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.

Acute and Chronic Effects of Crude Oil Water-Accommodated Fractions on the Early Life Stages of Marine Medaka (Oryzias melastigma, McClelland, 1839).

Oil spill is a major marine environmental pollution issue. Research regarding the long-term effects of oil spills on the early life stage of marine fish is still limited. In this study, the potential adverse impact of crude oil from one oil spill accident which occurred in the Bohai Sea on the early life stages of marine medaka (Oryzias melastigma, McClelland, 1839) was evaluated. A 96-h acute test (larvae) and a 21-d chronic test (embryo-larvae) of water-accommodated fractions (WAFs) from crude oil were conducted, respectively. The results of the acute test showed that only the highest concentration of WAFs (100.00%) significantly affected the mortality of larvae (p < 0.01) and that the 96 h-LC50 was 68.92% (4.11 mg·L-1 expressed as total petroleum hydrocarbons (TPHs)). Larval heart demonstrated histopathological alterations in all WAF-exposed groups. The chronic test results showed that, except for larval mortality, the total hatching success (%)/hatching time of embryos in WAF treatments was not significantly different from those of the control group (p > 0.05), and no malformation was found in surviving larvae after 21 d of exposure. Nevertheless, the exposed embryos and larvae in the highest concentration of WAFs (60.00%) demonstrated significantly reduced heart rate (p < 0.05) and increased mortality (p < 0.01), respectively. Overall, our results indicated that both acute and chronic WAF exposures had adverse impacts on the survival of marine medaka. In the early life stages, the heart of the marine medaka was the most sensitive organ which showed both structural alteration and cardiac dysfunction.

Polystyrene microplastics alleviate adverse effects of benzo[a]pyrene on tissues and cells of the marine mussel, Mytilus galloprovincialis.

As two major ubiquitous pollutants, microplastics (MPs) and polycyclic aromatic hydrocarbons (PAHs) coexist in the marine environment. However, the role of MPs in altering the toxicity of PAHs to marine organisms is poorly understood. We therefore investigated the accumulation and toxicity of benzo[a]pyrene (B[a]P, 0.4 nM), in the marine mussel Mytilus galloprovincialis over a 4-day of exposure with or without the presence of 10 µm polystyrene microplastics (PS MPs) (10 particles/mL). The presence of PS MPs significantly decreased B[a]P accumulation in soft tissues of M. galloprovincialis by approximately 6.7%. Single exposure of PS MPs or B[a]P decreased the mean epithelial thickness (MET) of digestive tubules and enhanced reactive oxygen species (ROS) levels in haemolymph, while upon co-exposure the adverse impacts were alleviated. Real-time q-PCR results showed that most selected genes involved in stress response (FKBP, HSP90), immune (MyD88a, NF-κB) and detoxification (CYP4Y1) were induced for both single exposure and co-exposure. The co-presence of PS MPs down-regulated the mRNA expression of NF-κB in gills compared with of B[a]P alone. The uptake and toxicity reductions of B[a]P might result from the decrease of its bioavailable concentrations caused by the adsorption of B[a]P by PS MPs and the strong affinity of B[a]P to PS MPs. Adverse outcomes for the co-existence of marine emerging pollutants under long-term conditions remain to be further validated.

Lethal, behavioral, growth and developmental toxicities of alkyl-PAHs and non-alkyl PAHs to early-life stage of brine shrimp, Artemia parthenogenetica.

Alkyl-PAHs are the predominant form of PAHs in crude oils which are supposed to demonstrate different toxicities compared to non-alkyl PAHs. Little information is available about the toxicity of alkyl-PAHs on marine Artemia. This study addressed and compared the lethal, behavioral, growth and developmental toxicities of three alkyl-PAHs, namely 3-methyl phenanthrene (3-mPhe), retene (Ret) and 2-methyl anthracene (2-mAnt), to their non-alkyl forms, phenanthrene (Phe) and anthracene (Ant) using Artemia parthenogenetica (nauplii, <24 h) as test organism following a 48 h and a 7 d of exposure, respectively. Benzo-a-pyrene (Bap) was selected as a reference toxicant for the comparison with the above alkyl-PAHs and non-alkyl PAHs. Results showed that for all tested endpoints, A. parthenogenetica nauplii had the highest sensitivity to Bap while Ant had no significant effect on nauplii survival or development within given concentrations. Considering the aqueous freely dissolved PAH concentrations, the 48 h-LC50 (survival), 48 h-EC50 (immobility) and 7 d-LC10 (survival) of Bap were calculated as 0.321, 0.285 and 0.027 µg/L, respectively, which were twofold to fivefold lower than those of Phe, 3-mPhe, Ret, Ant and 2-mAnt. A higher acute toxicity of alkyl-PAHs (3-mPhe and 2-mAnt) than their non-alkyl forms (Phe and Ant) was observed. Not limited to Phe, the common non-polar narcotic mode of action was also observed for Bap, 3-mPhe, Ret and 2-mAnt, which was evident by the inhibited mobility of nauplii. The decreased body lengths were found for all PAH treatments compared to the solvent control, whereas instar retardations were only found in nauplii exposed to Bap, Phe and Ret. Our findings emphasized the sensitivity differences of A. parthenogenetica nauplii to selected alkyl PAHs and non-alkyl PAHs and confirmed the application of lethal, behavioral and growth indicators in the toxicity evaluation of selected PAHs other than Ant. However, the distinct toxicities of these PAHs suggested other toxic modes of action may play more important roles apart from narcotic mode of action and need to be elucidated in future studies. In addition, a strong correlation between the body length and the instar of A. parthenogenetica nauplii was observed for each PAH exposure, suggesting that body length can be representative for both growth and developmental indicators during biological monitoring of PAH pollution in marine environment.

