The Effects of Single and Combined Stressors on Daphnids—Enzyme Markers of Physiology and Metabolomics Validate the Impact of Pollution

Abstract

The continuous global increase in population and consumption of resources due to human activities has had a significant impact on the environment. Therefore, assessment of environmental exposure to toxic chemicals as well as their impact on biological systems is of significant importance. Freshwater systems are currently under threat and monitored; however, current methods for pollution assessment can neither provide mechanistic insight nor predict adverse effects from complex pollution. Using daphnids as a bioindicator, we assessed the impact in acute exposures of eight individual chemicals and specifically two metals, four pharmaceuticals, a pesticide and a stimulant, and their composite mixture combining phenotypic, biochemical and metabolic markers of physiology. Toxicity levels were in the same order of magnitude and significantly enhanced in the composite mixture. Results from individual chemicals showed distinct biochemical responses for key enzyme activities such as phosphatases, lipase, peptidase, β-galactosidase and glutathione-S-transferase. Following this, a more realistic mixture scenario was assessed with the aforementioned enzyme markers and a metabolomic approach. A clear dose-dependent effect for the composite mixture was validated with enzyme markers of physiology, and the metabolomic analysis verified the effects observed, thus providing a sensitive metrics in metabolite perturbations. Our study highlights that sensitive enzyme markers can be used in advance on the design of metabolic and holistic assays to guide the selection of chemicals and the trajectory of the study, while providing mechanistic insight. In the future this could prove to become a useful tool for understanding and predicting freshwater pollution.

Molecular Responses of Daphnids to Chronic Exposures to Pharmaceuticals

“…Metabolomics specifically is useful in an ecotoxicology scenario, as an organism’s metabolism is their first line of defines to xenobiotics [ 58 , 59 ]. We recently highlighted the use of metabolic signatures in daphnids combined with physiologic markers as endpoints used in toxicity of mixtures of pollutants [ 60 ]. Our study employed a combination of physiological endpoints and a metabolomic analysis to identify targets of toxicity for several pharmaceuticals at a molecular level.…”

“…Recently, we highlighted the role of daphnids as key species in molecular ecotoxicology and the application of metabolomics in pollution monitoring as a sensitive endpoint [ 60 ]. This study revealed distinct changes in the polar metabolic profile of daphnids exposed to various pharmaceuticals at sub-lethal concentrations, as well as significant changes in multiple biochemical markers.…”

“…Daphnids were cultured in conformity with OECD guidelines in 4 L beakers in OECD media (final concentrations 0.29 g CaCl 2 .2H 2 O/l, 0.123 g MgSO 4 .7H 2 O/l, 0.065 g NaHCO 3 /l, 0.0058 g KCl/l, 2 μg Na 2 SeO 3 /l, pH 7.7) [ 60 ] under a 16h:8h of light:dark photoperiod at 21 °C. Breeding cultures of daphnnids were fed with an algal suspension ( Chlamydomonas rheinhartii ) and supplemented with dried baker’s yeast and an organic seaweed extract ( Ascophylum nodossum ) upon media renewal every four days.…”

Production of Silver Nano-Inks and Surface Coatings for Anti-Microbial Food Packaging and Its Ecological Impact

“…Daphniids were cultured in conformity with OECD guidelines in 4-litres beakers in OECD media (final concentrations 0.29 g CaCl 2 .2H 2 O /L, 0.123 g MgSO 4 .7H 2 O/L, 0.065 g NaHCO 3 /L, 0.0058 g KCl/L, 2 μg Na 2 SeO 3 /L, pH 7.7) using 16 h:8 h of light and dark photoperiod at 20 °C and a density of 100 adults per 4 litres of media [ 28 ]. Neonates (<24 h) were collected and exposed to 50 mL OECD media against unexposed controls, and mortality was measured after 24 and 48 h of exposure.…”

Daphnia as a model organism to probe biological responses to nanomaterials—from individual to population effects via adverse outcome pathways

“…Despite biochemical markers being promising tools for early aquatic toxicity assessment (Michalaki et al, 2022), the biochemical and physiological responses generally do not correlate and sometimes there is no consistency in the results. Therefore, it is essential to push the scientific community to harmonise experimental procedures to produce reliable data, following the Findable, Accessible, Interoperable, and Reusable (FAIR) principles (Wilkinson et al, 2016), which will later allow linking physiological responses with molecular patterns and (sub) organismal responses and facilitate computational modelling and predictive (nano) toxicology.…”