Effects of seawater acidification and warming on morphometrics and biomineralization-related gene expression during embryo-larval development of a lightly-calcified echinoderm.

CO2-induced ocean acidification and warming pose ecological threats to marine life, especially calcifying species such as echinoderms, who rely on biomineralization for skeleton formation. However, previous studies on echinoderm calcification amid climate change had a strong bias towards heavily calcified echinoderms, with little research on lightly calcified ones, such as sea cucumbers. Here, we analyzed the embryo-larval development and their biomineralization-related gene expression of a lightly calcified echinoderm, the sea cucumber (Apostichopus japonicus), under experimental seawater acidification (OA) and/or warming (OW). Results showed that OA (- 0.37 units) delayed development and decreased body size (8.58-56.25 % and 0.36-19.66 % decreases in stage duration and body length, respectively), whereas OW (+3.1 °C) accelerated development and increased body size (33.99-55.28 % increase in stage duration and 2.44-14.41 % enlargement in body length). OW buffered the negative effects of OA on the development timing and body size of A. japonicus. Additionally, no target genes were expressed in the blastula stage, and only two biomineralization genes (colp3α, cyp2) and five TFs (erg, tgif, foxN2/3, gata1/2/3, and tbr) were expressed throughout the embryo-larval development. Our findings suggest that the low calcification in A. japonicus larvae may be caused by biomineralization genes contraction, and low expression of those genes. Furthermore, this study indicated that seawater acidification and warming affect expression of biomineralization-related genes, and had an effect on body size and development rate during the embryo-larval stage in sea cucumbers. Our study is a first step toward a better understanding of the complexity of high pCO2 on calcification and helpful for revealing the adaptive strategy of less-calcified echinoderms amid climate change.

Toxicity assessment of a tebuconazole-based fungicide on the embryo-larval development of the common south American toad Rhinella arenarum.

Agrochemicals cause diverse effects on aquatic communities, and amphibian species are particularly threatened due the high susceptibility to contamination. Present study evaluates the toxicity of a widely used fungicide tebuconazole (Trigal®) by the assessment of mortality and developmental alterations at acute, subchronic, and chronic exposure during the embryo-larval development of the South American toad Rhinella arenarum. Also, the sensitivity of the different embryonic stages was evaluated with 24-h pulse exposure treatments. The results demonstrated that larvae were more sensitive than embryos at acute exposure (LC50-24 and 96 h = 74.62, 31.92 mg/L and 24.27, 16.81 mg/L for embryos and larvae, respectively). Nevertheless, embryos toxicity increased significantly achieving a sensitivity very similar to larvae at chronic exposure (LC50-168 and 504 h = 13.31, 4.35 mg/L and 14.47, 6.83 mg/L for embryos and larvae, respectively). Embryos exhibited several sublethal effects from 5 mg/L at 96 h onwards, such as delayed development, reduce body size, edemas, tail/axial flexures, weakness, and absence of movements. The teratogenic index at 96 h was 10.13, indicating the severe teratogenic potential of the fungicide. 24-h pulse exposure treatments showed an increased sensitivity in intermediate stages as S.11, S.18, S20, and S.23 (NOEC-96 h = 100, 200, 75, and 20 mg/L, respectively), while stage S.25 was the most sensitive to the fungicide (NOEC-96 h = 5 mg/L). About metamorphic process, tebuconazole caused an acceleration of metamorphosis at the lowest concentration (0.001 mg/L), but also an increase in mortality and in addition, significant differences in the weight in all treatments. The results obtained throughout this work indicate that tebuconazole cause several adverse effects in Rhinella arenarum embryo-larval development.

Stress response to trace elements mixture of different embryo-larval stages of Paracentrotus lividus.

This study investigated for the first time the oxidative biomarkers responses in all larval stages of sea urchin. The contamination effects were reproduced by using contaminated seawater to concentrations measured in the area adjacent to an old asbestos mine at factors of 5 and 10. The results suggested that the concentrations were not sufficiently high to induce a major oxidative stress. The biometric differences make this method a more sensitive approach for assessing the effects on sea urchin larvae. Measurements of specific activities of antioxidant enzymes at each stage suggested a high capacity of the larvae to respond to oxidative stress. This normal activity of the organism must be considered in future research. This work also highlighted the importance of spawners provenance in ecotoxicological studies. These data are essential to better understand the stress responses of sea urchin larvae and provide baseline information for later environmental assessment research.

Microscale Toxicity Identification Evaluation (TIE) for interstitial water of estuarine sediments affected by multiple sources of pollution.

