Comparing the effects of pristine and UV-VIS aged microplastics: Behavioural response of model terrestrial and freshwater crustaceans.

Physico-chemical properties of microplastics (MPs) change during weathering in the environment. There is a lack of knowledge about the effects of such environmentally relevant MPs on organisms. We investigated: 1) the physico-chemical changes of MPs due to UV-VIS weathering, and 2) compared the effect of pristine and aged MPs on the behaviour of the water flea Daphnia magna and terrestrial crustacean Porcellio scaber. Dry powders of MPs were produced from widely used polymer types: disposable three-layer polypropylene (PP) medical masks (inner, middle and outer), polyester textile fibres, car tires and low-density polyethylene (LDPE) bags and were subjected to accelerated ultraviolet-visible (UV-VIS) ageing. Our results show that the extent of transformation depends on the type of polymer, with PP showing the most changes, followed by LDPE, textile fibres and tire particles. Obvious fragmentation was observed in PP and textile fibres. In the case of PP, but not polyester textile fibres, changes in FTIR spectra and surface properties were observed. Tire particles and LDPE did not change in size, but clear changes were observed in their FTIR spectra. Most MPs, aged and pristine, did not affect the swimming of daphnids. The only effect observed was a significant increase in path length and swimming speed for the pristine tire particles when the recording was done with particles remaining in the wells. After transfer to a clean medium, this effect was no longer present, suggesting a physical rather than chemical effect. Similarly, woodlice showed no significant avoidance response to the MPs tested, although there was a noticeable trend to avoid soils contaminated with pristine polyester textile fibers and preference towards the soils contaminated with aged MP of the middle mask layer. Overall, the apparent changes in physico-chemical properties of MPs after accelerated ageing were not reflected in their effects on woodlice and daphnids.

Combining QSAR and SSD to predict aquatic toxicity and species sensitivity of pyrethroid and organophosphate pesticides.

The widespread use of pyrethroid and organophosphate pesticides necessitates accurate toxicity predictions for regulatory compliance. In this study QSAR and SSD models for six pyrethroid and four organophosphate compounds using QSAR Toolbox and SSD Toolbox have been developed. The QSAR models, described by the formula 48 h-EC50 or 96 h-LC50 = x + y * log Kow, were validated for predicting 48 h-EC50 values for acute Daphnia toxicity and 96 h-LC50 values for acute fish toxicity, meeting criteria of n ≥10, r2 ≥0.7, and Q2 >0.5. Predicted 48 h-EC50 values for pyrethroids ranged from 3.95 × 10-5 mg/L (permethrin) to 8.21 × 10-3 mg/L (fenpropathrin), and 96 h-LC50 values from 3.89 × 10-5 mg/L (permethrin) to 1.68 × 10-2 mg/L (metofluthrin). For organophosphates, 48 h-EC50 values ranged from 2.00 × 10-5 mg/L (carbophenothion) to 3.76 × 10-2 mg/L (crufomate) and 96 h-LC50 values from 3.81 × 10-3 mg/L (carbophenothion) to 12.3 mg/L (crufomate). These values show a good agreement with experimental data, though some, like Carbophenothion, overestimated toxicity. HC05 values, indicating hazardous concentrations for 5% of species, range from 0.029 to 0.061 µg/L for pyrethroids and 0.030 to 0.072 µg/L for organophosphates. These values aid in establishing environmental quality standards (EQS). Compared to existing EQS, HC05 values for pyrethroids were less conservative, while those for organophosphates were comparable.

Are UV filters better together? A comparison of the toxicity of individual ultraviolet filters and off-the-shelf sunscreens to Daphnia magna.

