Differential accumulation of PAHs within planarian cephalic and posterior body parts.

It has been observed that freshwater planarians can accumulate cadmium in their head portions, with neurotoxicity and head loss accompanying this phenomenon. Since planarians exhibit head loss and symptoms of neurotoxicity in response to PAHs, we investigated the differential accumulation of pyrene and B[a]P in the body and head portions of Girardia tigrina, a freshwater planarian. It is evidenced that planarian head fragments present higher amounts of pyrene- and B[a]P-equivalents than body fragments, indicating a differential distribution of these compounds within planarian tissues.

Invasive Species Mediate Insecticide Effects on Community and Ecosystem Functioning.

Anthropogenic activities increase pesticide contamination and biological invasions in freshwater ecosystems. Understanding their combined effects on community structure and on ecosystem functioning presents challenges for an improved ecological risk assessment. This study focuses on an artificial stream mesocosms experiment testing for direct and indirect effects of insecticide (chlorantraniliprole - CAP) exposure on the structure of a benthic macroinvertebrate freshwater community and on ecosystem functioning (leaf decomposition, primary production). To understand how predator identity and resource quality alter the community responses to chemical stress, the mediating effects of an invasive predator species (crayfish Procambarus clarkii) and detritus quality (tested by using leaves of the invasive Eucalyptus globulus) on insecticide toxicity were also investigated. Low concentrations of CAP reduced the abundance of shredders and grazers, decreasing leaf decomposition and increasing primary production. Replacement of autochthonous predators and leaf litter by invasive species decreased macroinvertebrate survival, reduced leaf decomposition, and enhanced primary production. Structural equation modeling (SEM) highlighted that CAP toxicity to macroinvertebrates was mediated by the presence of crayfish or eucalypt leaf litter which are now common in many Mediterranean freshwaters. In summary, our results demonstrate that the presence of these two invasive species alters the effects of insecticide exposure on benthic freshwater communities. The approach used here also allowed for a mechanistic evaluation of indirect effects of these stressors and of their interaction on ecosystem functional endpoint, emphasizing the value of incorporating biotic stressors in ecotoxicological experiments.

Planarian behavioural endpoints in ecotoxicology: A case study evaluating mercury and salinity effects.

Freshwater planarians can be useful for the evaluation of contaminant stress on behavioral endpoints. In this work, we studied the sensitivity of the freshwater planarian Girardia tigrina in response to two model stressors (Hg and NaCl) by evaluating mortality, feeding rate and locomotion. A simple feeding assay with G. tigrina was devised, and an automated tracking system was used to evaluate locomotion. The estimated 96 h LC50s were 176.8 µg L-1 of Hg and 6.79 g L-1 of NaCl. Acute effects of Hg also included the disintegration of tissues, and loss of pigmentation. Acute effects of NaCl included motionlessness and rupture of the tegument. Hg and NaCl sub-lethal exposures caused feeding inhibition and locomotion impairment. This study demonstrates the usefulness of planarians for ecotoxicological research and that sensitive behavioral endpoints can evaluate the sub-lethal impacts of stressors to freshwater invertebrates.

Effects of pyrene and benzo[a]pyrene on the reproduction and newborn morphology and behavior of the freshwater planarian Girardia tigrina.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of aquatic ecosystems. Because they are persistent, there is great potential for chronic toxicity to aquatic species, and the evaluation of reproductive effects is fundamental. In this context, planarians are interesting experimental animals, since they can be sensitive to environmental pollutants, and a wide range of reproductive-related endpoints can be assessed. In this work we evaluated fecundity (number of cocoons), fertility (number of newborns), newborn anomalies, adult weight, regenerative abilities and PAH-residues in tissues of the freshwater planarian Girardia tigrina, exposed to either pyrene or benzo[a]pyrene (B[a]P). Pyrene reduced planarian fecundity and fertility at 18.75 µg L-1 and 75.00 µg L-1, while B[a]P reduced planarian fecundity at the 37.50 µg L-1 treatment, which was accompanied by a 33.7% reduction in fertility. Cocoons were kept in clean media and newborns were evaluated for behavioral and morphological anomalies. Many of the newborns resulting from the B[a]P experiment revealed behavioral anomalies, such as spasms and uncoordinated movements. These behavioral anomalies were observed in 12.9% and 38.2% of newborns resulting from the exposure of adult planarians to 9.38 µg L-1 and 37.50 µg L-1 of B[a]P, respectively. This study is the first report on the effects of PAHs in freshwater planarians' sexual reproduction and a decreased reproductive output was evidenced. Moreover, the exposure of adults to B[a]P lead to defects in newborns, raising concern on the possible long-term consequences of these compounds for natural planarian populations.

Toxicity of different polycyclic aromatic hydrocarbons (PAHs) to the freshwater planarian Girardia tigrina.

Freshwater planarians have been gaining relevance as experimental animals for numerous research areas given their interesting features, such as high regeneration potential, shared features with the vertebrates' nervous system or the range of endpoints that can be easily evaluated in response to contaminants. Ecotoxicological research using these animals has been steadily increasing in the past decades, as planarians' potentialities for this research area are being recognized. In this work, we used polycyclic aromatic hydrocarbons (PAHs) as model contaminants and evaluated effects of exposure to phenanthrene, pyrene and benzo[a]pyrene (B[a]P) in planarians. The freshwater planarian Girardia tigrina was chosen and mortality, cephalic regeneration (during and post-exposure), behavioral endpoints and presence of PAHs in tissues, were evaluated. Mortality was only observed in planarians exposed to phenanthrene, with an estimated LC50 of 830 µg L-1. Results indicate that planarian behavioral endpoints were very sensitive in response to sub-lethal concentrations of PAHs, showing a greater sensitivity towards B[a]P and pyrene. Briefly, post-exposure locomotion and post-exposure feeding were significantly impaired by sub-lethal concentrations of all compounds, whereas regeneration of photoreceptors was only significantly delayed in planarians exposed to pyrene. Moreover, levels of PAH-type compounds in planarian tissues followed a concentration-dependent increase, showing uptake of compounds from experimental solutions. The present results highlight the importance of studying alternative and complementary endpoints, such as behavior, not only because these may be able to detect effects at lower levels of contamination, but also due to their ecological relevance. The simplicity of evaluating a wide range of responses to contaminants further demonstrates the utility of freshwater planarians for ecotoxicological research.