Estuaries in the world are affected by different contamination sources related to urbanisation and port/industrial activities. Identifying the substances responsible for the environmental toxicity in estuaries is challenging due to the multitude of stressors, both natural and anthropogenic. The Toxicity Identification and Evaluation (TIE) is a suitable way of determining causes of toxicity of sediments, but it poses difficulties since its application is labour intensive and time consuming. The aim of this study is to evaluate the diagnosis provided by a TIE based on microscale embryotoxicity tests with interstitial water (IW) to identify toxicants in estuarine sediments affected by multiple stressors. TIE showed toxicity due to different combinations of metals, apolar organic compounds, ammonia and sulphides, depending on the contamination source closest to the sampling station. The microscale TIE was able to discern different toxicants on sites subject to different contamination sources. There is good agreement between the results indicated in the TIE and the chemical analyses in whole sediment, although there are some disagreements, either due to the sensitivity of the test used, or due to the particularities of the use of interstitial water to assess the sediment toxicity. The improvement of TIE methods focused on identifying toxicants in multiple-stressed estuarine areas are crucial to discern contamination sources and subsidise management strategies.

Application of the Paracentrotus lividus sea-urchin embryo-larval bioassay to the marine pollution biomonitoring program in the Tunisian coast.

The pollution of the marine environment by treated and untreated effluents has increased due to human activities. Monitoring the marine ecosystem is nowadays a global concern. In this work, we evaluated the effect of contaminated and uncontaminated seawater, from different Tunisian coastal areas, on the fertilization, gastrulation, and embryo-larval development events of sea urchins (Paracentrotus lividus). The station of Salakta (SA) is considered as a control station, while the stations of Hamdoun Wadi (HW), Port of Monastir (PM), Karaia Monastir (KM), Teboulba (TE), and Khniss Lagoon (KL) are considered to be contaminated stations. The analysis of seawater physicochemical characteristics showed that levels of the total suspended matter (TSM), chemical oxygen demand (COD), biochemical oxygen demand (BOD), total organic carbon (TOC), and nitrate (NO3-) were lower in the seawater of the reference site Salakta (SA) when compared to those of the contaminated seawater sites. In addition, a very strong variation in the levels of trace metals in seawaters sampled in the studied sites was noted. In fact, the highest concentrations of Pb and Cu were observed in Hamdoun Wadi (HW), port of Monastir (PM), and Karaia Monastir (KM), while the highest concentration of Zn was noted in the Teboulba lagoon (TE) and Khniss (LK). Alterations in physicochemical characteristics as well as elevated trace metal levels in the studied seawater samples were correlated with reduced fertility rate, gastrulation rate, and the frequency of normal sea urchin larvae. The total absence of normal sea urchin pluteus larvae in the sea waters of heavily polluted sites proves the great sensitivity of the larval frequency to mixed pollution. This work recommends the utility of urchin fertilization and gastrulation rates and normal pluteus larval frequencies as useful bioassays to monitor the exposure of marine ecosystems to mixed pollution.

Generational effects of a chronic exposure to a low environmentally relevant concentration of glyphosate on rainbow trout, Oncorhynchus mykiss.

In the past few decades, glyphosate became the most used herbicide substance worldwide. As a result, the substance is ubiquitous in surface waters. Concerns have been raised about its ecotoxicological impact, but little is known about its generational toxicity. In this study, we investigate the impact of an environmentally relevant concentration of glyphosate and its co-formulants on an F2 generation issued from exposed generations F0 and F1. Trans, inter and multigenerational toxicity of 1 µgL-1 of the active substance was evaluated on early stages of development and juvenile rainbow trout (Oncorhynchus mykiss) using different molecular, biochemical, immuno-hematologic, and biometric parameters, behavior analysis, and a viral challenge. Reproductive parameters of generation F1 were not affected. However, developmental toxicity in generation F2 due to glyphosate alone or co-formulated was observed with head size changes (e.g. head surface up to +10%), and metabolic disruptions (e.g. 35% reduction in cytochrome-c-oxidase). Moreover, larvae exposed transgenerationally to Viaglif and intergenerationally to glyphosate and Roundup presented a reduced response to light, potentially indicating altered escape behavior. Overall methylation was, however, not altered and further experiments using gene-specific DNA metylation analyses are required. After several months, biochemical parameters measured in juvenile fish were no longer impacted, only intergenerational exposure to glyphosate drastically increased the susceptibility of rainbow trout to hematopoietic necrosis virus. This result might be due to a lower antibody response in exposed fish. In conclusion, our results show that generational exposure to glyphosate induces developmental toxicity and increases viral susceptibility. Co-formulants present in glyphosate-based herbicides can modulate the toxicity of the active substance. Further investigations are required to study the specific mechanisms of transmission but our results suggest that both non-genetic mechanisms and exposure during germinal stage could be involved.