Organic ultraviolet filters (UVFs) are known to contaminate many aquatic ecosystems, with much environmental contamination attributed to the use of UVF-containing skin care products such as sunscreens during aquatic recreation. Most studies addressing the impact of sunscreen contamination have focused on the effects of UVFs under the assumption that they are the primary contaminants of concern from sunscreen pollution; however, the extent to which the toxicity of UVFs is representative of the environmental impacts of the whole sunscreen mixture is unknown. To address this knowledge gap, this study compared the mixture toxicity of five off-the-shelf sunscreen spray products containing the UVFs avobenzone, homosalate, octisalate, octocrylene and oxybenzone to the toxicity of each UVF in isolation to the freshwater invertebrate Daphnia magna. It was found that sunscreen toxicity was not proportional to their total UVF content, as the sunscreen containing the fewest UVFs was approximately equivalent to the sunscreen with the most UVFs, causing ≥90 % mortality and inhibiting all daphnid reproduction over 21 d exposures. Sunscreen toxicity was typically lower than expected when compared to the toxicity of each individual UVF within the mixture, as some sunscreens causing ≤20 % mortality contained octocrylene and/or oxybenzone at concentrations exceeding those which caused 90 % mortality during exposure to the UVF alone. Despite sunscreens causing large impairments in reproduction, growth and metabolism, poor correlations existed between the severity of most sublethal endpoints with respect to the measured UVF content of each sunscreen. Overall, these results indicate that potential antagonistic relationships between sunscreen ingredients can greatly reduce the toxicity of UVFs, creating more uncertainty regarding the level of threat that UVFs pose to the environment as a result of sunscreen contamination.

Virulence and transmission biology of the widespread, ecologically important pathogen of zooplankton, Spirobacillus cienkowskii.

Spirobacillus cienkowskii (Spirobacillus, hereafter) is a widely distributed bacterial pathogen that has significant impacts on the population dynamics of zooplankton (Daphnia spp.), particularly in months when Daphnia are asexually reproducing. However, little is known about Spirobacillus' virulence, transmission mode, and dynamics. As a result, we cannot explain the dynamics of Spirobacillus epidemics in nature or use Spirobacillus as a model pathogen, despite Daphnia's tractability as a model host. Here, we work to fill these knowledge gaps experimentally. We found that Spirobacillus is among the most virulent of Daphnia pathogens, killing its host within a week and reducing host fecundity. We further found that Spirobacillus did not transmit horizontally among hosts unless the host died or was destroyed (i.e., it is an "obligate killer"). In experiments aimed at quantifying the dynamics of horizontal transmission among asexually reproducing Daphnia, we demonstrated that Spirobacillus transmits poorly in the laboratory. In mesocosms, Spirobacillus failed to generate epidemics; in experiments wherein individual Daphnia were exposed, Spirobacillus' transmission success was low. In the (limited) set of conditions we considered, Spirobacillus' transmission success did not change with host density or pathogen dose and declined following environmental incubation. Finally, we conducted a field survey of Spirobacillus' prevalence within egg cases (ephippia) made by sexually reproducing Daphnia. We found Spirobacillus DNA in ~40% of ephippia, suggesting that, in addition to transmitting horizontally among asexually reproducing Daphnia, Spirobacillus may transmit vertically from sexually reproducing Daphnia. Our work fills critical gaps in the biology of Spirobacillus and illuminates new hypotheses vis-à-vis its life history. IMPORTANCE: Spirobacillus cienkowskii is a bacterial pathogen of zooplankton, first described in the 19th century and recently placed in a new family of bacteria, the Silvanigrellaceae. Spirobacillus causes large epidemics in lake zooplankton populations and increases the probability that zooplankton will be eaten by predators. However, little is known about how Spirobacillus transmits among hosts, to what extent it reduces host survival and reproduction (i.e., how virulent it is), and what role virulence plays in Spirobacillus' life cycle. Here, we experimentally quantified Spirobacillus' virulence and showed that Spirobacillus must kill its host to transmit horizontally. We also found evidence that Spirobacillus may transmit vertically via Daphnia's seed-like egg sacks. Our work will help scientists to (i) understand Spirobacillus epidemics, (ii) use Spirobacillus as a model pathogen for the study of host-parasite interactions, and (iii) better understand the unusual group of bacteria to which Spirobacillus belongs.

ß-ionone inhibits the grazing of Daphnia sinensis by reducing the activity of acetylcholinesterase.