Acute and chronic combined effect of polystyrene microplastics and dibutyl phthalate on the marine copepod Tigriopus japonicus.

Dibutyl phthalate (DBP) is a commonly used additive in plastic products, so it may potentially coexist with microplastics (MPs) in marine environment. The ingestion of MPs might affect the accumulation of DBP in marine organisms. In this study, the marine copepod Tigriopus japonicus was applied to study the combined effect of DBP and polystyrene microplastics (mPS) on the copepod through both acute mortality tests and chronic reproduction tests. The LC50 of DBP was 1.23 mg L-1 (95% CI: 1.11-1.35 mg L-1), while exposure to mPS didn't have significant lethal effect on the copepods. Adsorption to MPs led to decreased bioavailability of DBP, resulting in decreased toxicity of DBP. In contrast to the results of acute toxicity tests, DBP didn't affect the reproduction of the copepods at lower exposure concentrations, while mPS reduced the number of nauplii and extended the time to hatch. Similar as acute toxicity tests, antagonistic interaction was observed for mPS and DBP in chronic reproduction tests, which might be attributed to promoted aggregation of mPS at presence of DBP. Overall, antagonistic toxicity effect between the two pollutants was observed for both acute and chronic tests, but the mechanisms of the interaction between DBP and mPS were different. Results of the present study highlighted the importance of long-term exposure when evaluating the toxic effect of MPs and their combined effect with other chemicals.

Transcriptomic responses of Artemia salina exposed to an environmentally relevant dose of Alexandrium minutum cells or Gonyautoxin2/3.

Toxicities of the marine algae Alexandrium minutum and its excreted gonyautoxins (GTXs) to the marine crustacean Artemia salina were investigated. Mortality was observed for neither larvae nor adult A. salina exposed to A. minutum at a density of 5000 cells/mL or 0.5 µM GTX2/3. After exposure, the full transcriptome of adult A. salina was assembled and functionally annotated. A total of 599,286 transcripts were obtained, which were clustered into 515,196 unigenes. Results of the transcriptional effect level index revealed that direct exposure to the toxic algae A. minutum caused greater alterations in the transcriptome than did exposure to the extracellular product GTX2/3. Mechanisms of effects were different between exposure of A. salina to A. minutum cells or GTX2/3. Exposure to A. minutum modulated formation of the ribonucleoprotein complex and metabolism of amino acids and lipids in A. salina. Exposure to GTX2/3 exposure inhibited expression of genes related to metabolism of chitin, which might result in disruption of molting process or disturbed sheath morphogenesis. Overall, effects on transcription observed in this study represent the first report based on application of next generation sequencing techniques to investigate the transcriptomic response of A. salina exposed to an environmentally realistic level of A. minutum or GTX2/3.

Combined effect of polystyrene microplastics and dibutyl phthalate on the microalgae Chlorella pyrenoidosa.

The combined effect of polystyrene microplastics (mPS) and dibutyl phthalate (DBP), a common plastic additive, on the microalgae Chlorella pyrenoidosa was investigated in the present study. The 96 h-IC50 value of DBP was 2.41 mg L-1. Polystyrene microplastics exhibited size-dependent inhibitory effect to C. pyrenoidosa, with the 96 h-IC50 at 6.90 and 7.19 mg L-1 for 0.1 and 0.55 µm mPS respectively, but little toxicity was observed for 5 µm mPS. The interaction parameter ρ based on the response additive response surface (RARS) model varied from -0.309 to 5.845, indicating the interaction pattern varying with exposure concentrations of chemical mixtures. A modified RARS model (taking ρ as a function of exposure concentration) was constructed and could well predict the combined toxicity of mPS and DBP. More than 20% reduction of DBP was observed at 20 mg L-1 mPS, while 1 mg L-1 mPS had no significant effect on the bioavailability of DBP at different sampling time points. Volume, morphological complexity and chlorophyll fluorescence intensity of microalgal cells were disturbed by both DBP and mPS. The antagonistic effect of high concentrations of mPS might be partially attributed to the combination of hetero- and homo-aggregation and the reduced bioavailability of DBP. The overall findings of the present study profiled the combined toxic effects of mPS and DBP on marine phytoplankton species which will be helpful for further evaluation of ecological risks of mPS and DBP in marine environment.