Developmental effect of parental or direct chronic exposure to environmental concentration of glyphosate on the larvae of rainbow trout, Oncorhynchus mykiss.

The environmental safety profile of glyphosate, the most commonly used herbicide worldwide, is still a subject of debate and little is known about the generational toxicity of this active substance (AS) and the associated commercial formulations called "glyphosate-based herbicides" (GBHs). This study investigated the impact of parental and direct exposure to 1µgL-1 of glyphosate using the AS alone or one of two GBH formulations (i.e. Roundup Innovert® and Viaglif Jardin®) in the early developmental stages of rainbow trout. Three different modes of exposure on the F1 generation were studied: (1) intergenerational (i.e. fish only exposed through their parents); (2) direct (i.e. fish exposed only directly) and (3) multigenerational (i.e. fish both exposed intergenerationally and directly). The impact of chemical treatments on embryo-larval development (survival, biometry and malformations), swimming behaviour, biochemical markers of oxidative stress equilibrium (TBARS and catalase), acetylcholine esterase (AChE) and energy metabolism (citrate synthase, CS; cytochrome-c oxidase, CCO; lactate dehydrogenase, LDH; glucose-6-phosphate dehydrogenase, G6PDH) was explored. Chemical exposure did not affect the survival of F1 embryos or malformation rates. Direct exposure to the AS induced some biometric changes, such as reduction in head size (with a 10% decrease in head length), independently of co-formulants. Intergenerational exposure to the AS or the Roundup GBH increased swimming activity of the larvae, with increase of between 78 and 102% in travel speeds. Viaglif co-formulants appear to have counteracted this behavioural change. The minor changes detected in the assayed biochemical markers suggested that observed effects were not due to oxidative damage, AChE inhibition or alterations to energy metabolism. Nonetheless, multi- and intergenerational exposure to Roundup increased CS:CCO and LDH:CS ratios by 46% and 9%, respectively, with a potential modification of the aerobic-to-anaerobic energy production balance. These biochemical effects were not correlated with those observed on individual level of biological organization. Therefore, further studies on generational toxicity of glyphosate and its co-formulants are needed to identify the other mechanisms of glyphosate toxicity at the cellular level.

Assessment of the individual and mixture toxicity of cadmium, copper and oxytetracycline, on the embryo-larval development of the sea urchin Paracentrotus lividus.

Multiple pollutions by trace metals and pharmaceuticals have become one of the most important problems in marine coastal areas because of its excessive toxicity on organisms living in this area. This study aimed to assess the individual and mixture toxicity of Cu, Cd, and oxytetracycline frequently existing in the contaminated marine areas and the embryo-larval development of the sea urchin Paracentrotus lividus. The individual contamination of the spermatozoid for 1 h with the increasing concentrations of Cd, Cu, and OTC decreases the fertility rate and increases larvae anomalies in the order Cu > Cd > OTC. Moreover, the normal larva frequency and the length of spicules were more sensitive than the fertilization rate and normal gastrula frequency endpoints. The mixture toxicity assessed by multiple experimental designs showed clearly that concentrations of Cd, Cu, and OTC superior to 338 µg/L, 0.56 µg/L, and 0.83 mg/L, respectively, cause significant larva malformations.

External gamma irradiation-induced effects in early-life stages of zebrafish, Danio rerio.

In the general context of validation of tools useful for the characterization of ecological risk linked to ionizing radiation, the effects of an external gamma irradiation were studied in zebrafish larvae irradiated for 96 h with two dose rates: 0.8 mGy/d, which is close to the level recommended to protect ecosystems from adverse effects of ionizing radiation (0.24 mGy/d) and a higher dose rate of 570 mGy/d. Several endpoints were investigated, such as mortality, hatching, and some parameters of embryo-larval development, immunotoxicity, apoptosis, genotoxicity, neurotoxicity and histological alterations. Results showed that an exposure to gamma rays induced an acceleration of hatching for both doses and a decrease of yolk bag diameter for the highest dose, which could indicate an increase of global metabolism. AChE activity decreased with the low dose rate of gamma irradiation and alterations were also shown in muscles of irradiated larvae. These results suggest that gamma irradiation can induce damages on larval neurotransmission, which could have repercussions on locomotion. DNA damages, basal ROS production and apoptosis were also induced by irradiation, while ROS stimulation index and EROD biotransformation activity were decreased and gene expression of acetylcholinesterase, choline acetyltransferase, cytochrome p450 and myeloperoxidase increased. These results showed that ionizing radiation induced an oxidative stress conducting to DNA damages. This study characterized further the modes of action of ionizing radiation in fish.