ß-ionone is a volatile metabolite of Microcystis aeruginosa that is toxic to aquatic organisms. Using Daphnia sinensis as model, our present study found that ß-ionone could significantly reduce heart rate and feeding rate, and induce intestinal emptying. Transcriptomic analysis showed that ß-ionone could significantly inhibit the expression of acetylcholinesterase (AchE) mRNA, while metabolomics further revealed that ß-ionone could significantly increase the level of acetylcholine (Ach) in D. sinensis. These results indicated that ß-ionone might act as an AchE inhibitor, resulting in an increase in Ach levels. To test this hypothesis, both in vivo and in vitro experiments demonstrated that ß-ionone could significantly reduce AchE activity. Furthermore, the inhibitory effects of ß-ionone on heart rate and feeding rate could be blocked by the M-type Ach receptor (mAchR) blocker. These findings confirm that ß-ionone is a novel AchE inhibitor. ß-ionone could inhibit the activity of AchE, which in turn resulted in an increase of Ach in D. sinensis. Consequently, elevated levels of Ach could suppress the heart rate and feeding rate of D. sinensis by activating the mAchR, while concurrently accelerating the rate of intestinal emptying by stimulating intestinal peristalsis, thereby obstructing the digestion of algae within the intestinal tract.

Photoperiod-dependent effects of zinc oxide nanoparticles on the growth and reproduction of Daphnia pulex.

The discharge of metal nanoparticles into the water inevitably poses a threat to aquatic organisms and the balance of the aquatic ecosystem. Photoperiod is one of the most important ecological factors for the development of cladocerans. In addition, different light conditions can also affect the toxicity of metal nanoparticles. In this study, we studied the effects of four photoperiods (8L/16D, 10L/14D, 14L/10D, and 16L/8D) combined with three concentrations of ZnO NPs (0 mg L-1, 0.05 mg L-1, and 0.10 mg L-1) on the growth and reproduction of Daphnia pulex. With the increase of photoperiod, the maternal body size and growth rate increased first and then decreased; the first time to reproduction was advanced, and broods and the total offspring also increased. Under the influence of ZnO NPs, growth rate and reproductive capacity were inhibited. The photoperiod 8L/16D and 16L/8D interacted with ZnO NPs on the growth of D. pulex, which significantly decreased the growth rate. Besides, the interaction between photoperiod 8L/16D and ZnO NPs decreased the reproduction ability of D. pulex. These results suggest that the effects of zinc oxide nanoparticles on the growth and reproduction of D. pulex is photoperiod dependent, which is useful for assessing the risk of pollutants to cladoceras under different light conditions.

Evaluation of arsenic induced genotoxicity and its impact on life processes of Daphnia magna.

Heavy metals like arsenic is ubiquitously present in the environment. Geologic and anthropogenic activities are the root cause behind high concentration of arsenic in natural water bodies demanding strict monitoring of water quality prior to human consumption and utilization. In the present study, we have employed Daphnia magna for studying the biological effects of environmentally relevant high concentration of arsenic in water. In acute toxicity study, the LC50 value for 24hr exposure was found to be 2.504 mg/L, which gradually decreased with increase in time period (24hr- 96hr) to 2.011 mg/ L at 96hr. Sub-chronic toxicity was evaluated over 12 days using sub-lethal concentrations (5 %, 10 %, 15 %, and 20 % of the 24-hr LC50). Survivability in Daphnia showed a decreasing trend from 96 % to 91 % with increase in arsenic concentrations from 5 % of LC50 24 hr value to 20 % of LC 50 24hr value respectively. Alongside decreased survivability, there was a significant reduction in body size, with organisms exposed to the highest concentration of arsenic measuring 0.87±0.01 mm compared to 1.51±0.10 mm in the control group. Reproductive potential declined concentration dependently with exposure, with the highest reduction observed at 20 % of LC50 24hr value, where offspring numbers decreased to 7±1 from 23±5 in the control. Heart rate decreased in concentration and time-dependent manners, with the lowest rates observed at the highest arsenic concentration (279±16 bpm after 24hr and 277±27 bpm after 48hr). Comet assay and micronucleus assay conducted after 48 hrs of exposure revealed concentration-dependent genotoxic effects in Daphnia magna. Our results indicate negative impact on physiology and reproduction of Daphnia magna at environmentally existent concentration of arsenic. Also Daphnia magna could serve as a sensitive test system for investigating arsenic contamination in water bodies.

Rapid Response of Daphnia magna Motor Behavior to Mercury Chloride Toxicity Based on Target Tracking.

