Enchytraeus crypticus Avoid Soil Spiked with Microplastic.

Microplastics (MPs) of varying sizes are widespread pollutants in our environment. The general opinion is that the smaller the size, the more dangerous the MPs are due to enhanced uptake possibilities. It would be of considerably ecological significance to understand the response of biota to microplastic contamination both physically and physiologically. Here, we report on an area choice experiment (avoidance test) using Enchytraeus crypticus, in which we mixed different amounts of high-density polyethylene microplastic particles into the soil. In all experimental scenarios, more Enchytraeids moved to the unspiked sections or chose a lower MP-concentration. Worms in contact with MP exhibited an enhanced oxidative stress status, measured as the induced activity of the antioxidative enzymes catalase and glutathione S-transferase. As plastic polymers per se are nontoxic, the exposure time employed was too short for chemicals to leach from the microplastic, and as the microplastic particles used in these experiments were too large (4 mm) to be consumed by the Enchytraeids, the likely cause for the avoidance and oxidative stress could be linked to altered soil properties.

Self-contamination from clothing in microplastics research.

Self-contamination should not be underestimated when quantifying microplastics (MPs) in environmental matrices. Standardised and validated methodologies for MP sampling, extraction, and analysis are lacking. The various applications of plastics in our society have made them ubiquitous, even in clothing, rendering MP self-contamination inevitable. In the present study, we sampled lake sediment, snow, and ice, purposefully wearing red overalls composed of cotton; fibres from which we could quantify using Fourier-Transform Infrared Spectroscopy (FTIR), serving as an indication of possible self-contamination from clothes. The suitability of cotton as a representation of MP contamination was also evaluated. For all detected fibres, 25 ± 1%, 20 ± 7%, and 8 ± 6% for snow, ice, and sediment, respectively, originated from sampling attire. These findings demonstrate that self-contamination can play a significant role when quantifying MP pollution, highlighting that sampling conducted to date might have overestimated the presence of MP or even contaminated MP-free samples.

Correction to: Assessment of microplastic pollution: occurrence and characterisation in Vesijärvi lake and Pikku Vesijärvi pond, Finland.

In the original publication, Figure 5 and the related text incorrectly referred to a polyurethane fibre; however, it was a possible PET-polyurethane blend.

Uptake, Growth, and Pigment Changes in Lemna minor L. Exposed to Environmental Concentrations of Cylindrospermopsin.

Cylindrospermopsin (CYN)-producing cyanobacterial blooms such as Raphidiopsis, Aphanizomenon, Anabaena, Umezakia, and Lyngbya spp. are occurring more commonly and frequently worldwide. CYN is an environmentally stable extracellular toxin, which inhibits protein synthesis, and, therefore, can potentially affect a wide variety of aquatic biota. Submerged and floating macrophytes, as primary producers in oligotrophic habitats, are at risk of exposure and information on the effects of CYN exposure at environmentally relevant concentrations is limited. In the present study, we investigated CYN uptake in the floating macrophyte Lemna minor with exposure to reported environmental concentrations. The effects were evaluated in terms of bioaccumulation, relative plant growth, and number of fronds per day. Variations in the concentrations and ratios of the chlorophylls as stress markers and carotenoids as markers of oxidative stress defense were measured. With exposure to 25 µg/L, L. minor could remove 43% of CYN within 24 h but CYN was not bioaccumulated. Generally, the pigment concentrations were elevated with exposure to 0.025, 0.25, and 2.5 µg/L CYN after 24 h, but normalized quickly thereafter. Changes in relative plant growth were observed with exposure to 0.25 and 2.5 µg/L CYN. Adverse effects were seen with these environmentally realistic concentrations within 24 h; however, L. minor successfully recovered within the next 48-96 h.

Assessment of microplastic pollution: occurrence and characterisation in Vesijärvi lake and Pikku Vesijärvi pond, Finland.