The effect of polystyrene plastics on the toxicity of triphenyltin to the marine diatom Skeletonema costatum-influence of plastic particle size.

The effect of polystyrene (PS) particles on the toxicity of triphenyltin (TPT) to the marine diatom Skeletonema costatum was investigated. The 0.1-µm PS particles attached to the cell walls of S. costatum but did not cause adverse effects on the growth of the diatom. The adsorption of TPT to PS particles was negligible in seawater systems, but the presence of 0.1-µm PS significantly reduced the bioavailable concentrations of TPT in f/2-Si medium, indicating a potential three-way interaction between TPT, PS particles, and components of f/2-Si medium. The adsorption of TPT to PS of smaller size (i.e., 0.1 µm) was stronger than that of PS of larger size (i.e., 5 µm), which was probably attributed to larger surface areas of smaller PS particles. The presence of PS could reduce the toxicity of TPT. IC50 values of TPT increased from 0.56 to 0.85 and 0.71 µg/L at the presence of 20 mg/L 0.1-µm PS and 5-µm PS, respectively. The overall results of this study profiled the combined toxic effects of PS and TPT on marine phytoplankton species and highlighted the difference in adsorption of organic pollutants by microplastics in different ambient mediums.

Effect of Multi-walled Carbon Nanotubes on the Toxicity of Triphenyltin to the Marine Copepod Tigriopus japonicus.

Marine organisms are often exposed to a mixture of various pollutants in marine environment (i.e., nanoparticles, organic pollutants). The present study investigated the potential effects of multi-walled carbon nanotubes (MWCNTs) on the toxicity of triphenyltin chloride (TPTCl). The results revealed an antagonistic interaction between MWCNTs and TPTCl on the copepod through 96 h acute exposure, which was attributed to the adsorption of TPTCl to MWCNTs and aggregation of MWCNTs in the test solutions. Results of 21 days' chronic exposure showed that the effect concentration of MWCNTs could be 100 times lower than that of acute exposure. The exposure to binary mixture of MWCNT (1.0 mg/L) and TPTCl (0.3 µg/L) caused a reduction by 94% for the 3rd time spawning and 83% for the total number of hatched nauplii. The ingestion and exterior attachment of MWCNTs to the copepod might be the main reasons causing the adverse effect in reproduction.

Combined effect of polystyrene plastics and triphenyltin chloride on the green algae Chlorella pyrenoidosa.

The combined effect of polystyrene (PS) particles and triphenyltin chloride (TPTCl) to the green algae Chlorella pyrenoidosa was studied. The 96 h IC50 of TPTCl to the green algae C. pyrenoidosa was 30.64 µg/L. The toxicity of PS particles to C. pyrenoidosa was size-dependent, with the 96 h IC50 at 9.10 mg/L for 0.55 µm PS but no toxicity observed for 5.0 µm PS. The exposure to 0.55 µm PS led to damage on structure of algal cells, which could in turn cause inhibition on photosynthesis and population growth of the green algae. TPTCl concentrations in test medium were lowered by 15-19% at presence of 0.55 µm PS particles, indicating a reduced bioavailability of TPTCl. In spite of this reduced bioavailability, the presence of PS increased the toxicity of TPTCl, which might be attributed to facilitated uptake of TPTCl by the green algae after the damage of cell structure. The overall results of the present study provided important information on the effect of PS on the bioavailability and toxicity of TPTCl to phytoplankton species.

Toxic effect of triphenyltin in the presence of nano zinc oxide to marine copepod Tigriopus japonicus.

Marine organisms are naturally exposed to different environmental pollutants including organic pollutants and nanoparticles. The interactive effects between nanoparticles and other chemicals on aquatic organisms have raised concerns regarding the potential of nanomaterials as the vector for other chemicals. In the present study, the effect of nano zinc oxide (nZnO) on the bioavailability of triphenyltin chloride (TPTCl) was studied, and their combined acute and reproductive toxicity to the marine copepod Tigriopus japonicus were evaluated. At experimental concentration ranges of nZnO in this study, the percentage of dissolution of Zn2+ was relative stable (from 62% to 66%), and nZnO did not affect the bioavailability of TPTCl to the copepods. The acute toxicity of binary mixtures of nZnO/TPT was equivalent to that of the mixture of Zn2+/TPT. In agreement with the decrease in TPTCl's LC50 values at the presence of nZnO, their interacting effect was synergistic based on response addition response surface model, and the interacting parameter was modelled to be -1.43. In addition to acute toxicity test, reproductive toxicity tests revealed that exposure to nZnO and TPTCl didn't affect the successful mating rate and the number of nauplii in the 1st brood, but they extended the time for the eggs to hatch from 2.53 days to 3.94 and 3.64 days, respectively. The exposure to nZnO/TPTCl mixture delayed the time to hatch to 5.78 days.