A rapid and timely response to the impacts of mercury chloride, which is indispensable to the chemical industry, on aquatic organisms is of great significance. Here, we investigated whether the YOLOX (improvements to the YOLO series, forming a new high-performance detector) observation system can be used for the rapid detection of the response of Daphnia magna targets to mercury chloride stress. Thus, we used this system for the real-time tracking and observation of the multidimensional motional behavior of D. magna. The results obtained showed that the average velocity (v¯), average acceleration (a¯), and cumulative travel (L) values of D. magna exposed to mercury chloride stress changed significantly under different exposure times and concentrations. Further, we observed that v¯, a¯ and L values of D. magna could be used as indexes of toxicity response. Analysis also showed evident D. magna inhibition at exposure concentrations of 0.08 and 0.02 mg/L after exposure for 10 and 25 min, respectively. However, under 0.06 and 0.04 mg/L toxic stress, v¯ and L showed faster toxic response than a¯, and overall, v¯ was identified as the most sensitive index for the rapid detection of D. magna response to toxicity stress. Therefore, we provide a strategy for tracking the motile behavior of D. magna in response to toxic stress and lay the foundations for the comprehensive screening of toxicity in water based on motile behavior.

A proof-of-concept multi-tiered Bayesian approach for the integration of physiochemical properties and toxicokinetic time-course data for Daphnia magna.

The use of in silico and in vitro methods, commonly referred to as New Approach Methodologies (NAMs), has been proposed to support environmental (and human) chemical safety decisions, ensuring enhanced environmental protection. Toxicokinetic models developed for environmentally relevant species are fundamental to the deployment of a NAMs-based safety strategy, enabling the conversion between external and internal chemical concentrations, although they require historical toxicokinetic data and robust physical models to be considered a viable solution. Daphnia magna is a key model organism in ecotoxicology albeit with limited and scattered quantitative toxicokinetic data, as for most invertebrates, resulting in a lack of robust toxicokinetic models. Moreover, current D. magna models are chemical specific, which restricts their applicability domain. One aim of this study was to address the current data availability limitations by collecting toxicokinetic time-course data for D. magna covering a broad chemical space and assessing the dataset's uniqueness. The collated toxicokinetic dataset included 48 time-courses for 30 chemicals from 17 studies, which was developed into an R package named AquaTK, with 11 studies unique to our work when compared to existing databases. Subsequently, a proof-of-concept Bayesian analysis was developed to estimate the steady-state concentration ratio (internal concentration / external concentration) from the data at three different levels of precision given three different data availability scenarios for environmental risk assessment. Specifically, an atrazine case study illustrates the multi-level modelling approach providing improvements (uncertainty reductions) in predictions of ratios for increasing amounts of data availability. Our work provides a consistent and self-contained Bayesian framework that irrespective of the hierarchy or resolution of individual experiments, can utilise the available information to generate optimal predictions of steady-state concentration ratios in D. magna. This approach is paramount to supporting the implementation of a NAMs based environmental safety paradigm shift in environmental risk assessment.

Ecotoxicological assessment of waste foundry sands and the application of different classification systems.

The application of a battery of bioassays is widely recognized as a useful tool for assessing environmental hazard samples. However, the integration of different toxicity data is a key aspect of this assessment and remains a challenge. The evaluation of industrial waste leachates did not initially undergo any of the proposed integration procedures. This research addressed this knowledge gap. Twenty-five samples of waste foundry sands were subjected to a leaching test (UNI EN 12457-2) to evaluate waste recovery and landfill disposal. The leachates were evaluated using a battery of standardized toxicity bioassays composed of Aliivibrio fischeri (EN ISO 11348-3), Daphnia magna (UNI EN ISO 6341), and Pseudokirchneriella subcapitata (UNI EN ISO 8692), both undiluted and diluted. Daphnia magna and P. subcapitata were the most affected organisms, with significant effects caused by 68% and 64% of undiluted samples, respectively. The dilution of samples facilitates the calculation of EC50 values, which ranged from greater than the highest concentration tested to 2.5 g/L for P. subcapitata. The data on single-organism toxicity were integrated using three methods: the Toxicity Classification System, the toxicity test battery integrated index, and the EcoScore system. The three classifications were strongly similar. According to all applied systems, three samples were clearly nontoxic (from iron casting plants) and two were highly toxic (from steel casting plants). Moreover, the similar ranking between undiluted and diluted leachates suggests the possibility of using only undiluted leachates for a more cost-effective and time-efficient screening of waste materials. The findings of this study highlight the usefulness of integrating ecotoxicological waste assessment. Integr Environ Assess Manag 2024;00:1-18. © 2024 The Author(s). Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Ecotoxicological assessment, in freshwater environment, of wastewater sludge coupled and uncoupled with micro-polyvinyl chloride on algae and water fleas.