In the last few years, several studies have investigated microplastics (MPs) in marine ecosystems, but data monitoring and assessing the occurrence in freshwater environments are still scarce. The present study aims to investigate the occurrence, distribution, and chemical composition of MP pollution in Vesijärvi lake and Pikku Vesijärvi pond close to the city of Lahti (Finland) in winter. Sediment, snow, and ice core samples were collected near the shore of these two aquatic systems. MPs were analysed and identified by a non-destructive method using Fourier transform infrared spectroscopy (FTIR) 2D imaging. The mean concentrations of MPs detected in sediment, snow, and ice samples were 395.5 ± 90.7 MPs/kg, 117.1 ± 18.4 MPs/L, and 7.8 ± 1.2 MPs/L, respectively. FTIR results showed the predominant abundance of microplastics, such as polyamides (up to 53.3%), polyethylene and polypropylene (up to 17.1%), and natural fragments such as cellulose (up to 45.8%) and wool (up 18.8%) in the same size range. The potential release of MPs arising from stormwaters and sport and recreational activities was evidenced.

Translocation of the cyanobacterial toxin microcystin-LR into guttation drops of Triticum aestivum and remaining toxicity.

Uptake of the commonly occurring cyanobacterial toxin microcystin-LR (MC-LR) into crop plants via spray irrigation has been demonstrated. As other hazardous compounds such as pesticides were shown to be transported within plants, it was essential to understand the transport and fate of MC-LR in plants and the risks posed to grazers and other consumers. Of specific interest was to investigate if MC-LR could be detected in guttation drops and the toxicity thereof. Triticum aestivum (wheat) seedlings were exposed to 100 µg L-1 MC-LR in two separate experiments during which guttation drops were collected at various time points. The plants of one experiment were sectioned to investigate MC-LR distribution to the various plant appendages via liquid chromatography-tandem mass spectrometry analysis. After exposure, MC-LR could be detected in the roots, stems, leaves, and the guttation drops. However, the guttation drops were not toxic to Daphnia. As the environmentally relevant toxin concentration used was not sufficient to promote mortality in Daphnia, the physiological effect in insects, which rely on guttation drops as a water source, remains unknown. Combined with other contaminants that insects may be exposed to, the additional MC-LR exposure could contribute to the overall toxicity through the "tears of death".

Desmodesmus subspicatus co-cultured with microcystin producing (PCC 7806) and the non-producing (PCC 7005) strains of Microcystis aeruginosa.

Although microcystins (MCs) are the most commonly studied cyanotoxins, their significance to the producing organisms remains unclear. MCs are known as endotoxins, but they can be found in the surrounding environment due to cell lysis, designated as extracellular MCs. In the present study, the interactions between MC producing and the non-producing strains of Microcystis aeruginosa, PCC 7806 and PCC 7005, respectively, and a green alga, Desmodesmus subspicatus, were studied to better understand the probable ecological importance of MCs at the collapse phase of cyanobacterial blooms. We applied a dialysis co-cultivation system where M. aeruginosa was grown inside dialysis tubing for one month. Then, D. subspicatus was added to the culture system on the outside of the membrane. Consequently, the growth of D. subspicatus and MC contents were measured over a 14-day co-exposure period. The results showed that Microcystis negatively affected the green alga as the growth of D. subspicatus was significantly inhibited in co-cultivation with both the MC-producing and -deficient strains. However, the inhibitory effect of the MC-producing strain was greater and observed earlier compared to the MC-deficient strain. Thus, MCs might be considered as an assistant factor that, in combination with other secondary metabolites of Microcystis, reinforce the ability to outcompete co-existing species.

Interspecies interactions between Microcystis aeruginosa PCC 7806 and Desmodesmus subspicatus SAG 86.81 in a co-cultivation system at various growth phases.

