Different responses of marine microalgae Phaeodactylum tricornutum upon exposures to WAF and CEWAF of crude oil: A case study coupled with stable isotopic signatures.

Increasing use of chemical dispersants for oil spills highlights the need to understand their adverse effects on marine microalgae and nutrient assimilation because the toxic components of crude oil can be more bioavailable. We employed the crude oil water-accommodated fraction (WAF) and chemically enhanced WAF (CEWAF) to compare different responses in marine microalgae (Phaeodactylum tricornutum) coupled with stable isotopic signatures. The concentration and proportion of high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs), which are key toxic components in crude oil, increased after dispersant addition. CEWAF exposure caused higher percent growth inhibition and a lower chlorophyll-a level of microalgae than those after WAF exposure. Compared with WAF exposure, CEWAF led to an enhancement in the self-defense mechanism of P. tricornutum, accompanied by an increased content of extracellular polymeric substances. 13C-depletion and carbon assimilation were altered in P. tricornutum, suggesting more HMW PAHs could be utilized as carbon sources by microalgae under CEWAF. CEWAF had no significant effects on the isotopic fractionation or assimilation of nitrogen in P. tricornutum. Our study unveiled the impact on the growth, physiological response, and nutrient assimilation of microalgae upon WAF and CEWAF exposures. Our data provide new insights into the ecological effects of dispersant applications for coastal oil spills.

Bisphenol A decreases the developmental toxicity and histopathological alterations caused by polystyrene nanoplastics in developing marine medaka Oryzias melastigma.

Nanoplastics (NPs) are emerging pollutants posing risks to marine biota and human health due to their small size and high bioavailability. However, there are still knowledge gaps regarding effects of co-existing pollutants on NPs toxicity to marine organisms at their respective environmentally relevant concentrations. Herein we investigated developmental toxicity and histopathological alterations caused by co-exposure of polystyrene nanoplastics (PS-NPs) and bisphenol A (BPA) to marine medaka, Oryzias melastigma. Embryos at 6 h post-fertilization were exposed to 50-nm PS-NPs (55 µg/L) or BPA (100 µg/L) or co-exposed to a combination of both. Results showed that PS-NPs exhibited decreased embryonic heart rate, larval body length, and embryonic survival as well as larval deformities such as hemorrhaging and craniofacial abnormality. When co-exposed, BPA mitigated all the adverse developmental effects caused by PS-NPs. PS-NPs also led to an increase in histopathological condition index of liver with early inflammatory responses, while co-exposure of BPA with PS-NPs did not. Our data suggest that the toxicity reduction of PS-NPs in the presence of BPA might result from the decreased bioaccumulation of PS-NPs caused by the interaction between BPA and PS-NPs. This study unveiled the impact of BPA on the toxicity of nanoplastics in marine fish during early developmental stages and highlight the need of more research on the long-term effects of complex mixtures in the marine environment by applying omics approaches to better understand the toxicity mechanism.

Long-Term Toxicity of 50-nm and 1-µm Surface-Charged Polystyrene Microbeads in the Brine Shrimp Artemia parthenogenetica and Role of Food Availability.

Micro and nanoplastics (MNPs) as emerging contaminants have become a global environmental issue due to their small size and high bioavailability. However, very little information is available regarding their impact on zooplankton, especially when food availability is a limiting factor. Therefore, the present study aims at evaluating the long-term effects of two different sizes (50 nm and 1 µm) of amnio-modified polystyrene (PS-NH2) particles on brine shrimp, Artemia parthenogenetica, by providing different levels of food (microalgae) supply. Larvae were exposed to three environmentally relevant concentrations (5.5, 55, and 550 µg/L) of MNPs over a 14-days of exposure with two food levels, high (3 × 105~1 × 107 cells/mL), and low (1 × 105 cells/mL) food conditions. When exposed to high food levels, the survival, growth, and development of A. parthenogenetica were not negatively affected at the studied exposure concentrations. By comparison, when exposed to a low food level, a U shape trend was observed for the three measured effects (survival rate, body length, and instar). Significant interactions between food level and exposure concentration were found for all three measured effects (three-way ANOVA, p < 0.05). The activities of additives extracted from 50 nm PS-NH2 suspensions were below toxic levels, while those from 1-µm PS-NH2 showed an impact on artemia growth and development. Our results demonstrate the long-term risks posed by MNPs when zooplankton have low levels of food intake.

