Laboratory of Environmental Toxicology and Aquatic Ecology Environmental Toxicology Unit - GhEnToxLab Ghent University - Faculty of Bioscience Engineering
Emerging contaminants in Belgian marine waters: Single toxicant and mixture risks of pharmaceuticals
Knowledge on the effects of pharmaceuticals on aquatic marine ecosystems is limited. The aim of this study was therefore to establish the effect thresholds of pharmaceutical compounds occurring in the Belgian marine environment for the marine diatom Phaeodactylum tricornutum, and subsequently perform an environmental risk assessment for these substances. Additionally, a screening-level risk assessment was performed for the pharmaceutical mixtures.
No immediate risk for acute toxic effects of these compounds on P. tricornutum were apparent at the concentrations observed in the Belgian marine environment. In two Belgian coastal harbours however, a potential chronic risk was observed for the ?-blocker propranolol. No additional risks arising from the exposure to mixtures of pharmaceuticals present in the sampling area could be detected. However, as risk characterization ratios for mixtures of up to 0.5 were observed, mixture effects could emerge should more compounds be taken into account.
Claessens M, Vanhaecke L, Wille K, Janssen CR. 2013. Emerging contaminants in Belgian marine waters: Single toxicant and mixture risks of pharmaceuticals. Marine Pollution Bulletin. In press.
Cloning and functional analysis of the ecdysteroid receptor complex in the opossum shrimp Neomysis integer (Leach, 1814)
In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan.
Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage = 75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein–protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100 μg/L, and no effects against the molting process and nymphal development were scored.
In conclusion, the in vitro cell reporter assay, based on NiEcR-LBD/CrcRXR-LBD heterodimerization in Hi5 cells and validated with the natural ecdysteroid hormone PonA, represents a useful tool for the screening of putative EDCs. As a test example for non-steroidal ecdysone agonist insecticides, tebufenozide had no negative effects on NiEcR/RXR receptor dimerization in vitro, nor on the molting process and nymphal development of N. integer at the tested concentration (100 μg/L) in vivo.
De Wilde R, Swevers L, Soina T, Christiaens O, Rougé P, Cooreman K, Janssen CR, Smagghe G. 2013. Cloning and functional analysis of the ecdysteroid receptor complex in the opossum shrimp Neomysis integer (Leach, 1814). Aquatic Toxicology 130–131:31–40.
Modelling the effects of copper on soil organisms and processes using the free ion approach: Towards a multi-species toxicity model
Our lab has a long history of metal bioavailability research. Here, the results of previous terrestrial studies performed by our lab was used and integrated with other datasets to develop a general biovailability model for copper. The free ion approach has been previously used to calculate critical limit concentrations for soil metals based on point estimates of toxicity. In this study, the approach was applied to dose–response data for copper effects on seven biological endpoints in each of 19 European soils. The approach was applied using the concept of an effective dose, comprising a function of the concentrations of free copper and 'protective' major cations, including H+. A significant influence of H+ on the toxicity of Cu2+ was found, while the effects of other cations were inconsistent. The model could be generalised by forcing the effect of H+ and the slope of the dose–response relationship to be equal for all endpoints. This suggests the possibility of a general bioavailability model for copper effects on organisms. Furthermore, the possibility of such a model could be explored for other cationic metals such as nickel, zinc, cadmium and lead.
Lofts S, Criel P, Janssen CR, Lock K, McGrath SP, Oorts K, Rooney CP, Smolders E, Spurgeon DJ, Svendsen C, Van Eeckhout H, Zhao F-Z. 2013. Modelling the effects of copper on soil organisms and processes using the free ion approach: Towards a multi-species toxicity model. Environmental Pollution 178:244–253.
New techniques for the detection of microplastics in sediments and field collected organisms
Microplastics have been reported in marine environments worldwide. Accurate assessment of quantity and type is therefore needed. Here, we propose new techniques for extracting microplastics from sediment and invertebrate tissue. The method developed for sediments involves a volume reduction of the sample by elutriation, followed by density separation using a high density NaI solution. Comparison of this methods' efficiency to that of a widely used technique indicated that the new method has a considerably higher extraction efficiency. For fibres and granules an increase of 23% and 39% was noted, extraction efficiency of PVC increased by 100%. The second method aimed at extracting microplastics from animal tissues based on chemical digestion. Extraction of microspheres yielded high efficiencies (94–98%). For fibres, efficiencies were highly variable (0–98%), depending on polymer type. The use of these two techniques will result in a more complete assessment of marine microplastic concentrations.
