We investigated the biological response of soluble organic fraction (SOF) and water-soluble fraction (WSF) extracted from particulate matter (PM) emitted by an automotive diesel engine operating in a representative urban driving condition. The engine was fueled with ultra-low sulfur diesel (ULSD), and its binary blends by volume with 13% of butanol (Bu13), and with hydrotreated vegetable oil (HVO) at 13% (HVO13) and 20% (HVO20). Cytotoxicity, genotoxicity, oxidative DNA damage and ecotoxicity tests were carried out, and 16 polycyclic aromatic hydrocarbons (PAH) expressed as tbenzo(a)pyrene total toxicity equivalent (BaP-TEQ) were also analyzed. The Hepatocarcinoma epithelial cell line (HepG2) was exposed to SOF for 24 h and analyzed using comet assay, with the inclusion of formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (Endo III) to recognize oxidized DNA bases. The WSF was evaluated through acute ecotoxicity tests with the aquatic microcrustacean Daphnia pulex (D. Pulex). Results showed that there was no cytotoxic activity for all tested SOF concentrations. Genotoxic responses by all the SOF samples were at same level, except for the HVO13 which was weaker in the absence of the enzymes. The addition of the FPG and Endo III enzymes resulted in a significant increase in the comet tail, indicating that the DNA damage from SOF for all tested fuel blends involves oxidative damage including a higher level of oxidized purines for ULSD and Bu13 in comparison with HVO blends, but the oxidized pyrimidines for HVO blends were slightly higher compared to Bu13. The WSF did not show acute ecotoxicity for any of the fuels. Unlike other samples, Bu13-derived particles significantly increase the BaP-TEQ. The contribution to the genotoxic activity and oxidative DNA from SOF was not correlated to BaP-TEQ, which means that the biological activity of PM might be affected also by other toxic compounds present in particulate phase.

The biological effects of nanoplastics are a growing concern. However, most studies have focused on exposure to high concentrations or short-term exposure. The potential effects of exposure to low environmental nanoplastic concentrations over the long-term and across multiple generations remain unclear. In the present study, Daphnia pulex was exposed over three 21-day generations to a typical environmental nanoplastic concentration (1 μg/L) and the effects were investigated at physiological (growth and reproduction), gene transcription and enzyme activity levels. Chronic exposure did not affect the survival or body length of D. pulex, whereas the growth rate and reproduction were influenced in the F2 generation. Molecular responses indicated that environmental nanoplastic concentrations can modulate the response of antioxidant defenses, vitellogenin synthesis, development, and energy production in the F0-F1 generations, and prolongation resulted in inhibitory effects on antioxidant responses in F2 individuals. Some recovery was observed in the recovery group, but reproduction and stress defenses were significantly induced. Taken together, these results suggest that D. pulex recovery from chronic exposure to nanoplastic may take several generations, and that nanoplastics have potent long-term toxic effects on D. pulex. The findings highlight the importance of multigenerational and chronic biological evaluations to assess risks of emerging pollution.

Microplastics (<5 mm) are distributed ubiquitously in natural environments. The majority of microplastics in aquatic environments are shown to have rough surfaces due to various weathering processes (secondary microplastics; SMP), while laboratory studies predominantly utilise pristine microplastics (primary microplastics; PMP). Here we present the results from a study comparing the chronic effects of pristine PMP and artificially weathered SMP to three different Cladoceran species (Daphnia magna, Daphnia pulex, Ceriodaphnia dubia). We assessed the impact of PMP and SMP on reproductive output using various measured parameters, including time of first brood, size of first brood, size of first three broods, cumulative number of neonates, total number of broods and terminal length of test animals. Our results show that reproductive output of all species declined in a dose-dependent manner. The No Observed Effect Concentration (NOEC) was less than the lowest tested concentration (102 p/mL) for at least one measured endpoint for all species and both PMP and SMP. Further, it was inferred that species sensitivity varied inversely with body size for most endpoints, resulting in C. dubia being the most sensitive species; and D. magna being the least sensitive species under study. In addition, PMP appeared to have greater toxic potential as compared to SMP. This study is the first to directly compare the chronic toxicity of both pristine and weathered microplastic particles on three freshwater toxicological model organisms. Our results indicate that sensitivity in reproduction and growth to microplastics may differ between species and type of microplastic exposed; highlighting the importance of using multiple species and structural types of particles.