Synthesis and release of fatty acids under the interaction of Ulva pertusa and Heterosigma akashiwo by stable isotope analysis.

Symbiosis of marine algae is inevitable in the marine environment, and species may occur interaction on the growth. In this study, the macroalgae Ulva pertusa and marine microalgae Heterosigma akashiwo were selected as target species to study the interaction mechanism between them. After the 8 days of co-cultivation, the inhibition on growth was observed for both of U. pertusa and H. akashiwo. Eight fatty acids in U. pertusa was detected, with the significant decrease in contents of polyunsaturated fatty acids (PUFAs) especially for C18:2, C18:3n-3 and C18:3n-6. Twelve fatty acids in H. akashiwo was detected, with the significant change for PUFAs. PUFA concentrations in the co-culture group were less than those in the mono-culture. Meanwhile the principal component analysis was conducted to insight into the interaction between U. pertusa and H. akashiwo by fatty acids content and carbon stable isotope ratio of fatty acids (δ13CFAs). Fatty acid content could not distinguish mono and co-culture. However, δ13CFAs could distinguish not only the culture time of algae, but also the living environment of algae. In addition, this study combined fatty acids content and δ13CFAs to explore the release of fatty acids by algae into the seawater. The C18:3n-3 was identified as the allelochemical released by U. pertusa to inhibit the growth of H. akashiwo. The ratio of δ13CFAs in seawater decreased. This study provides a theoretical basis for the symbiosis of marine algae, and a new method of compound-specific stable carbon isotopes was used to better explore the metabolism of fatty acids in algae.

Influence of fuel oil on Platymonas helgolandica: An acute toxicity evaluation to amino acids.

It is highly likely that the toxicity of water accommodated fractions (WAF) will influence marine microalgae, and consequently lead to potential risk for the marine ecological environment. However, it was often neglected whether WAF can influence the transformation of relative compounds in organisms. The metabolism of amino acids (AAs) can be used to track physiological changes in microalgae because amino acids are the basis of proteins and enzymes. In this study, using marine Chlorophyta Platymonas helgolandica as the test organism, the effects of different concentrations of WAF on AA compositions and stable carbon isotope ratios (δ13C) of individual AAs of Platymonas helgolandica were investigated. The results showed that the WAF of #180 fuel oil had an obvious suppressing effect on the growth and chlorophyll a content of microalgae. The growth inhibitory rate at 96 h was 80.66% at a WAF concentration of 0.50 mg L-1 compared with the control. Furthermore, seven among the 16 AAs, including alanine, cysteine, proline, aspartic acid, lysine, histidine and tyrosine, had relatively high abundance. Under the glycolysis pathway, the cysteine abundance was higher than control, meaning that the biosynthesized pathway of alanine through cysteine as a precursor could be damaged. Phosphoenolpyruvate (PEP) was an important synthesis precursor of alanine (leucine) and aromatic AA family (Phenylalanine and tyrosine), and played an important role in δ13CAAs fractionation under the WAF stress. Under the TCA pathway, to protect cell metabolism activities under WAF stress, the δ13C value of threonine and proline abundance in microalgae with the increase in WAF stress. Therefore, δ13CAAs fractionation can be used as a novel method for toxicity evaluation of WAF on future.

Response mechanisms of domoic acid in Pseudo-nitzschia multiseries under copper stress.