Claessens M, Van Cauwenberghe L, Vandegehuchte M, Janssen CR. 2013. New techniques for the detection of microplastics in sediments and field collected organisms. Marine Pollution Bulletin, In press.
Monitoring micropollutants in marine waters, can quality standards be met?
The environmental risks of 33 micropollutants occurring in Belgian coastal zone were assessed as single substances and as mixtures. Water and sediment samples were taken in harbors, coastal waters and the Scheldt estuary during 2007–2009. Measured environmental concentrations were compared to quality standards such as Predicted No Effect Concentrations (PNECs), Environmental Quality Standards (EQSs), and Ecotoxicological Assessment Criteria (EAC). Out of a total of 2547 samples analyzed, 232 and 126 samples exceeded the EQS and EAC, respectively. Highest risks were observed for TBT, PBDEs, PCBs and the PAHs anthracene, indeno(1,2,3-cd)pyrene, benzo(g,h,i)perylene, benzo(k)fluoranthene, and benzo(b)fluoranthene in the water compartment and for TBT and PCBs in the sediment compartment. Samples taken at all stations during the April 2008 campaign indicate a potential risk of the contaminant mixtures to the aquatic environment (except W06 station). This study argues the need to revise quality standards when appropriate and hence the overall regulatory implication of these standards.
Ghekiere A, Verdonck F, Claessens M, Monteyne E, Roose P, Wille K, Goffin A, Rappé K, Janssen CR. 2013. Monitoring micropollutants in marine waters, can quality standards be met? Marine Pollution Bulletin 69:243-250.
Application of a silicone rubber passive sampling technique for monitoring PAHs and PCBs at three Belgian coastal harbours
A 4-year monitoring study - coordinated by our lab - was performed to examine the freely dissolved water concentrations of PAHs and PCBs in three coastal harbours and at an offshore station in the North Sea. The results are part of a more extensive study to provide information on occurrence, distribution and effects of pollutants in the Belgian coastal zone. In the present study silicone rubber passive samplers were used. We found that the non-linear least-square (NLS) method proved to be suitable for estimating sampling rates when using the following performance reference compounds: fluorene-d10, phenanthrene-d10, fluoranthene-d10, benzo(e)pyrened12, coronene-d12, CB10, CB14, CB50, CB104, CB145 and CB204. The application of two NLS methods for estimating the sampling rate (Rs) resulted in significant differences for freely dissolved concentrations for individual compounds of up to 30% between the two methods. A model that takes into account the decrease of sampling rate for compounds with higher molecular weight should give a more accurate Rs and was the preferred estimation method. Rs varied from 0.9 to 34.8 L/d for the different target compounds, while estimated freely dissolved concentrations for sum 15 PAHs varied between 3.9 and170 ng/L and for sum 14 PCBs between 0.030 and 3.1 ng/L. The stations located within marinas showed the highest level of contamination, while the offshore station (5 mile from coastline) exhibited the lowest level. The implications of the use of passive samplers for monitoring programs are discussed.
Monteyne E, Roose R, Janssen CR. 2013. Application of a silicone rubber passive sampling technique for monitoring PAHs and PCBs at three Belgian coastal harbours. Chemosphere 91:390-398.
Whole sediment toxicity tests for metal risk assessments: On the importance of equilibration and test design to increase ecological relevance
Current laboratory-based approaches for predicting metal toxicity in sediments exhibit a number of limitations. The most important are (1) a lack of sufficient equilibration resulting in unrealistically low pH values or unnaturally high pore water metal concentrations and (2) an inadequate test design regarding the metal concentrations selected for spiking. This study illustrates that by explicitly accounting for these limitations, one obtains reliable and environmentally realistic toxicity data, thus advancing the metal risk assessments of sediments. To this end, a toxicity test design with natural sediments was developed in which the administered metal concentrations were selected to comprise a range of [SEM-AVS] (the difference between the molar concentration of simultaneously extracted metals and acid volatile sulfides) closely surrounding zero. In addition, the presented test design includes a 35 or 40 day equilibration period with overlying water renewal during which conductivity, pH and metal concentrations in the overlying water are monitored. This allows toxicity testing to start after equilibrium for these parameters has been reached. This test design was applied to Ephoron virgo (Olivier, 1791), Gammarus pulex (Linnaeus, 1758) and Lumbriculus variegatus (Mueller, 1774) exposed to Zn and Pb. These tests indicated that the general concept of absence of toxicity when [SEM-AVS]<0 could not be rejected. However, the onset of Zn toxicity occurred at lower concentrations than generally assumed.
