RESEARCH

Our laboratory is specializing in aquatic ecotoxicology, behavioural ecotoxicology, eco-neurotoxicology and environmental risk assessment studies.

We are particularly interested in how pollutants and neuroactive chemicals impact animal behaviour and neuro-development. We are also investigating mechanistic neurobiological foundations underlying behavioural changes in response to contaminants.

Eco-neurotoxicology

We are particularly interested in emerging aspects of eco-neurotoxicology. This new branch of ecotoxicology studies how pollutants impact animal behaviour, neuro-development as well as tries to elucidate neurobiological foundations underlying changes in animal behaviour.

During the last decade there has been a significant increase in awareness of how the anthropogenic pollution can alter behavioural traits of diverse aquatic fauna as well as human health.

There are still no regulatory requirements to assess the eco-neurotoxic properties of production chemicals with less than one percent of all chemicals sold on global markets so far evaluated against their neurotoxic properties.

Matrix heatmap analysis of high-throughput time-resolved behavioural phenotyping screening test.

Behavioural barcoding demonstrates dynamic alterations upon exposure of Artemia sp. to organophosphate pesticide.

To assess impact of chemical on nervous system we are using diverse alternative aquatic model organisms from rotifers to embryonal and early larval stages of zebrafish.

We are applying advanced neuro-behavioral phenotyping approaches.

Particularly we are using new bioassays amenable for high-throughput and thus rapid prioritisation of chemicals with neurotoxic and/or neuro-modulating properties. 

In mechanistic studies we apply transgenic zebrafish models as well as cell-based and molecular bioassays to quantify programmed cell death, assess discrete neuronal sub-populations and perform functional analysis of neuronal transmission. 

Expertise

We have significant know-how in eco-neurotoxicology, behavioural ecotoxicology and development of sensitive neuro-behavioural biotests in:

 

  • Environmental risk assessment
  • Characterisation of new production chemicals
  • Discovery of new neuroactive drugs
  • Water quality sensing

 

We are also developing new advanced behavioural biotests to test impact of toxicants on higher behavioural functions such as sensory-motor assays, decision-making and cognitive behaviours.

Research Project Examples

  • Applications of advanced neuro-behavioural tests in aquatic ecotoxicology
  • Embryo assays in rapid aquatic risk assessment
  • Neurotoxicity of industrial pollutants
  • Neurotoxicity of plastic additives
  • Neuro-behavioural alterations induced by emerging pollutants
  • Real-time, early-warning water biomonitoring systems
  • High-throughout behavioural video analytics for rapid discovery of nerve poison countermeasures
  • Automation of biotests in aquatic ecotoxicology

Neurotoxicity of plastic additives

Recent studies have demonstrated that small molecular weight toxic chemicals can leach from common recyclable and non-recyclable plastics used in manufacturing and food industry. Those include compounds such as bisphenol A (BPA) and its analogues, polybrominated diphenyl ethers (PBDE), vinyl polymers (high-density polyethylene, HDPE; low-density polyethylene, LDPE; polypropylene, PP; polyvinyl chloride, PVC), polyesters (polycarbonate, PC; polyethylene terephthalate, PET) and aromatic polymers (polystyrene, PS).

Many studies have demonstrated that additives from manufacturing plastics can readily migrate to water and have significant impact on aquatic organisms. The long-term physiological impact of exposure to such compounds still remains unknown. Since many low molecular weight plastic additives are used ubiquitously in the manufacturing of various plastic polymers and appear to exhibit phenotypes associated with toxicity, they may represent a nascent ecotoxicological risk for both freshwater and marine organisms as well as human health 

At present very little is known about neurotoxicity and eco-neurotoxicity of plastic additives. There a paucity of data on how exposure to such chemicals during neuro-development can impact behaviour in later stages of life. 

We have recently discovered that additives such as plastic photoinitiators can leach out of 3D printed plastics to water and can induce significant neuro-behavioural perturbations during development of zebrafish.

Extracts from 3D printed plastic induced rapid retardation of locomotion, changes in photomotor response and habituation to photic stimuli with progressive paralysis in 120 hpf larvae.

Significantly decreased acetylcholinesterase (AChE) activity with lack of any CNS-specific apoptotic phenotypes as well as lack of changes in motor neuron density, axonal growth, muscle segment integrity or presence of myoseptal defects were detected upon exposure to plastic extracts during embryogenesis.

Considering implications of our discovery for environmental risk assessment and the growing usage of 3D-printing technologies, we speculate that some 3D-printed plastic waste may represent a significant and yet very poorly uncharacterized environmental hazard that merits further investigation on a range of aquatic and terrestrial species.

3D-printed plastic waste may represent new and very poorly uncharacterised environmental hazard

Toxic additives from some manufacturing and 3D printed plastics can readily migrate to water and have significant impact on aquatic organisms. The long-term physiological impact of exposure to highly mobile plastic additive compounds still remains unknown.