In lakes, cyanobacterial blooms are frequently associated with green algae and dominate the phytoplankton community in successive waves. In the present study, the interactions between Microcystis aeruginosa PCC 7806 and Desmodesmus subspicatus were studied to clarify the probable ecological significance of algal secondary metabolites; focusing on the role of cyanotoxin 'microcystin-LR' (MC-LR). A dialysis co-cultivation technique was applied where M. aeruginosa was grown inside and D. subspicatus was cultured outside of the dialysis tubing. The concentration of the intra- and extracellular MC-LR and the growth of two species were measured at different time points over a period of one month. Additionally, the growth of the two species in the culture filtrate of one another and the effect of the purified MC-LR on the growth of the green alga were studied. The results indicated that the co-existing species could affect each other depending on the growth phases. Despite the early dominance of D. subspicatus during the logarithmic phase, M. aeruginosa suppressed the growth of the green alga at the stationary phase, which coincided with increased MC production and release. However, the inhibitory effects of Microcystis might be related to its other extracellular metabolites rather than, or possibly in addition to, MC.

Phytoremediation: green technology for the removal of mixed contaminants of a water supply reservoir.

The Iraí Reservoir, a water supply in Brazil, is constantly impacted by anthropogenic activities such as waste inputs from agriculture, hospitals and urbanization, resulting toxic cyanobacterial blooms causing economic, social and environmental problems. This study assessed the concentration of some common contaminants of the Iraí Reservoir, namely paracetamol, diclofenac and microcystin-LR and tested whether a laboratory scale Green Liver System® would serve as a suitable technology to remove these contaminants. Further, the study investigated whether the pollutants caused adverse effects to the macrophytes using catalase as a biomarker for oxidative stress and investigated whether biotransformation (glutathione S-transferase) was a main route for detoxification. Egeria densa, Ceratophyllum demersum and Myriophyllum aquaticum were exposed to a mixture of the three contaminants for 14 days in a concentration range similar to those detected in the reservoir. The plants removed 93% of diclofenac and 100% of MC-LR after 14 days. Paracetamol could not be detected. Catalase and glutathione S-transferase enzyme activities remained unaltered after the 14-day exposure, indicating that the mixture did not cause oxidative stress. The study showed that the aquatic macrophytes used are suitable tools to apply in a Green Liver System® for the remediation of mixed pollutants.

Vegetables cultivated with exposure to pure and naturally occurring ß-N-methylamino-L-alanine (BMAA) via irrigation.

Bioaccumulation and biomagnification of ß-N-methylamino-L-alanine (BMAA), a potent neurotoxin, has been demonstrated in various food webs. It is alarming as this intensification of BMAA will result in exposure to higher concentrations from a direct cyanobacterial source. As more food items are being identified as a source of BMAA and with the large variations in BMAA content, the aim of the present study was to evaluate BMAA uptake by, and accumulation in, two commonly consumed vegetables, Lactuca sativa and Allium fistulosum. Plants exposed to pure BMAA in controlled laboratory experiments, as well as vegetables naturally irrigated with water containing a BMAA producing cyanobacterial bloom were evaluated during growth and ripening. In the laboratory exposures, free BMAA was detected in both the edible ripe parts of L. sativa and A. fistulosum after 60 days of exposure to a total of 4.5 µg BMAA. However, in the bloom exposure samples no BMAA could be detected in the ripe vegetables of A. fistulosum, Cucurbita pepo, or Brassica rapa chinensis. The study emphasises the need to further screen items for BMAA to understand the human exposure risk as well as the difference between BMAA uptake patterns with free BMAA and that contained in cyanobacterial cells.

Protein association of ß-N-methylamino-L-alanine in Triticum aestivum via irrigation.