A complex relationship exists between copper stress and the accumulation and release of domoic acid (DA) in toxin-producing Pseudo-nitzschia cells. To clarify the changes and role of DA in this process, we exposed the toxin-producing P. multiseries and the non-toxin-producing P. pungens to copper stress (5 and 9 µM) for 96 h. Results showed that P. multiseries grew better than P. pungens under the two aforementioned copper concentrations. DA content in the cells of P. multiseries increased with increased copper stress, and the dissolved DA in the medium under the 9 µM copper treatment increased. DA addition at a 9 µM copper concentration reduced the copper content in P. multiseries cells and cell walls, but did not change the free copper ion content in culture medium. Adding DA to the medium reduced the malondialdehyde (MDA) content in the cells of P. multiseries under copper stress, DA addition also reduced the activities of catalase (CAT) and superoxide dismutase (SOD) at 5 µM Cu, and the activity of peroxidase (POD) at 9 µM Cu. This suggests that DA may not alleviate copper stress by improving the antioxidant defense system of algal cells, nor can it be complexed with copper ions in the medium to alleviate copper stress. Furthermore, the reactive oxygen species (ROS) scavenger N-tert-butyl-α-phenylnitrone (BPN) was used to study the DA accumulated in cells. The BPN addition significantly reduced the accumulation of DA in the cells under copper stress, suggesting that DA content in cells was closely related to ROS. Moreover, further experiments demonstrated that DA addition can improve the growth of P. multiseries under hydrogen peroxide stress. Our results indicate that DA alleviates P. multiseries oxidative damage when expose to copper stress.

Field study on bioaccumulation and translocation of polybrominated diphenyl ethers in the sediment-plant system of a national nature reserve, North China.

Polybrominated diphenyl ethers (PBDEs) are the ubiquitous contaminants in the coastal wetlands, with high persistence and toxicity. Environmental behaviors of PBDEs in sediment-plant system is a hot research area, where much uncertainties still occurred in field environment. In this study, the sediments and Suaeda heteroptera were synchronously collected to investigate the bioaccumulation and translocation of PBDEs in Liaohe coastal wetland. Mean concentrations of PBDEs in sediments, roots, stems and leaves were 8.37, 6.64, 2.42 and 1.40 ng/g d.w., respectively. Tissue-specific accumulation of PBDEs were detected in Suaeda heteroptera, with predominant accumulation in roots. Congener patterns of PBDEs were similar between sediments and roots, demonstrating root uptake as the key pathway of PBDE bioaccumulation. The proportions of lower brominated congeners increased from roots to leaves, implying the congener-specific translocation. Meanwhile, the lower brominated congeners exhibited higher sediment-tissue bioaccumulation (AFs) and translocation factors (TFs) compared to higher brominated congeners in Suaeda heteroptera, further verifying their preferential translocation. AFs and TFs of PBDEs were both not correlated with their log Kow, which was inconsistent with those of laboratory studies, reflecting the complicated behaviors of PBDEs in field environment. This is the first comprehensive report on bioaccumulation and translocation of PBDEs within Suaeda heteroptera in Liaohe coastal wetland.

Effect of water accommodated fractions of fuel oil on fixed carbon and nitrogen by microalgae: Implication by stable isotope analysis.

Effect of water accommodated fractions (WAF) of #180 fuel oil on fixed carbon and nitrogen in microalgae was studied by stable isotopes. Platymonas helgolandica, Heterosigma akashiwo and Nitzschia closterium were exposed to five WAF concentrations for 96 h. The δ13C value of microalgae was significantly lower than that of the control group, indicated that carbon was limited in the WAF concentrations. The δ13C value of microalgae appeared peak valley at 48 h in control group, corresponding to the enhanced capacity in carbon fixation during microalgae photosynthesis. The physiological acclimation capacity of microalgae was revealed by the occurrence time when the δ13C value was in peak valley, and thus the physiological acclimation capacity of microalgae decreased in the order of Nitzschia closterium > Heterosigma akashiwo > Platymonas helgolandica. Principal component analysis (PCA) were applied to the δ13C value in order to verify the "hormesis" phenomenon in microalgae. The δ13C value could discriminate between stimulatory effects at low doses and inhibitory effects at high doses. In addition, the present study also investigated the effect of the nitrogen on microalgae growth. Because microalgae could still absorb the NO3-N and release of NO2-N and NH4-N in present study, the nitrogen cycle in microalgae was in the equilibrium status. The δ15N value in microalgae exhibited no obvious change with the increasing of WAF concentrations at the same time. However, due to the enrichment of nitrogen, the δ15N value first increased gradually with the time and finally was stable. Overall, the fractionation of carbon and nitrogen stable isotopes illustrated that the effect of carbon on the growth of microalgae was more prominent than nitrogen. Stable isotopes was used to investigate the influence of WAF on fixed carbon and nitrogen in microalgae growth, providing a fundamental theoretical guidance for risk assessment of marine ecological environment.