In the frame of bioeconomy and circular economy, wastewater sludge (WS) could be a good candidate for its use in agriculture as fertilizer, due to its high content of organic matter, N and P, but on the other hand, it is full of toxicants such as heavy metal, microplastics, detergent, antibiotics, and so on that can reach groundwater and water bodies in leachate form. In this study, we have investigated different sludge concentrations in the eluate form, combined and not with PVC on two different freshwater organisms Selenastrum capricornutum and Daphnia magna, using ecotoxicity tests. At the endpoint, we have evaluated inhibition growth rate, oxidative stress, and pigments production for S. capricornutum, while in case of D. magna, we have assessed organism immobilization and development. From our results, it emerged that at the higher WS concentration, there was not inhibition growth rate, while at oxidative stress, it was higher in algae treated with WS and PVC. Higher Chl-a production was shown for algae treated with 0.3 g/L of sludge coupled with PVC, where higher phaeopigments production were recorded for algae treated with 0.3 g/L of WS. D. magna has shown an opposite trend when compared with algae, where at the highest WS concentrations supplied was corresponding to an increased mortality explaned as the highest immobility percentage. PRACTITIONER POINTS: Wastewater sludge is used in agriculture as fertilizer. PVC microplastic presence and associate ecotoxicity was tested. PVC presence increased oxidative stress in S. capricornutum. D. magna was significantly affected by sludge concentrations supplied.

Feeding inhibition in daphnids - A sensitive and rapid toxicity endpoint for chemical stress?

The planktonic Crustacea Daphnia are among the most employed organisms in ecotoxicology, mainly in regulatory assays that follow OECD/ISO protocols. The most common endpoint for acute testing (24-48 h) without feeding of organisms is usually monitored as mortality or immobilization. A rapid and physiologically and environmentally more relevant toxicity endpoint could be the impaired feeding of daphnids. Decreased feeding of test organisms upon exposure to toxicants has been used to evaluate sub-lethal effects occurring already in minutes to hours. This endpoint, however, has not been used systematically and the respective data are inconsistent due to heterogeneity of experimental design. The aim of this review is to evaluate the scientific literature where impaired Daphnia feeding has been used in ecotoxicological research. The search made in WoS (June 5, 2024) using combination of keywords "Daphni* AND feed* yielded 152 articles. Out of these 152 papers 46 addressed feeding of d aphnids upon exposure to various toxicants (insecticides, heavy metals, pharmaceuticals, contaminated environmental samples and toxic cyanobacteria; in total 59 different chemicals/combinations). These 46 papers formed the basis of the critical analysis presented in the current review. For 18 chemicals it was possible to compare the sensitivity of the feeding and mortality endpoints. We conclude that although the feeding inhibition of Daphnia sp. did not prove systematically more sensitive than mortality/immobilization, it is a sub-lethal endpoint that allows rapid evaluation of toxic effects of chemicals to aquatic crustaceans - important and sensitive organisms in the aquatic food web.

Screening of morphology-related genes based on predator-induced transcriptome sequencing and the functional analysis of Dagcut gene in Daphnia galeata.

High fish predation pressure can trigger "induced defense" in Daphnia species, resulting in phenotypic plasticity in morphology, behavior, or life-history traits. The molecular mechanisms of defense morphogenesis (e.g., the tail spine and helmet) in Daphnia remain unclear. In the present study, the tail spine, helmet, and body of Daphnia galeata under fish and non-fish kairomones conditions were collected for transcriptome analysis. A total of 24 candidate genes related to the morphological defense of D. galeata were identified, including 2 trypsin, one cuticle protein, 1 C1qDC protein, and 2 ferritin genes. The function of the Dagcut gene (D. galeata cuticle protein gene) in relation to tail spine morphology was assessed using RNA interference (RNAi). Compared with the EGFP (Enhanced green fluorescent protein) treatment, after RNAi, the expression levels of the Dagcut gene (D. galeata cuticle protein gene) showed a significant decrease. Correspondingly, the tail spines of the offspring produced by D. galeata after RNAi of the Dagcut gene appeared curved during the experiment. In whole-mount in situ hybridization, a clear signal site was detected on the tail spine of D. galeata before RNAi which disappeared after RNAi. Our results suggest that the Dagcut gene may play an important role in tail spine formation of D. galeata, and will provide a theoretical basis for studying the molecular mechanisms of the morphological plasticity in cladocera in the future.