Bioaccumulation of several cyanotoxins has been observed in numerous food webs. More recently, the neurotoxic, non-proteinogenic amino acid ß-N-methylamino-L-alanine (BMAA) was shown to biomagnify in marine food webs. It was thus necessary to assess whether a human exposure risk via a terrestrial food source could exist. As shown for other cyanotoxins, spray irrigation of crop plants with cyanobacterial bloom-contaminated surface water poses the risk of toxin transfer into edible plant parts. Therefore, in the present study, we evaluated a possible transfer of BMAA via spray irrigation into the seeds of one of the world's most widely cultivated crop plants, Triticum aestivum. Wheat plants were irrigated with water containing 10 µg L-1 BMAA until they reached maturity and seed-bearing stage (205 days). Several morphological characteristics, such as germination rate, number of roots per seedling, length of primary root and cotyledon, and diameter of the stems were evaluated to assess the effects of chronic exposure. After 205 days, BMAA bioaccumulation was quantified in roots, shoots, and mature seeds of T. aestivum. No adverse morphology effects were observed and no free intracellular BMAA was detected in any of the exposed plants. However, in mature seeds, protein-associated BMAA was detected at 217 ± 150 ng g FW-1; significantly more than in roots and shoots. This result demonstrates the unexpected bioaccumulation of a hydrophilic compound and highlights the demand to specify in addition to limit values for drinking water, tolerable daily intake rates for the cyanobacterial-neurotoxin BMAA.

Uptake and biotransformation of pure commercial microcystin-LR versus microcystin-LR from a natural cyanobacterial bloom extract in the aquatic fungus Mucor hiemalis.

OBJECTIVES: To evaluate the remediation efficiency of Mucor hiemalis by comparing media elimination, uptake, and biotransformation of microcystin-LR with exposure to pure toxin versus a crude bloom extract. RESULTS: With exposure to the extract, the elimination rate of microcystin-LR from the media, which was 0.28 ng MC-LR l-1 h-1, was significantly higher compared to that achieved with exposure to the pure toxin (0.16 ng MC-LR l-1 h-1) after 24 h. However, intracellular breakdown of microcystin-LR was significantly lower in the extract exposed pellets compared to the pure toxin treated fungal pellets over time. This coincided with reduced intracellular glutathione S-transferase activity with crude extract exposure which could be responsible for the detection of only the glutathione conjugate of microcystin-LR. CONCLUSION: This paper signifies the importance of using laboratory exposure scenarios which resemble conditions in nature to fully understand and evaluate remediation efficiency. There is merit in using M. hiemalis for mycoremediation of cyanotoxins in surface waters.

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins.

OBJECTIVES: To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications. RESULTS: Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l-1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l-1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. ß-N-methylamino-L-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected. CONCLUSIONS: M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

Inability to detect free cylindrospermopsin in spiked aquatic organism extracts plausibly suggests protein binding.

Even though the frequency and prevalence of cylindrospermopsin producing cyanobacteria are increasing, several publications have reported the absence of free cylindrospermopsin bioaccumulation in aquatic food chains. Cylindrospermopsin modification by protein binding has been suggested, however, only one publication has investigated this with eukaryotic reticulocyte lysate and concluded that cylindrospermopsin binds non-covalently to soluble proteins larger than 100 kDa associated with eukaryotic translation. With this as the extent of knowledge regarding cylindrospermopsin binding, the present study aimed to determine whether cylindrospermopsin binding also occurred with other proteins. In the present study, proteins from various organisms were extracted, incubated with cylindrospermopsin, and the amount of free cylindrospermopsin was determined by liquid chromatography tandem mass spectroscopy. Additionally, cylindrospermopsin binding to various ammonium sulfate precipitation fractions of Egeria densa protein, as well as with selected amino acids was investigated. We find that the percentage of free cylindrospermopsin varied with exposure to various animal and plant proteins as well as with various fractions of proteins but found no binding with single amino acids.

Oxidative stress responses in the animal model, Daphnia pulex exposed to a natural bloom extract versus artificial cyanotoxin mixtures.

In the natural environment, Daphnia spp. are constantly exposed to a complex matrix of biomolecules, especially during cyanobacterial bloom events. When cyanobacterial cells decay, not only are toxic secondary metabolites known as cyanotoxins released, but also multiple other secondary metabolites, some of which act as enzyme inhibitors. The present study examined the effects of such a natural toxin matrix (crude extract from a bloom) versus artificial toxin mixtures in terms of oxidative stress in Daphnia pulex. The results indicate that there is no significant effect on the survival of D. pulex. However, exposure to the bloom extract resulted in increased lipid peroxidation over a shorter exposure period and reduced antioxidative enzyme activities when compared to the artificial mixtures. The daphnids also needed a longer recovery time to reduce the increased cellular hydrogen peroxide concentration associated with the exposure to the crude extract than with the artificial mixtures. The results indicate a significant difference between the bloom crude extract and the two synthetic mixtures for all stress markers tested, indicating enhanced toxicity of the bloom extract.

