Temperature and food quantity effects on the harpacticoid copepod Nitocra spinipes : combining in vivo bioassays with population modeling

The harpacticoid copepod Nitocra spinipes has become a popular model species for toxicity testing over the past few decades. However, the combined influence of temperature and food shortage, two climate change-related stressors, has never been assessed in this species. Consequently, effects of three temperatures (15, 20 and 25˚C) and six food regimes (between 0 and 5 × 10^5 algal cells/mL) on the life cycle of N. spinipes were examined in this study. Similarly to other copepod species, development times and brood sizes decreased with rising temperatures. Mortality was lowest in the 20˚C temperature setup, indicating a close-by temperature optimum for this species. Decreasing food concentrations led to increased development times, higher mortality and a reduction in brood size. A sex ratio shift toward more females per male was observed for increasing temperatures, while no significant relationship with food concentration was found. Temperature and food functions for each endpoint were integrated into an existing individual-based population model for N. spinipes which in the future may serve as an extrapolation tool in environmental risk assessment. The model was able to accurately reproduce the experimental data in subsequent verification simulations. We suggest that temperature, food shortage, and potentially other climate change-related stressors should be considered in environmental risk assessment of chemicals to account for non-optimal exposure conditions that may occur in the field. Furthermore, we advocate combining in vivo bioassays with population modeling as a cost effective higher tier approach to assess such considerations

Population modeling using harpacticoid copepods : Bridging the gap between individual-level effects and protection goals of environmental risk assessment

To protect the environment from contaminants, environmental risk assessment (ERA) evaluates the risk of adverse effects to populations, communities and ecosystems. Environmental management decisions rely on ERAs, which commonly are based on a few endpoints at the individual organism level. To bridge the gap between what is measured and what is intended for protection, individual-level effects can be integrated in population models, and translated to the population level. The general aim of this doctoral thesis was to extrapolate individual-level effects of harpacticoid copepods to the population level by developing and using population models. Matrix models and individual based models were developed and applied to life-history data of Nitocra spinipes and Amphiascus tenuiremis, and demographic equations were used to calculate population-level effects in low- and high-density populations. As a basis for the population models, individual-level processes were studied. Development was found to be more sensitive compared to reproduction in standard ecotoxicity tests measuring life-history data. Additional experimental animals would improve statistical power for reproductive endpoints, but at high labor and cost. Therefore, a new test-design was developed in this thesis. Exposing animals in groups included a higher number of animals without increased workload. The number of reproducing females was increased, and the statistical power of reproduction was improved. Individual-level effects were more or equally sensitive compared to population-level effects, and individual-level effects were translated to the population level to various degrees by population models of different complexities. More complex models showed stronger effects at the population level compared to the simpler models. Density dependence affected N. spinipes populations negatively so that toxicant effects were stronger at higher population densities. The tools presented here can be used to assess the toxicity of environmental contaminants at the individual and population level, improve ERA, and thereby the basis for environmental management.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Manuscript.</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

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

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