A test for microbiome-mediated rescue via host phenotypic plasticity in Daphnia.

Phenotypic plasticity is a primary mechanism by which organismal phenotypes shift in response to the environment. Host-associated microbiomes often exhibit considerable shifts in response to environmental variation and these shifts could facilitate host phenotypic plasticity, adaptation, or rescue populations from extinction. However, it is unclear how much shifts in microbiome composition contribute to host phenotypic plasticity, limiting our knowledge of the underlying mechanisms of plasticity and, ultimately, the fate of populations inhabiting changing environments. In this study, we examined phenotypic responses and microbiome composition in 20 genetically distinct Daphnia magna clones exposed to non-toxic and toxic diets containing Microcystis, a cosmopolitan cyanobacteria and common stressor for Daphnia. Daphnia exhibited significant plasticity in survival, reproduction, and population growth rates in response to Microcystis exposure. However, the effects of Microcystis exposure on the Daphnia microbiome were limited, with the primary effect being differences in abundance observed across five bacterial families. Moreover, there was no significant correlation between the magnitude of microbiome shifts and host phenotypic plasticity. Our results suggest that microbiome composition played a negligible role in driving host phenotypic plasticity or microbiome-mediated rescue.

Accumulation, depuration, and potential effects of environmentally representative microplastics towards Daphnia magna.

Microplastic risk assessment often characterizes primary plastics, plastics intentionally manufactured at the micro- and nanoscale, or plastics collected within the natural environment, which often lack repeatability and the volume necessary for reliable hazard characterization. There is limited understanding of how environmentally representative plastics prepared at the microscale impact aquatic organisms. The aim of the present study was to create environmentally representative microplastics and characterize their toxicokinetics and hazards. Plastic cups and forks were micronized to <120 µm particles and Daphnia magna were exposed for 48 h at concentrations ranging from 0.01 mg/L to 100 mg/L. Uptake and depuration experiments were conducted at the highest concentration where accumulation was confirmed. Raman spectroscopy identified that both plastics were polystyrene and had similar size distributions. Microplastics were not acutely toxic but accumulated and rapidly depurated. Toxicokinetics demonstrated that cup MPs were consumed at higher rates than fork MPs despite similar physical characteristics. Daphnia magna preferentially selected smaller particles from the heterogenous suspensions. Future research will need to further explore the relationship between physicochemical properties, particularly size, and ecotoxicity. The study focused on mortality as the primary hazard endpoint. However alternative, sublethal biomarkers may be more appropriate in describing the effects of microplastic exposure.

Dragon fruit-derived oligosaccharides alter hemocyte-mediated immunity and expression of genes related to innate immunity and oxidative stress in Daphnia magna.

Dragon fruit oligosaccharide (DFO) is an indigestible prebiotic that enhances the growth and reproduction of Daphnia magna, increases the expression of genes involved in immunity, and reduces oxidative stress. This study investigated the effects of DFO on the expression of innate immunity- (Toll, Pelle, proPO, A2M, and CTL), oxidative stress- (Mn-SOD), and nitric oxide (NO) synthesis-related genes (NOS1, NOS2, and arginase) as well as NO localization and number of hemocytes in D. magna. For this ten-day-old D. magna were treated with 0 or 9 mg l-1 of DFO for 24 and 85 h. Gene expression levels, NO intensity and localization, and total hemocytes were evaluated. After 24 h, the expression of Toll and proPO increased significantly (p < 0.05), while that of C-type lectins (CTL) was reduced (p < 0.05). At 85 h, Mn-SOD and CTL expressions were markedly suppressed (p < 0.05). NO was mostly localized in the foregut, midgut, hindgut, and carapace. The expression of NOS1 was reduced after 24 h (p < 0.05). In addition, NO intensity at 24 h was insignificantly lower than the control (p > 0.05). At 85 h, the expression of NOS1, NOS2, and arginase was higher than control, but NO intensity did not differ significantly (p > 0.05). Furthermore, the total hemocyte count elevated remarkably at 85 h (p < 0.05). Our study suggested that 9 mg l-1 of DFO could alter the expression of the genes related to innate immunity, oxidative stress, and NO synthesis in D. magna and significantly stimulate hemocyte production.