Developmental toxicity and risk assessment of nonylphenol to the South American toad, Rhinella arenarum.

The toxicity of Nonylphenol, an emerging pollutant, on the common South American toad Rhinella arenarum was stage and time dependent, thus Median Lethal Concentrations (LC50) for acute (96h), short-term chronic (168h) and chronic exposure (336h) were 1.06; 0.96 and 0.17mgNP/L from embryonic period (S.4), whereas for exposure from larvae (S.25), LC50 remained constant at 0.37mgNP/L from 96h to 168h, decreasing to 0.11mgNP/L at 336h. NOEC-168h for exposure from embryos was 0.025mgNP/L. The Teratogenic Potential (NOEC-lethality/NOEC-sublethal effects) was 23 times higher than the threshold value, indicating a high risk for embryos to be malformed in absence of significant lethality and representing a threat for the species conservation. By comparing with other amphibians, the early development of R. arenarum was very sensitive to NP. The results highlight the relevance of extending the exposure time and look for the most sensitive stage in order to perform the bioassays for conservation purposes.

Developmental toxicity of endocrine disruptors in early life stages of zebrafish, a genetic and embryogenesis study.

Endocrine disrupting compounds (EDCs) are capable of interfering with the endocrine system and are increasingly widespread in the aquatic environments. In the present study, zebrafish (Danio rerio) embryos and larvae were used to assess how EDCs may interfere with embryogenesis. Therefore, zebrafish embryos were exposed to 17α-ethinylestradiol (EE2: 0.4, 2, 4 and 20 ng/L), genistein (Gen: 2, 20, 200 and 2000 ng/L) and fadrozole (Fad: 2, 10, 50 and 250 µg/L), between 2 and 144 h post-fertilization (hpf). Somite development, heartbeat, malformations, mortality and hatching rates were evaluated. In parallel, the expression patterns of hormone receptors (esr1, esr2a, esr2b and ar) and apoptotic pathways related genes (p53 and c-jun) were determined using quantitative real-time PCR. Results showed that EE2, Gen and Fad caused a higher mortality and also malformations in larvae compared with control. A significant toxic effect was observed in the heartbeat rate, at 144 hpf, in larvae exposed to EE2 and Fad. QPCR revealed alterations in the expression levels of all the evaluated genes, at different time points. esr1 and c-jun genes were upregulated by EE2 and Gen exposure while the expression of esr2a, esr2b and ar genes was downregulated. Fad exposure decreased esr1, p53 and c-jun expression levels. This study shows a toxic effect of EE2, Gen and Fad to vertebrate embryogenesis and a relation between hormones action and apoptosis pathways.

Differential toxicity and uptake of Diazinon on embryo-larval development of Rhinella arenarum.

Diazinon, an anti-cholinesterase organophosphate, is an extensively used pesticide. The main objective of this work was to assess the lethal and sublethal effects of Diazinon and its comparison with the uptake by embryos and larvae of the common South American toad Rhinella arenarum by means of standardized bioassays during acute (96 h), short-term chronic (168 h) and chronic (504 h) exposures. Toxicity resulted time- and stage-dependent, thus the lethal concentration 50 for 96 h, 168 h and 504 h were 27.2; 20.1 and 6.8 mg Diazinon L(-1) for embryos and 8, 6.7 and 1.9 mg Diazinon L(-1) for larvae. It is noteworthy the remarkable differences found in the concentration which caused lethality with those causing adverse effects on development such as malformations (teratogenic effects). Therefore, the teratogenic index from 144 h was greater than two; the main adverse effects were axial flexures, irregular borders, wavy tail, microcephaly, malformed mouth and adhesive structures, gut miscoiling, underdeveloped gills, cloacal edema, desquamation and severe hydropsy. Moreover, the characteristic sublethal effect of Diazinon on larvae was abnormal behavior related to neurotoxicity with a NOEC-168 h of 4.5 mg Diazinon L(-1). Diazinon contents in R. arenarum were time-dependent and significantly related to exposure concentration for both embryos and larvae. Diazinon contents were also stage-dependent, as it was up to 27 times higher for organisms exposed from blastula stage onwards than early larvae. These facts and the Hazard Quotients, a numerical expression of ecological risk, of 2.73, which is above USEPA's Level of Concern, showed the threat that Diazinon represents for R. arenarum populations.