Microplastics (<5 mm, MP) are ubiquitously distributed in the environment, causing increasing concern regarding their potential toxicity to organisms. To date, most research has focussed on the impacts of MPs on marine and estuarine organisms, with fewer studies focussing on the effects of microplastics on freshwater ecosystems, especially under different environmental conditions. In the present study, the sensitivity of two temperate Cladoceran species, Daphnia magna and Daphnia pulex, and a smaller tropical species Ceriodaphnia dubia, to primary microplastics (PMP) and secondary (weathered) microplastics (SMP) was assessed. A prolonged acute toxicity assay (up to 72 or 96 h) was performed at 18°, 22°, and 26 °C, to determine the influence of temperature as an additional stressor and survival data were analysed using toxicokinetic-toxicodynamic (TK-TD) model. Acute sensitivity of D. magna and D. pulex to both PMP and SMP increased sharply with temperature, whereas that of C. dubia remained relatively stable across temperatures. C. dubia was the most sensitive species at 18 °C, followed by D. pulex and D. magna, which were of comparable sensitivity. However, this ranking was reversed at 26 °C as could be seen from the No Effect Concentration (NEC) estimates of the TK-TD model. In addition, SMP and PMP had a similar effect on D. magna and D. pulex, but PMP was more toxic to C. dubia. Effects on survival were strongly time-dependent and became substantially more severe after the standard 48 h test period. Our results indicate that sensitivity to microplastics may differ between species for different types of microplastics, and could be drastically influenced by temperature albeit at high exposure concentrations.

Organisms around the globe are experiencing novel environments created by human activities. One such disturbance of growing concern is the salinization of freshwater habitats from the application of road deicing salts, which creates salinity levels not experienced within the recent evolutionary history of most freshwater organisms. Moreover, salinization can induce trophic cascades and alter the structure of freshwater communities, but knowledge is still scarce about the ability of freshwater organisms to adapt to elevated salinity. We examined if a common zooplankton of freshwater lakes (Daphnia pulex) could evolve a tolerance to the most commonly used road deicing salt (sodium chloride, NaCl). Using a mesocosm experiment, we exposed freshwater communities containing Daphnia to five levels of NaCl (15, 100, 200, 500, and 1000 mg Cl− L−1). After 2.5 months, we collected Daphnia from each mesocosm and raised them in the lab for three generations under low salt conditions (15 mg Cl− L−1). We then conducted a time-to-death experiment with varying concentrations of NaCl (30, 1300, 1500, 1700, 1900 mg Cl− L−1) to test for evolved tolerance. All Daphnia populations exhibited high survival when subsequently exposed to the lowest salt concentration (30 mg Cl− L−1). At the intermediate concentration (1300 mg Cl− L−1), however, populations previously exposed to elevated concentrations (i.e.100–1000 mg Cl− L−1) had higher survival than populations previously exposed to natural background levels (15 mg Cl− L−1). All populations survived poorly when subsequently exposed to the highest concentrations (1500, 1700, and 1900 mg Cl− L−1). Our results show that the evolution of tolerance to moderate levels of salt can occur within 2.5 months, or 5–10 generations, in Daphnia. Given the importance of Daphnia in freshwater food webs, such evolved tolerance might allow Daphnia to buffer food webs from the impacts of freshwater salinization.

Bioassays using Daphnia pulex and Moina micrura were designed to detect cyanobacterial neurotoxins in raw water samples. Phytoplankton and cyanotoxins from seston were analyzed during 15 months in a eutrophic reservoir. Effective time to immobilize 50% of the exposed individuals (ET50) was adopted as the endpoint. Paralysis of swimming movements was observed between ∼0.5-3 h of exposure to lake water containing toxic cyanobacteria, followed by an almost complete recovery of the swimming activity within 24 h after being placed in control water. The same effects were observed in bioassays with a saxitoxin-producer strain of Cylindrospermopsis raciborskii isolated from the reservoir. Regression analysis showed significant relationships between ET50vs. cell density, biomass and saxitoxins content, suggesting that the paralysis of Daphnia in lake water samples was caused by saxitoxins found in C. raciborskii. Daphnia bioassay was found to be a sensitive method for detecting fast-acting neurotoxins in natural samples, with important advantages over mouse bioassays.