Neurotoxicity of additives migrating from 3D printed plastics

We have recently discovered that additives such as plastic photoinitiators can leach out of 3D printed plastics to water and can induce significant neuro-developmental perturbations in zebrafish.

Plastic leachates induce neuro-behavioural perturbations

Locomotory trajectories of zebrafish larvae from our studies demonstrate that additives leaching out of some 3D printed plastics to water can induce acute neuro-behavioural perturbations. Video-based behavioural analysis was performed using a ZebraBox system from ViewPoint Behavior Technology

Neurotoxicity of nano- and microplastics

Pollution by microplastics and nanoplastics  is increasingly recognised worldwide as a very significant environmental risk factor for terrestrial as well as freshwater and marine ecosystems.

Micro- and nanoplastics ingestion is the main route of exposure, however, aquatic organisms can also uptake nanoplatics via gils. Upon ingestion extremely small particles of plastic can be transported within the body of the organisms reportedly inducing diverse wide-ranging physiological effects.

At present their long-term biological impacts are still relatively unknown. Importantly, both micro- and nanoplastics can become carries for other pollutants. This is because especially hydrophobic chemicals can readily adsorb to the surface of plastic. The uptake of contaminated plastic particles can expose organisms to significant concentrations of toxicants.

Interestingly, despite a large number of studies on ecotoxicological aspects of plastic pollution there is a paucity of data about neurotoxic effects of micro- and nanoplastics.

Recent data indicate that plastic microparticles alone as well as through acting as carriers for known neurotoxicants can induce eco-neurotoxic effects.

Our laboratory was recently involved in the study of microplastics  generated from phenol-formaldehyde plastics are used globally as floral foam and generate microplastics that can enter the environment. This study is the first to describe how aquatic animals interact with this type of microplastic, and the resultant physiological responses. We analysed “regular foam” microplastics generated from petroleum-derived phenol-formaldehyde plastic, and “biofoam” microplastics generated from plant-derived phenol-formaldehyde plastic.

Our results demonstrated that phenol-formaldehyde microplastics can interact with a range of aquatic animals, and affect sublethal endpoints by leaching toxic compounds, and through the physical presence of the microplastics themselves.

Despite a large number of studies on ecotoxicological aspects of plastic pollution there is a paucity of data about neurotoxic effects of micro- and nanoplastics.ecent data indicate that

Plastic micro- and nano- particles can induce sublethal ecotoxic effects through the physical presence, by leaching toxic compounds,  as well as through acting as carriers for known neurotoxicants.

To understand the eco-neurotoxic impact of micro- and nano-plastics we can employ a battery of neurobehavioral as well as mechanistic biotets on a wide range of aquatic model organisms.

Neurotoxicity of pharmaceuticals and other emerging micro-pollutants

Pollution of aquifers by emerging micro-pollutants such as pharmaceuticals, neuroactive substances and diverse non-prescription “brain enhancers” is increasingly recognised as potential environmental risk factors.

Wastewater treatment plants are increasingly indicated as emitters of pharmaceutical micro-pollutants, pesticides, personal care products, surfactants, industrial additives, perfluorinated compounds or endocrine disrupters. The currently employed technologies underlying the wastewater treatment processes (WWTP) are largely incapable of their complete removal and/or inactivation. For instance, rate of removal of pharmaceuticals such as carbamazepine or diclofenac have been reported to be less than 25%  while the case of other emerging contaminants such as personal care products are already reaching chronic levels (i.e. microgram/L range) in some countries.

Better analysis strategies, new biotests and management of micro-pollutants and Contaminants of Emerging Concern (CECs i.e. chemicals that have only recently been identified as potentially harmful to the environment) in wastewater will be required including new additional guidelines for management and testing of euro-active micro-pollutants.

This will need to include revisions to the current EU Urban Wastewater Treatment Directive (UWWTD) that at present specifies requirements for acceptable levels and the removal only for nutrients and organic matter.

Furthermore inclusion of micro-pollutants and CEC should be expanded within current REACH strategy. In particular there needs to be a broader implementation and definition of hazardous substances and inclusion of persistent, mobile and toxic substances (PMTs) and very persistent and very mobile substances (vPvM) within the to be updated EU Chemicals Strategy for Sustainability Towards a Toxic-Free Environment.

At present there are still no regulatory guidelines available for the assessment of eco-neurotoxicity, i.e. neurotoxicity resulting from exposure to environmental chemicals in species other than humans (e.g. fish, birds, invertebrates).

As such, neurotoxicity effects of industrial contaminants are currently under investigated and require innovative analytical approaches to assess aquatic risks at individual, population and ecosystem levels.