Granulated rubber in playgrounds and sports fields: A potential source of atmospheric plastic-related contaminants and plastic additives after runoff events.

The use of "crumb rubber" coming from recycling materials in outdoor floors like playgrounds has been a frequent practice during the last years. However, these surfaces are object of abrasion and weathering being a potential source of micro and nanoplastics (MNPLs) to the atmosphere and a potential source of human exposure to them. Our main goal has been to expose different crumb rubber materials to summer weathering effects. The released inhalable fractions were sampled for two months with passive samplers and the composition of MNPLs and plastic additives (organic and inorganic) were evaluated. The ecotoxicological effects of leached materials emulating runoff events was evaluated in freshwater micro crustacean Daphnia magna and the green algae Chlorella vulgaris. The analysis of MNPLs showed the presence of polyethylene, polypropylene, polybutadiene, polysiloxanes and polybutylene at concentrations up to 30,426 ng/m3. In the same fraction, we also identified up to 56 plastic additives, including antioxidants, pigments, copolymers, flame retardants, fungicides, lubricants, plasticizers, UV filters and metal ions. Finally, runoff ecotoxicological effects on D. magna and C. vulgaris showed that leached compounds, either from virgin or aged material, would be toxicants for exposed organisms although at concentrations much higher than those expected to be released to the media.

Non-chemical stresses do not strongly induce male offspring in Daphnia magna ascertained using the short-term juvenile hormone activity screening assay.

Juvenile hormone (JH), together with ecdysone, regulates molting, metamorphosis, growth, and reproduction in arthropods. The effects of its analogs used as insecticides on nontarget species are of concern. Since JH and JH analogs (JHAs) induce male offspring in daphnids, which generally reproduce by parthenogenesis, short-term JH activity screening assay (JHASA) using the male offspring ratio as an endpoint has been developed as a detection method for JHA. However, the production of male offspring is also induced by environmental stresses such as temperature, short-day length, overcrowding, and food limitation. Thus, it is vital to prevent non-chemical stresses from inducing male offspring during the test to detect chemicals with potential JH activity accurately. Therefore, we investigated the effects of temperature (low and high), hardness, high density with low feeding, and day length on male production utilizing JHASA. Male offspring were not strongly induced by any stresses in JHASA, although the male ratios of 4-12% were observed in the preculture under high density (≥70 daphnid/L) and constant darkness. The Clone A strain was relatively more sensitive to high density and day length compared with the strain from National Institute for Environmental Studies (NIES). The selection of strains that rarely produce males under non-chemical stresses and finding the culturing conditions for each strain appropriate for not-inducing male offspring are recommended to control and prevent male offspring induction during JHASA.

Trehalose mediates salinity-stress tolerance in natural populations of a freshwater crustacean.

Salinization poses an increasing problem worldwide, threatening freshwater organisms and raising questions about their ability to adapt. We explored the mechanisms enabling a planktonic crustacean to tolerate elevated salinity. By gradually raising water salinity in clonal cultures from 185 Daphnia magna populations, we showed that salt tolerance strongly correlates with native habitat salinity, indicating local adaptation. A genome-wide association study (GWAS) further revealed a major effect of the Alpha,alpha-trehalose-phosphate synthase (TPS) gene, suggesting that trehalose production facilitates salinity tolerance. Salinity-tolerant animals showed a positive correlation between water salinity and trehalose concentrations, while intolerant animals failed to produce trehalose. Animals with a non-functional TPS gene, generated through CRISPR-Cas9, supported the trehalose role in salinity stress. Our study highlights how a keystone freshwater animal adapts to salinity stress using an evolutionary mechanism known in bacteria, plants, and arthropods.

A molecular mechanism for environmental sex determination.

Daphnia produce genetically identical males and females; their sex is determined by environmental conditions. Recently, Kato et al. identified isoform switching events in Daphnia as a gene regulatory mechanism for sex-specific development. This finding uncovers the impact of alternative usage of gene isoforms on this extreme phenotypic plasticity trait.