Using aquatic fungi for pharmaceutical bioremediation: Uptake of acetaminophen by Mucor hiemalis does not result in an enzymatic oxidative stress response.

The increasing anthropogenic pollution of aquatic environments and fresh water scarcity worldwide have prompted the development of low-cost and effective water treatment alternatives. One example of a highly released anthropogenic xenobiotics is acetaminophen (APAP), which has been detected in surface waters at concentrations as high as 5 µg L(-1). To date, traditional water treatment plants were unable to remove all pharmaceutical xenobiotics and as in the case with APAP, the breakdown products are toxic. Phytoremediation has proved to remove xenobiotics efficiently producing no toxic breakdown products, however, they are often restrained in their application range. Therefore, it was necessary to find alternate remediation tools to extend and complement the application ranges of existing bioremediation techniques. With the success of mycoremediation as well as the adaptability of fungi, Mucor hiemalis was investigated in terms of its APAP uptake capabilities. The investigation included the examination of concentration- and time-dependent uptake studies to examine the effects of each of these parameters independently. Additionally, the extracellular peroxidase activity of M. hiemalis was measured with exposure to APAP to evaluate possible breakdown and the antioxidative stress enzymes, catalase, glutathione peroxidase, and glutathione reductase, were assayed to investigate whether APAP caused oxidative stress. The results showed that M. hiemalis was able to internalize between 1 and 2 µg APAP per g dried fungal biomass when exposed to 5, 10, 50 and 100 ng mL(-1) APAP for 24-48 h, but not beyond this time frame. Further, exposure to APAP did not result in elevated extracellular peroxidase activity or oxidative stress. The findings led to the conclusion that M. hiemalis could be integrated in bioremediation systems, for short-term degradation at low concentrations of APAP with effective management.

LC-MS/MS method development for quantitative analysis of acetaminophen uptake by the aquatic fungus Mucor hiemalis.

Acetaminophen is a pharmaceutical, frequently found in surface water as a contaminant. Bioremediation, in particular, mycoremediation of acetaminophen is a method to remove this compound from waters. Owing to the lack of quantitative analytical method for acetaminophen in aquatic organisms, the present study aimed to develop a method for the determination of acetaminophen using LC-MS/MS in the aquatic fungus Mucor hiemalis. The method was then applied to evaluate the uptake of acetaminophen by M. hiemalis, cultured in pellet morphology. The method was robust, sensitive and reproducible with a lower limit of quantification of 5 pg acetaminophen on column. It was found that M. hiemalis internalize the pharmaceutical, and bioaccumulate it with time. Therefore, M. hiemalis was deemed a suitable candidate for further studies to elucidate its pharmaceutical tolerance and the longevity in mycoremediation applications.

Antioxidative stress responses in the floating macrophyte Lemna minor L. with cylindrospermopsin exposure.

