Feeding Activity and Xenobiotics Modulate Oxidative Status in Daphnia magna: Implications for Ecotoxicological Testing

To apply biomarkers of oxidative stress in laboratory and field settings, an understanding of their responses to changes in physiological rates is important. The evidence is accumulating that caloric intake can increase production of reactive oxygen species and thus affect background variability of oxidative stress biomarkers in ecotoxicological testing. This study aimed to delineate effects of food intake and xenobiotics on oxidative biomarkers in Daphnia magna. Antioxidant capacity measured as oxygen radical absorbance capacity (ORAC) and lipid peroxidation assayed as thiobarbituric acid reactive substances (TBARS) were measured. Food intake was manipulated by varying food densities or by exposing the animals to chemicals inhibiting feeding rate (pharmaceutical haloperidol and pesticide lindane). Feeding rate proved to affect both protein, ORAC, and TBARS in unexposed daphnids. However, there was no significant effect of feeding rate on the protein-specific ORAC values. Both substances affected individual protein and ORAC levels and changed their relationship to feeding rate. Our results show that inhibition of feeding rate influenced the interpretation of biomarker response and further emphasize the importance of understanding (1) baseline variability in potential biomarkers due to variations in metabolic state and (2) the contribution of feeding rate on toxic response of biomarkers

Effect concentrations obtained in acute toxicity test (OECD 202) (LC₅₀; mg L⁻¹), reproduction test (OECD 211) (LOEC reproduction and mortality; mg L⁻¹) and feeding inhibition test (LOEC; mg L⁻¹), and concentration range (Range, mg L⁻¹) for the pharmaceuticals tested.

<p>Feeding inhibition test with miconazole could not be conducted due to high toxic effects on the algae.</p

Are Pharmaceuticals with Evolutionary Conserved Molecular Drug Targets More Potent to Cause Toxic Effects in Non-Target Organisms?

<div><p>The ubiquitous use of pharmaceuticals has resulted in a continuous discharge into wastewater and pharmaceuticals and their metabolites are found in the environment. Due to their design towards specific drug targets, pharmaceuticals may be therapeutically active already at low environmental concentrations. Several human drug targets are evolutionary conserved in aquatic organisms, raising concerns about effects of these pharmaceuticals in non-target organisms. In this study, we hypothesized that the toxicity of a pharmaceutical towards a non-target invertebrate depends on the presence of the human drug target orthologs in this species. This was tested by assessing toxicity of pharmaceuticals with (miconazole and promethazine) and without (levonorgestrel) identified drug target orthologs in the cladoceran <i>Daphnia magna</i>. The toxicity was evaluated using general toxicity endpoints at individual (immobility, reproduction and development), biochemical (RNA and DNA content) and molecular (gene expression) levels. The results provide evidence for higher toxicity of miconazole and promethazine, i.e. the drugs with identified drug target orthologs. At the individual level, miconazole had the lowest effect concentrations for immobility and reproduction (0.3 and 0.022 mg L⁻¹, respectively) followed by promethazine (1.6 and 0.18 mg L⁻¹, respectively). At the biochemical level, individual RNA content was affected by miconazole and promethazine already at 0.0023 and 0.059 mg L⁻¹, respectively. At the molecular level, gene expression for cuticle protein was significantly suppressed by exposure to both miconazole and promethazine; moreover, daphnids exposed to miconazole had significantly lower vitellogenin expression. Levonorgestrel did not have any effects on any endpoints in the concentrations tested. These results highlight the importance of considering drug target conservation in environmental risk assessments of pharmaceuticals.</p></div

General linear models testing treatment effects on the RNA – body length (BL) and DNA-BL relationships.

<p>Tested concentrations were for miconazole 0.0023 mg L⁻¹, for promethazine 0.059 mg L⁻¹ and for levonorgestrel 1.02 mg L⁻¹. Asterisks indicate significant level: p≤0.05 (*); p≤0.01 (**); p≤0.001 (***). All treatments were compared against the control.</p

Gene expression changes.

<p>Change in gene expression of cuticle protein 12 and vitellogenin for <i>D. magna</i>, instar 3, exposed to miconazole (0.0023 mg L⁻¹), promethazine (0.059 mg L⁻¹) or levonorgestrel (1.02 mg L⁻¹). The fold change (mean ± SD; <i>n</i> = 3) is shown in relation to the respective controls. Asterisks indicate significance level: p≤0.01 (**) determined by an unpaired t-test.</p

Development and mean instar.

<p>Mean instar (with 95% confidence intervals) for the 2-d incubation (n = 10). Test concentrations were 0.0023 mg L⁻¹ for miconazole, 0.059 mg L⁻¹ for promethazine and 1.02 mg L⁻¹ for levonorgestrel. Black bars represent controls and white are the treatments. Asterisk indicates significant level: p≤0.05 (*) determined by an unpaired t-test.</p

Bacteria-Mediated Effects of Antibiotics on <i>Daphnia</i> Nutrition

In polluted environments, contaminant effects may be manifested via both direct toxicity to the host and changes in its microbiota, affecting bacteria–host interactions. In this context, particularly relevant is exposure to antibiotics released into environment. We examined effects of the antibiotic trimethoprim on microbiota of Daphnia magna and concomitant changes in the host feeding. In daphnids exposed to 0.25 mg L^–1 trimethoprim for 24 h, the microbiota was strongly affected, with (1) up to 21-fold decrease in 16S rRNA gene abundance and (2) a shift from balanced communities dominated by <i>Curvibacter</i>, <i>Aquabacterium,</i> and <i>Limnohabitans</i> in controls to significantly lower diversity under dominance of <i>Pelomonas</i> in the exposed animals. Moreover, decreased feeding and digestion was observed in the animals exposed to 0.25–2 mg L^–1 trimethoprim for 48 h and then fed ¹⁴C-labeled algae. Whereas the proportion of intact algal cells in the guts increased with increased trimethoprim concentration, ingestion and incorporation rates as well as digestion and incorporation efficiencies decreased significantly. Thus, antibiotics may impact nontarget species via changes in their microbiota leading to compromised nutrition and, ultimately, growth. These bacteria-mediated effects in nontarget organisms may not be unique for antibiotics, but also relevant for environmental pollutants of various nature