Vandegehuchte MB, Nguyen LTH, De Laender F, Muyssen BTA, Janssen CR. 2013. Whole sediment toxicity tests for metal risk assessments: On the importance of equilibration and test design to increase ecological relevance. Environmental Toxicology and Chemistry. Accepted. DOI: 10.1002/etc.2156.
Using additive modelling to quantify the effect of chemicals on phytoplankton diversity and biomass
Environmental authorities require the protection of biodiversity and other ecosystem properties such as biomass production. However, the endpoints listed in available ecotoxicological datasets generally do not contain these two ecosystem descriptors. Inferring the effects of chemicals on such descriptors from micro- or mesocosm experiments is often hampered by inherent differences in the initial biodiversity levels between experimental units or by delayed community responses. Here we introduce additive modelling to establish the effects of a chronic application of the herbicide linuron on 10 biodiversity indices and phytoplankton biomass in microcosms. We found that communities with a low (high) initial biodiversity subsequently became more (less) diverse, indicating an equilibrium biodiversity status in the communities considered here. Linuron adversely affected richness and evenness while dominance increased but no biodiversity indices were different from the control treatment at linuron concentrations below 2.4 μg/L. Richness-related indices changed at lower linuron concentrations (effects noticeable from 2.4 μg/L) than other biodiversity indices (effects noticeable from 14.4 μg/L) and, in contrast to the other indices, showed no signs of recovery following chronic exposure. Phytoplankton biomass was unaffected by linuron due to functional redundancy within the phytoplankton community. Comparing thresholds for biodiversity with conventional toxicity test results showed that standard ecological risk assessments also protect biodiversity in the case of linuron.
Viaene K, De Laender F, Van den Brink PJ, Janssen C. 2013. Using additive modelling to quantify the effect of chemicals on phytoplankton diversity and biomass Science of the Total Environment 449:71-80.
Interactive effects of a bacterial parasite and the insecticide carbaryl to life-history and physiology of two Daphnia magna clones differing in carbaryl sensitivity
Natural and chemical stressors occur simultaneously in the aquatic environment. Their combined effects on biota are usually difficult to predict from their individual effects due to interactions between the different stressors. Several recent studies have suggested that synergistic effects of multiple stressors on organisms may be more common at high compared to low overall levels of stress. In this study, we used a three-way full factorial design to investigate whether interactive effects between a natural stressor, the bacterial parasite Pasteuria ramosa, and a chemical stressor, the insecticide carbaryl, were different between two genetically distinct clones of Daphnia magna that strongly differ in their sensitivity to carbaryl. Interactive effects on various life-history and physiological endpoints were assessed as significant deviations from the reference Independent Action (IA) model, which was implemented by testing the significance of the two-way carbaryl × parasite interaction term in two-way ANOVA's on log-transformed observational data for each clone separately. Interactive effects (and thus significant deviations from IA) were detected in both the carbaryl-sensitive clone (on survival, early reproduction and growth) and in the non-sensitive clone (on growth, electron transport activity and prophenoloxidase activity). No interactions were found for maturation rate, filtration rate, and energy reserve fractions (carbohydrate, protein, lipid). Furthermore, only antagonistic interactions were detected in the non-sensitive clone, while only synergistic interactions were observed in the carbaryl sensitive clone. Our data clearly show that there are genetically determined differences in the interactive effects following combined exposure to carbaryl and Pasteuria in D. magna.
De Coninck DIM, De Schamphelaere KAC, Jansen M, De Meester L, Janssen CR. 2013. Interactive effects of a bacterial parasite and the insecticide carbaryl to life-history and physiology of two Daphnia magna clones differing in carbaryl sensitivity. Aquatic Toxicology 130-131: 149-159.