Cylindrospermopsin toxicity and oxidative stress have been examined in aquatic animals, however, only a few studies with aquatic plants have been conducted focusing on the potential for bioaccumulation of cylindrospermopsin. The oxidative stress effects caused by cylindrospermopsin on macrophytes have not yet been specifically studied. The oxidative stress response of Lemna minor L. with exposure to cylindrospermopsin, was therefore tested in this study. The hydrogen peroxide concentration together with the activities of the antioxidant enzymes (catalase, peroxidase, glutathione reductase and glutathione S-transferase) were determined after 24h (hours) of exposure to varying concentrations (0.025, 0.25, 2.5 and 25µg/L) of cylindrospermopsin. Responses with longer exposure periods (48, 96, 168h) were tested only with exposure to 2.5 and 25µg/L cylindrospermopsin. Additionally, the content of the carotenoids was determined as a possible non-enzymatic antioxidant defence mechanism against cylindrospermopsin. The levels of hydrogen peroxide increased after 24h even at the lowest cylindrospermopsin exposure concentrations. Catalase showed the most representative antioxidant response observed after 24h and maintained its activity throughout the experiment. Catalase activity corresponded with the contents of hydrogen peroxide at 2.5 and 25µg/L cylindrospermopsin. The data suggest that glutathione S-transferase, glutathione reductase and the carotenoid content act together with catalase but are more sensitive to higher concentrations of cylindrospermopsin and after a longer exposure period (168h). The results indicate that cylindrospermopsin promotes oxidative stress in L. minor at concentrations of 2.5 and 25µg/L. However, L. minor has sufficient defence mechanisms in place against this cyanobacterial toxin. Even though L. minor exhibits the potential to managing and control cylindrospermopsin contamination in aquatic systems, further studies in tolerance limits to cylindrospermopsin, uptake and experiments with prolonged exposure periods of more than 7 days are required.

Development and validation of an in-house quantitative analysis method for cylindrospermopsin using hydrophilic interaction liquid chromatography-tandem mass spectrometry: Quantification demonstrated in 4 aquatic organisms.

The cyanobacterial toxin cylindrospermopsin (CYN) is of great concern in aquatic environments because of its incidence, multiple toxicity endpoints, and, therefore, the severity of health implications. It may bioaccumulate in aquatic food webs, resulting in high exposure concentrations to higher-order trophic levels, particularly humans. Because of accumulation at primary levels resulting from exposure to trace amounts of toxin, a sensitive analytical technique with proven aquatic applications is required. In the present study, a hydrophilic interaction liquid chromatographic-tandem mass spectrometric method with a lower limit of detection of 200 fg on column (signal-to-noise ratio = 3, n = 9) and a lower limit of quantification of 1 pg on column (signal-to-noise ratio = 11, n = 9) with demonstrated application in 4 aquatic organisms is described. The analytical method was optimized and validated with a linear range (r(2) = 0.999) from 0.1 ng mL(-1) to 100 ng mL(-1) CYN. Mean recovery of the extraction method was 98 ± 2%. Application of the method was demonstrated by quantifying CYN uptake in Scenedesmus subspicatus (green algae), Egeria densa (Brazilian waterweed), Daphnia magna (water flea), and Lumbriculus variegatus (blackworm) after 24 h of static exposure to 50 µg L(-1) CYN. Uptake ranged from 0.05% to 0.11% of the nominal CYN exposure amount. This constitutes a sensitive and reproducible method for extraction and quantification of unconjugated CYN with demonstrated application in 4 aquatic organisms, which can be used in further aquatic toxicological investigations.

ß-N-methylamino-L-alanine (BMAA) metabolism in the aquatic macrophyte Ceratophyllum demersum.

The cyanobacterial neurotoxin, ß-N-methylamino-l-alanine (BMAA) bioaccumulates and biomagnifies within the environment. However, most reports on the environmental presence of BMAA focus on the presence of BMAA in animals rather than in plants. Various laboratory studies have reported that this neurotoxin, implicated in neurodegenerative disease, is rapidly taken up by various aquatic and terrestrial plants, including crop plants. In this study the metabolism of BMAA in the aquatic macrophyte, Ceratophyllum demersum, was investigated using stable isotopically labelled BMAA. Data show that the toxin is rapidly removed from the environment by the plant. However, during depuration cellular BMAA concentrations decrease considerably, without excretion of the toxin back into the environment and without catabolism of BMAA, evidenced by the absence of label transfer to other amino acids. This strongly suggests that BMAA is metabolised via covalent modification and sequestered inside the plant as a BMAA-derivative. This modification may be reversed in humans following consumption of BMAA-containing plant material. These data therefore impact on the assessment of the risk of human exposure to this neurotoxin.