Our laboratory is currently involved in several large international projects aimed at development of new chemo-behavioural test strategies that are amenable for higher-throughout as well as assessing impact of anti-depressant drugs such as fluoxetine, diazepam and other neuro-modulating micro-pollutants on aquatic species. We are also involved in projects evaluating eco-neurotoxicological connotations of interactions between emerging contaminants such as neuro-active and nonsteroidal anti-inflammatory drugs.

Wastewater treatment plants (WWTP) are major indicated as emitters of pharmaceutical micro-pollutants, pesticides, personal care products, surfactants, industrial additives, perfluorinated compounds or endocrine disrupters.

Current WWTP technologies (WWTP) are largely incapable of their complete removal and/or inactivation. Source: Baltic Sea Centre, Stockholm University.

To understand the eco-neurotoxic impact of aquatic pollution, new test strategies need to integrate both physiologically and ecologically relevant end-points while high-throughput chemo-behavioural prioritising strategies for rapid screening of chemicals need to be developed.

High-throughput chemo-behavioural screening strategies for prioritisation and benchmarking of chemicals with eco-neurotoxic properties.

New test strategies with small aquatic bioindicators are emerging as distinctively advantageous for high-throughput neuro-behavioural neurotoxicity testing.

Neuroactive drug discovery

Apart from eco-neurotoxicology we are also very interested in rapid discovery and characterisation of neuro-active drug candidates. In particular drug candidates derived from medicinal plants as well as tropical and marine organisms. 

Our biological models such as planarians and zebrafish and established high-throughput behavioural phenotyping capabilities are particularly suitable for neuro-active drug discovery.

Majority of even most complex attempts at organotypic neuronal cell cultures do not represent truly integrated neural networks. As a result they are inherently unable to recapitulate the structural and functional integration of an intact CNS that manifests itself in complex endpoints such as behavior and cognitive responses. Neuro-active therapeutics such as anxiolytics, antipsychotics and antidepressants, alter the CNS function and thus manifestation of behavioral phenotypes and the latter can only be discovered using behavioral phenotyping 

In this context, chemobehavioral phenotyping using innovative small organism models represent powerful and highly integrative approaches to study impact of new chemicals on central and peripheral nervous systems. Their advantages include relative low cost per screen, fast life cycles, ease of obtaining ethical approval, amenability to non-invasive, whole-animal in vivo imaging as well behavioural phenotypes that can be rapidly analysed 

At present we are collaborating with several partners in Europe, Japan and Australia on research projects employing our high-throughput chemo-behavioural phenotyping strategies in discovery of neuro-modulating chemicals derived from medicinal plants and marine organisms.

Chemo-behavioural phenotyping in euro-active drug discovery.

Small aquatic model organisms such as planarians and zebrafish are particularly suitable for neuro-active drug discovery when combined with innovative high-throughput behavioural phenotyping capabilities.

Behavioural biotests performed on early life stages of zebrafish such as embryos and larvae are convenient, high-throughput tools in neuro-active drug discovery.

Zebrafish embryo photomotory responses (PMR, left) and larval photomotory response (LPR, right) assays for rapid assessment of chemicals with neuro-modulating mode of action.

References

Bownik A and Wlodkowic D 2021 Applications of advanced neuro-behavioural analysis strategies in aquatic ecotoxicology, Science of The Total Environment, 772, 145577 DOI

Bai Y, Henry J, Campana O, Wlodkowic D 2021 Emerging prospects of integrated bioanalytical systems in neuro-behavioral toxicology, Science of The Total Environment, 756, 143922 DOI

Wlodkowic D and Campana O 2021 Toward High-Throughput Fish Embryo Toxicity Tests in Aquatic Toxicology, Environmental Science & Technology, 55(6), 3505–3513DOI

Henry J and Wlodkowic D 2020 High-throughput animal tracking in chemobehavioral phenotyping: Current limitations and future perspectives, Behavioural Processes, 180, 104226 DOI

Trestrail C, Walpitagama M, Hedges C, Truskewycz A, Miranda A, Wlodkowic D, Shimeta J, Nugegoda D 2020 Foaming at the mouth: Ingestion of floral foam microplastics by aquatic animals, Science of The Total Environment, 705, 135826 DOI

Walpitagama M, Carve M, Douek AM, Trestrail C, Bai Y, Kaslin J, Wlodkowic D 2019 Additives migrating from 3D-printed plastic induce developmental toxicity and neuro-behavioural alterations in early life zebrafish (Danio rerio), Aquatic Toxicology, 213, 105227 DOI

Carve M and Wlodkowic D 2018 3D-Printed Chips: Compatibility of Additive Manufacturing Photopolymeric Substrata with Biological Applications, Micromachines, 9(2), 91 DOI

Macdonald NM,  Zhu F, Hall CJ, Reboud J, Crosier PS, Patton EE, Wlodkowic D, Cooper JM 2016 Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays, Lab Chip, 16, 291-297 DOI