Ocean acidification challenges copepod phenotypic plasticity

Ocean acidification is challenging phenotypic plasticity of individuals and populations. Calanoid copepods (zooplankton) are shown to be fairly plastic against altered pH conditions, and laboratory studies indicate that transgenerational effects are one mechanism behind this plasticity. We studied phenotypic plasticity of the copepod Acartia sp. in the course of a pelagic, large-volume mesocosm study that was conducted to investigate ecosystem and biogeochemical responses to ocean acidification. We measured copepod egg production rate, egg-hatching success, adult female size and adult female antioxidant capacity (ORAC) as a function of acidification (fCO(2) similar to 365-1231 mu atm) and as a function of quantity and quality of their diet. We used an egg transplant experiment to reveal whether transgenerational effects can alleviate the possible negative effects of ocean acidification on offspring development. We found significant negative effects of ocean acidification on adult female size. In addition, we found signs of a possible threshold at high fCO(2), above which adaptive maternal effects cannot alleviate the negative effects of acidification on egg-hatching and nauplii development. We did not find support for the hypothesis that insufficient food quantity (total particulate carbonPeer reviewe

Negligible effects of ocean acidification on Eurytemora affinis (Copepoda) offspring production

Ocean acidification is caused by increasing amounts of carbon dioxide dissolving in the oceans leading to lower seawater pH. We studied the effects of lowered pH on the calanoid copepod Eurytemora affinis during a mesocosm experiment conducted in a coastal area of the Baltic Sea. We measured copepod reproductive success as a function of pH, chlorophyll a concentration, diatom and dinoflagellate biomass, carbon to nitrogen (C : N) ratio of suspended particulate organic matter, as well as copepod fatty acid composition. The laboratory-based experiment was repeated four times during 4 consecutive weeks, with water and copepods sampled from pelagic mesocosms enriched with different CO2 concentrations. In addition, oxygen radical absorbance capacity (ORAC) of animals from the mesocosms was measured weekly to test whether the copepod's defence against oxidative stress was affected by pH. We found no effect of pH on offspring production. Phytoplankton biomass, as indicated by chlorophyll a concentration and dinoflagellate biomass, had a positive effect. The concentration of polyunsaturated fatty acids in the females was reflected in the eggs and had a positive effect on offspring production, whereas monounsaturated fatty acids of the females were reflected in their eggs but had no significant effect. ORAC was not affected by pH. From these experiments we conclude that E. affinis seems robust against direct exposure to ocean acidification on a physiological level, for the variables covered in the study. E. affinis may not have faced acute pH stress in the treatments as the species naturally face large pH fluctuations.Peer reviewe

Integrative approaches in ecotoxicological testing: Implications for biomarker development and application

Ecotoxicology aims to understand toxic effects of chemicals in the environment. Effects can be observed at different levels of the biological organization, from molecular to ecosystem level. Biomarkers on molecular and biochemical levels are used in ecotoxicology as early warning signals of chemical exposure, possible toxic effects and underlying mechanisms. As methods and technologies improve, more biomarkers are being implemented in ecotoxicological studies, due to the general interest in early detection and thus efficient prevention of environmental risks. However, to be of value in ecotoxicological assessment, a connection between biomarker response and effects at higher levels of biological organization should be established. Also, baseline variability for the biomarker in question as well as response to natural fluctuations of environmental factors should be evaluated. The aim of this thesis was to increase value and understanding of biomarkers in ecotoxicological assessment by (1) linking responses across different levels of biological organization, and (2) gaining better understanding of the relative importance of ecological and physiological factors affecting oxidative biomarkers. Paper I is focused on evolutionary conserved drug targets and the toxicity of pharmaceuticals for non-target organisms. The main conclusion from this study is that pharmaceuticals with conserved drug targets in non-target organisms have a higher toxicity than pharmaceuticals for which drug-targets have not been identified in the species. The effects were evaluated using end points at molecular, biochemical and individual levels. Consistent with the expected higher sensitivity of molecular and biochemical end points, the effects on the low-level biomarkers were observed at lower concentrations than at the individual level.   Paper II is focused on delineating effects of feeding and toxic exposure on oxidative biomarkers commonly used in ecotoxicology. The results are in agreement with the theory of caloric restriction that links enhanced caloric intake to increased pro-oxidative processes in animals. In our experiments with the cladoceran Daphnia magna, we observed positive effects for both antioxidant capacity and oxidized lipids in response to enhanced feeding rates. This have implications for the use of oxidative stress biomarkers in ecotoxicology as many substances have inhibitory effects on feeding rate and thus, changes in oxidative biomarkers can result from the altered feeding rate rather than other toxic mechanisms. Therefore, possible changes in feeding rate should be assessed when conducting exposure experiments or interpreting field data in studies employing oxidative stress biomarkers. However, it was concluded that the ratio between antioxidative capacity and protein content was independent of feeding rate. Thus, this biomarker is suitable for xenobiotic exposure in D. magna. This thesis have contributed to better understanding of molecular and biochemical biomarkers in ecotoxicological studies in regard to the connections between effects at different biological levels and confounding factors in biomarker response.At the tiem of defence the following papers wer unpublished and had a status as follows:Paper 1: Manuscript; Paper 2: Manuscript</p