Combined and interactive effects of global climate change and toxicants on populations and communities
Increased temperature and other environmental effects of global climate change (GCC) have documented impacts on many species (e.g., polar bears, amphibians, coral reefs) as well as on ecosystem processes and species interactions (e.g., the timing of predator–prey interactions). A challenge for ecotoxicologists is to predict how joint effects of climatic stress and toxicants measured at the individual level (e.g., reduced survival and reproduction) will be manifested at the population level (e.g., population growth rate, extinction risk) and community level (e.g., species richness, food-web structure). The authors discuss how population- and community-level responses to toxicants under GCC are likely to be influenced by various ecological mechanisms. Stress due to GCC may reduce the potential for resistance to and recovery from toxicant exposure. Long-term toxicant exposure can result in acquired tolerance to this stressor at the population or community level, but an associated cost of tolerance may be the reduced potential for tolerance to subsequent climatic stress (or vice versa). Moreover, GCC can induce large-scale shifts in community composition, which may affect the vulnerability of communities to other stressors. Ecological modeling based on species traits (representing life-history traits, population vulnerability, sensitivity to toxicants, and sensitivity to climate change) can be a promising approach for predicting combined impacts of GCC and toxicants on populations and communities.
Moe SJ, De Schamphelaere K, Clements WH, Sorensen MT, Van den Brink PJ, Liess M. 2013. Combined and interactive effects of global climate change and toxicants on populations and communities. Environ Tox Chem 32(1): 49-61.
Laboratory of Environmental Toxicology and Aquatic Ecology
Knowledge on the effects of pharmaceuticals on aquatic marine ecosystems is limited. The aim of this study was therefore to establish the effect thresholds of pharmaceutical compounds occurring in the Belgian marine environment for the marine diatom Phaeodactylum tricornutum, and subsequently perform an environmental risk assessment for these substances. Additionally, a screening-level risk assessment was performed for the pharmaceutical mixtures. 
In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. 
Our lab has a long history of metal bioavailability research. Here, the results of previous terrestrial studies performed by our lab was used and integrated with other datasets to develop a general biovailability model for copper. The free ion approach has been previously used to calculate critical limit concentrations for soil metals based on point estimates of toxicity. In this study, the approach was applied to dose–response data for copper effects on seven biological endpoints in each of 19 European soils. The approach was applied using the concept of an effective dose, comprising a function of the concentrations of free copper and 'protective' major cations, including H+. A significant influence of H+ on the toxicity of Cu2+ was found, while the effects of other cations were inconsistent. The model could be generalised by forcing the effect of H+ and the slope of the dose–response relationship to be equal for all endpoints. This suggests the possibility of a general bioavailability model for copper effects on organisms. Furthermore, the possibility of such a model could be explored for other cationic metals such as nickel, zinc, cadmium and lead.
A 4-year monitoring study - coordinated by our lab - was performed to examine the freely dissolved water concentrations of PAHs and PCBs in three coastal harbours and at an offshore station in the North Sea. The results are part of a more extensive study to provide information on occurrence, distribution and effects of pollutants in the Belgian coastal zone. In the present study silicone rubber passive samplers were used. We found that the non-linear least-square (NLS) method proved to be suitable for estimating sampling rates when using the following performance reference compounds: fluorene-d10, phenanthrene-d10, fluoranthene-d10, benzo(e)pyrened12, coronene-d12, CB10, CB14, CB50, CB104, CB145 and CB204. The application of two NLS methods for estimating the sampling rate (Rs) resulted in significant differences for freely dissolved concentrations for individual compounds of up to 30% between the two methods. A model that takes into account the decrease of sampling rate for compounds with higher molecular weight should give a more accurate Rs and was the preferred estimation method. Rs varied from 0.9 to 34.8 L/d for the different target compounds, while estimated freely dissolved concentrations for sum 15 PAHs varied between 3.9 and170 ng/L and for sum 14 PCBs between 0.030 and 3.1 ng/L. The stations located within marinas showed the highest level of contamination, while the offshore station (5 mile from coastline) exhibited the lowest level. The implications of the use of passive samplers for monitoring programs are discussed.
