase2, caspase9 and bax) were also time- and concentration-dependent. Genes related to antioxidant responses such as gst and catalase were generally decreased after 6 h of incubation and increased after 24 h. The same pattern was observed for cyp1a and cyp3a, both related to xenobiotic metabolism. The expression of genes related to cryoprotective responses anti ROS (akt and pi3k) decreased at almost all HBCD concentrations tested after 6 h but remained unaltered after 24 h. Overall, we demonstrated that the cytotoxic effect of HBCD in fathead minnow liver explant was not proportional to its concentration in the culture media. Cytotoxicity was highly dynamic and did not follow a typical concentration-response pattern, complicating its toxicological characterization.