Eutrophication and warming lead to frequent occurrence of cyanobacterial blooms, which significantly impact on zooplankton. Freshwater zooplankton Daphnia adopts two distinct ways of reproduction: asexual (parthenogenetic) reproduction for rapidly reproducing many offspring in favorable environment and sexual reproduction for producing resting eggs as seed bank to survive in harsh environments. Daphnia pulex has worse performance in growth and reproduction under the exposure to toxic cyanobacteria Microcystis aeruginosa and tends to allocate less energy to reproduction in the case of insufficient food. However, the relative reproductive allocation strategy (energy allocation) of D. pulex individuals exposed to toxic M. aeruginosa is still unclear. Here we tested the relative reproductive performance of D. pulex fed on solely Chlorella pyrenoidosa (high quality food) or Chlorella mixed with toxic M. aeruginosa (low quality food), based on the parthenogenetic reproduction (life-history experiments) and sexual reproduction (population experiments). The results showed that under low quality food conditions, D. pulex reproduced fewer offspring which were also smaller and thus led to a reduced absolute output in parthenogenetic reproduction, but produced ephippia in the same size and quantity compared to those cultured under high quality food conditions. However, as the body size of maternal D. pulex cultured under low quality food conditions decreased, the relative reproductive allocation significantly increased in both parthenogenetic and sexual reproduction, compared to those cultured under high quality food conditions. In conclusion, D. pulex tend to allocate relatively more energy to reproduction under Microcystis conditions, which is a reasonable strategy for it to decentralize the risks from low-quality food.

This study evaluated hormetic effect of oxidative stress exerted by fullerene crystals (nC₆₀) on Daphnia pulex, employing transcriptomics and metabolomics. D. pulex were exposed to various concentrations of nC₆₀ for 21 days. Hormetic effect of oxidative stress was most evident after 7 days, with markedly increased L-Glutathione (GSH) concentration and Superoxide Dismutase (SOD) activity at low doses of nC₆₀ exposure, and oppositely at high doses. The transcriptomics and metabolomics were used to elucidate the molecular mechanism underlying the hormesis in oxidative stress. There were significant alterations in major pathways involving oxidative stress and energy metabolism in D. pulex. Some important intermediates and the expression of their regulatory genes coincided with each other with first up-regulated and then down-regulated with the concentration increased, consistent with the hormesis description. The nC₆₀ interfered the TCA cycle of D. pulex. The synthesis of L-cysteine and glutamate was directly affected, and further disturbed the synthesis of GSH. This work is of great significance to provide the molecular-level evidence into the hormetic effect in oxidative stress of D. pulex exposed to nC₆₀.

The worldwide use of pesticides has led to increases in agricultural yields by reducing crop losses. However, increased pesticide use has resulted in pesticide-resistant pest species and recent studies have discovered pesticide-resistance in non-target species living close to farms. Such increased tolerance not only affects the species, but can alter the entire food web. Given that some species can evolve not only resistance to a single pesticide, but also cross-resistance to other pesticides that share the same mode of action, one would predict that cross-resistance to pesticides would also have effects on the entire community and affect community stability. To address this hypothesis, we conducted an outdoor mesocosm experiment comprised of 200 identical aquatic communities with phytoplankton, periphyton, and leopard frog (Lithobates pipiens) tadpoles. To these communities, we added one of four Daphnia pulex populations that we previously discovered were either resistant or sensitive to the insecticide of chlorpyrifos as a result of living close to or far from agriculture, respectively. We then exposed the communities to either no insecticide or three different concentrations of AChE-inhibiting insecticides (chlorpyrifos, malathion or carbaryl) or sodium channel-inhibiting insecticides (permethrin or cypermethrin). We discovered that communities containing sensitive Daphnia pulex experienced phytoplankton blooms and subsequent cascades through all trophic groups including amphibians at moderate to high concentrations of all five insecticides. However, communities containing resistant D. pulex were buffered from these effects at low to moderate concentrations of all AChE-inhibiting insecticides, but were not buffered against the pyrethroid insecticides. These data suggest that a simple change in the population-level resistance of zooplankton to a single insecticide can have widespread consequences for community stability and that the effects can be extrapolated to a wide variety of pesticides that share the same mode of action.

In aquatic ecosystems, mixtures of chemical and natural stressors can occur which may significantly complicate risk assessment approaches. Here, we show that effects of binary combinations of four different insecticides and Microcystis aeruginosa, a toxic cyanobacteria, on Daphnia pulex exhibited distinct interaction patterns. Combinations with chlorpyrifos and tetradifon caused non-interactive effects, tebufenpyrad caused an antagonistic interaction and fenoyxcarb yielded patterns that depended on the reference model used (i.e. synergistic with independent action, additive with concentration addition). Our results demonstrate that interactive effects cannot be generalised across different insecticides, not even for those targeting the same biological pathway (i.e. tebufenpyrad and tetradifon both target oxidative phosphorylation). Also, the concentration addition reference model provided conservative predictions of effects in all investigated combinations for risk assessment. These predictions could, in absence of a full mechanistic understanding, provide a meaningful solution for managing water quality in systems impacted by both insecticides and cyanobacterial blooms.