Application and interpretation of biomarkers in ecotoxicology - from molecular to individual level responses

The use of biomarkers is considered a promising alternative, or complement, to traditional ecotoxicological assays. Toxic effects are often initially manifested at the molecular or biochemical level, biomarkers are therefore used as sensitive indicators of toxic exposure. Ideally, biomarkers would also indicate reduced fitness and possible later effects at the individual or population levels. However, implementing biomarkers in ecotoxicology is challenging and few biomarkers have an established connection to reduced individual fitness. The aim of this thesis was to increase the value and improve the interpretation of biomarker responses in ecotoxicological studies by examining the impact of confounding factors and the relationship between oxidative biomarkers and reproductive effects in crustaceans. The sensitivity of biomarkers was confirmed in paper I as toxic effects of pharmaceuticals with conserved drug target orthologs were observed at the molecular and biochemical levels both earlier and at lower concentrations than effects on mortality and reproduction. No toxic effects were observed for the pharmaceutical without identified drug target orthologs, thus stressing the importance of considering toxic mechanisms and being aware of the most likely target when evaluating toxic effects also in non-target species. Many xenobiotics and environmental stressors interfere with oxidative processes, making oxidative biomarkers interesting to study in ecotoxicology and stress ecology. Still, feeding rate was identified as a confounding factor for antioxidant capacity (assayed as oxygen radical absorbance capacity, ORAC) and lipid peroxidation in ecotoxicological studies (paper II). However, ORAC normalized to protein was independent of altered feeding rates, hence it can be applied as a suitable exposure biomarker without considering alterations and effects of feeding rate. The connection between reproduction and oxidative stress is dual, as reproduction both can be inhibited by oxidative stress and induce pro-oxidative processes. Further, a positive association was found between ORAC and the occurrence of embryo aberrations in the benthic amphipod Monoporeia affinis (paper III). An association between antioxidant defense and reproduction was also observed for Daphnia magna (paper IV). Threshold values for identification of exposed individuals and prediction of possible later reproductive effects were established for ORAC. This thesis has contributed to diminishing some of the knowledge gaps limiting the use of oxidative biomarkers in ecotoxicology, by contributing to increased understanding of how oxidative biomarkers relate to important life-traits. Moreover, ORAC has been identified as a suitable biomarker of not only exposure, but also reproductive effects. Future research should continue to establish connections between biomarker responses and effects at higher levels, and focus on providing defined threshold values to enable predictions about later effects.   At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p

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

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

KOSMOS Finland 2012 mesocosm study: Acartia sp. egg production rate, adult female size, adult female antioxidant capacity, and egg-hatching succes and nauplii development index of the egg transplant experiment

Ocean acidification is challenging phenotypic plasticity of individuals and populations. Calanoid copepods (zooplankton) are shown to be fairly plastic against altered pH conditions, and laboratory studies indicate that transgenerational effects are one mechanism behind this plasticity. We studied phenotypic plasticity of the copepod Acartia sp. in the course of a pelagic, large-volume mesocosm study that was conducted to investigate ecosystem and biogeochemical responses to ocean acidification. We measured copepod egg production rate, egg-hatching success, adult female size and adult female antioxidant capacity (ORAC) as a function of acidification (fCO2 ~ 365-1231 µatm) and as a function of quantity and quality of their diet. We used an egg transplant experiment to reveal whether transgenerational effects can alleviate the possible negative effects of ocean acidification on offspring development. We found significant negative effects of ocean acidification on adult female size. In addition, we found signs of a possible threshold at high fCO2, above which adaptive maternal effects cannot alleviate the negative effects of acidification on egg-hatching and nauplii development. We did not find support for the hypothesis that insufficient food quantity (total particulate carbon < 55 µm) or quality (C : N) weakens the transgenerational effects. However, females with high-ORAC-produced eggs with high hatching success. Overall, these results indicate that Acartia sp. could be affected by projected near-future CO2 levels