Assessing microbial degradation degree and bioavailability of BDE-153 in natural wetland soils: Implication by compound-specific stable isotope analysis.

Microbial degradation is an important pathway for the attenuation of polybrominated diphenyl ethers (PBDEs) in natural soils. In this study, the compound-specific stable isotope analysis (CSIA) was applied to characterize microbial degradation of BDE-153, one of the prevailing and toxic PBDE congeners, in natural wetland soils. During the 45-day incubation, the residual percentages of BDE-153 decreased to 67.9% and 73.6% in non-sterilized soils spiked with 1.0 and 5.0 µg/g, respectively, which were both much lower than those in sterilized soils (96.0% and 97.2%). This result indicated that microbial degradation could accelerate BDE-153 elimination in wetland soils. Meanwhile, the significant carbon isotope fractionation was observed in non-sterilized soils, with δ13C of BDE-153 shifting from -29.4‰ to -26.7‰ for 1.0 µg/g and to -27.2‰ for 5.0 µg/g, respectively, whilst not in sterilized soils. This phenomenon indicated microbial degradation could induce stable carbon isotope fractionation of BDE-153. The carbon isotope enrichment factor (εc) for BDE-153 microbial degradation was first determined as -7.58‰, which could be used to assess the microbial degradation and bioavailability of BDE-153 in wetland soils. Based on δ13C and εc, the new methods were developed to dynamically and quantitatively estimate degradation degree and bioavailability of BDE-153 during degradation process, respectively, which could exclude interference of physical processes. This work revealed that CSIA was a promising method to investigate in situ microbial degradation of PBDEs in field studies.

The influence of carbon limitation on growth of Heterosigma akashiwo: A case study in fatty acids composition.

Heterosigma akashiwo is an algal blooms species, and thus selected as the target microalgae in this work. This study attempted to investigate the influence of carbon limitation on the growth of H. akashiwo. Experiments were carried out in CO2-unlimited and CO2-limited systems (both include three nutritional groups). The stable isotope signatures and compound-specific stable three nutritional groups carbon isotopic composition of fatty acids were measured. Here we hypothesized that the carbon limitation could lead to the enriched of stable isotope ratios in the H. akashiwo. The results showed that carbon limitation made δ13C and δ13CFAs values more and more enriched in H. akashiwo. δ13C values were enriched in normal group of H. akashiwo within CO2-limited. δ15N values were enriched in nitrogen deficiency of H. akashiwo within both CO2-unlimited and CO2-limited. Furthermore, compared with the exponential phase, the enriched in δ13C was detected during the stationary phase in H. akashiwo within CO2-limited. A total of 8 major FAs were detected in H. akashiwo. Within CO2-unlimited, nitrogen deficiency promoted the synthesis of 4 FAs (14:1n-5c, 16:0, 18:0 and 18:3n-6c) in exponential phase. Within CO2-limited, nitrogen deficiency promoted the synthesis of FAs 14:0, 16:0 and 18:3n-3c, while phosphorus deficiency promoted the synthesis of all 6 FAs in exponential phase. δ13CFAs of H. akashiwo within CO2-limited showed the valley values and were significantly greater than those within CO2-unlimited. Furthermore, δ13CFAs of stationary phase were greater than those of exponential phase. δ13C14:1n-5c in nitrogen deficiency were the highest of the three nutrient groups within CO2-unlimited. This may point out that δ13CFAs could become an indicator of marine phytoplankton blooms. Overall, the present study may provide a novel approach to investigate the physiology and lipid metabolism of H. akashiwo blooms by using stable isotope ratios coupled with FAs profiles.

Effects of CO2 restriction on growth of Nitzschia closterium: Evidence from stable isotopes and fatty acids.

Algae blooms frequently occur in the coastal areas of China, and pose numbers of adverse effects to marine environment. Nitzschia closterium is one of the algal species associated with algae blooms and was selected as the target algae of this work. This study was the first attempt to investigate the effect of carbon source restriction on the growth of N. closterium during marine phytoplankton blooms. Experiments were implemented in the CO2-restricted (include three nutritional conditions) and CO2-unrestricted systems, respectively. The stable isotope ratios (δ13C and δ15N) and fatty acids (FAs) profiles were measured. Here we hypothesized that the deficient carbon source could lead to the changes of stable isotope ratios and FAs profiles in the N. closterium. The results showed that the δ13C of N. closterium enriched under the CO2 restriction during the culture time (the isotopic difference greater than 27.8‰), whereas depleted under CO2-unrestricted system. Furthermore, within the CO2 restriction, δ15N showed the enrichment trend with increasing culture time under the nitrogen deficiency conditions, where leveled off under the nitrogen non-deficiency conditions. A total of 12 FAs in N. closterium were detected. Within CO2 restriction, FA 20:5n-3c was the predominant congener in all the three conditions. In addition, FA 16:1n-7c was highest in nitrogen deficient, while FA 16:3 was highest in phosphorus deficient condition. Consequently, the deficient carbon sources leaded to inhibit the FA (14:0, 16:0, 16:1n-7c, 16:3, 18:0, 18:3n-3c) synthesis. Overall, the present study provided the new approach to investigate the forming mechanisms of marine phytoplankton blooms by using stable carbon and nitrogen isotope compositions as well as FAs profiles.

Effect of oil spill on compound-specific stable carbon isotope composition of amino acid in Nitzschia closterium and Heterosigma akashiwo.

Marine microalgae, the most important primary producers in marine ecosystems, are susceptible to toxicants and usually selected as the promising organisms for chemical risk testing. The stable carbon isotope ratios (δ13C) of amino acids (AAs) were measured to determine if compound-specific stable isotope analysis could be used to provide insight into the toxic effects of oil spill on the marine microalgae. The water accommodated fractions (WAF) of #180 fuel oil were selected as the toxic targets with different concentrations in acute (96 h) toxic tests. Naphthalene, phenanthrene, anthracene, and fluoranthene were detected as the predominant congeners of polycyclic aromatic hydrocarbons in the WAF, and may be the main toxic components. At the same WAF concentration, the δ13C values of leucine in Nitzschia closterium increased with the culture time, whereas decreased in Heterosigma akashiwo. However, with the increasing WAF concentrations, the δ13C values of glutamic acid exhibited the increasing trend in both of the two kinds of microalgae at the same culture time. The similar phenomenon was also observed for histidine in H. akashiwo, while opposite in N. closterium, but both enriching in δ13C compared to the controlled experiments without the WAF. Under the different culture times and WAF concentrations, the δ13C values of alanine and methionine showed unexpected trend. These findings indicated that the synthesis pathways of AA in microalgae cells were affected by the WAF, thus leading to the carbon isotopic fractionation in AAs. The present study provide a novel approach to explore the toxicity mechanism of primary producers under oil spill stress, and monitor and evaluate the marine ecological risk of oil spill pollution.

Effect of oil spill stress on fatty acid stable carbon isotope composition of Ulva pertusa.

Petroleum is one of the most important pollutants in the marine ecosystem, and oil spills have a long-term effect on the marine environment. After an oil spill, petroleum continues to diffuse, flowing to the intertidal zone where it accumulates. Ulva pertusa is the main macroalgae species in the intertidal zone of the ocean, and petroleum is toxic to marine macroalgae. Fatty acids, which are involved in various physiological activities, including metabolism, are indispensable substances in organisms. Thus, an organism's fatty acid composition reflects its physiological state. Because fatty acids are carbon-rich compounds, their stable carbon isotope composition can be measured to assess the state of the organism. In this study, the effects of the water accommodated fraction (WAF) of different concentrations of 180# fuel oil and 0# diesel oil on the chlorophyll a and fatty acid carbon stable isotope composition (δ13CFAs) of U. pertusa were assessed. The fatty acid carbon stable isotope value varied among the different petroleum treatments, and high WAF concentrations significantly inhibited the synthesis of unsaturated fatty acids. Thus, exposure to petroleum products affected the synthesis of fatty acids in U. pertusa and also caused carbon stable isotope fractionation during the fatty acid synthesis process. The results indicate that the saturated fatty acid δ13C16:0 and the unsaturated fatty acid δ13C18:1 can be used as biomarkers to evaluate the degree of petroleum-induced stress in U. pertusa and that carbon stable isotope analysis of U. pertusa can be used as an environmental risk assessment tool in the intertidal zone.