Strategies of cellular energy allocation to cope with paraquat-induced oxidative stress: Chironomids vs Planarians and the importance of using different species.

Paraquat (PQ) is still used in several countries worldwide as an herbicide for weed control in agricultural production, ponds, reservoirs and irrigation canals. Thus, PQ is frequently found in surface water systems and is potentially toxic to aquatic organisms, since it can cause mitochondrial dysfunction altering in the redox state of cells. This study aimed to investigate the chronic effects of PQ to Chironomus riparius and Girardia tigrina, and compare their physiological strategies to cope with environmental stress. The mean emergence time was the most sensitive endpoint for Chironomids, with the lowest observed effect concentrations (LOEC) being 0.02 for males and 0.1 mg PQ L-1 for females. Moreover, PQ reduced the body weight of male and female imagoes, with LOECs of 0.5 and 2.5 mg PQ L-1, respectively. Paraquat also decreased the respiration rate (LOEC = 2.5 mg PQ L-1) and total glutathione (tGSH) content (LOEC = 0.5 mg PQ L-1). Thus, the aerobic production of energy was not affected and allowed chironomids to cope with oxidative stress induced by PQ, but with consequent physiological costs in terms of development rates and weight of adults. In planarians, PQ decreased the locomotion and feeding activity, and delayed photoreceptor regeneration (LOECs = 2.5 mg PQ L-1 for all endpoints). Despite increased aerobic energy production (LOEC = 0.5 mg PQ L-1), planarians were not able to cope with oxidative stress induced by the highest PQ concentrations, since lipid peroxidation levels were significantly increased (LOEC = 2.5 mg PQ L-1) concomitantly with a significant decrease of tGSH (LOEC = 2.5 mg PQ L-1). These results showed that planarians were unable to cope with oxidative stress induced by PQ with consequent impairments of behavior and regeneration despite an increased aerobic energy production.

Effects of the organic UV-filter, 3-(4-methylbenzylidene) camphor, on benthic invertebrates and ecosystem function in artificial streams.

In the last decades, the use of organic ultraviolet-filters (UV-filters) has increased worldwide, and these compounds are now considered emerging contaminants of many freshwater ecosystems. The present study aimed to assess the effects of 3-(4-methylbenzylidene) camphor (4-MBC) on a freshwater invertebrate community and on associated ecological functions. For that, artificial streams were used, and a natural invertebrate benthic community was exposed to sediments contaminated with two concentrations of 4-MBC. Effects were evaluated regarding macroinvertebrate abundance and community structure, as well as leaf decomposition and primary production. Results showed that the macroinvertebrate community parameters and leaf decomposition rates were not affected by 4-MBC exposure. On the other hand, primary production was strongly reduced. This study highlights the importance of higher tier ecotoxicity experiments for the assessment of the effects of low concentrations of organic UV-filters on freshwater invertebrate community structure and ecosystem functioning.

Using a new high-throughput video-tracking platform to assess behavioural changes in Daphnia magna exposed to neuro-active drugs.

Recent advances in imaging allow to monitor in real time the behaviour of individuals under a given stress. Light is a common stressor that alters the behaviour of fish larvae and many aquatic invertebrate species. The water flea Daphnia magna exhibits a vertical negative phototaxis, swimming against light trying to avoid fish predation. The aim of this study was to develop a high-throughput image analysis system to study changes in the vertical negative phototaxis of D. magna first reproductive adult females exposed to 0.1 and 1 µg/L of four neuro-active drugs: diazepam, fluoxetine, propranolol and carbamazepine. Experiments were conducted using a custom designed experimental chamber containing four independent arenas and infrared illumination. The apical-located visible light and the GigE camera located in front of the arenas were controlled by the Ethovision XT 11.5 sofware (Noldus Information Technology, Leesburg, VA). Total distance moved, time spent per zone (bottom vs upper zones) and distance among individuals were analyzed in dark and light conditions, and the effect of different intensities of the apical-located visible light was also investigated. Results indicated that light intensity increased the locomotor activity and low light intensities allowed to better discriminate individual responses to the studied drugs. The four tested drugs decreased the response of exposed organisms to light: individuals moved less, were closer to the bottom and at low light intensities were closer each other. At high light intensities, however, exposed individuals were less aggregated. Propranolol, carbamazepine and fluoxetine induced the most severe behavioural effects. The tested drugs at environmental relevant concentrations altered locomotor activity, geotaxis, phototaxis and aggregation in D. magna individuals in the lab. Therefore the new image analysis system presented here was proven to be sensitive and versatile enough to detect changes in diel vertical migration across light intensities and low concentration levels of neuro-active drugs.

Toxicity of tributyltin (TBT) to the freshwater planarian Schmidtea mediterranea.

The freshwater planarian Schmidtea mediterranea, one of the best characterized animal models for regeneration research and developmental biology, is being recognised as a useful species for ecotoxicological studies. Sensitive endpoints related to planarians' behaviour and regeneration can be easily evaluated after exposure to environmental stressors. In this work the sensitivity of S. mediterranea to a gradient of environmentally relevant concentrations of TBT was studied using multiple endpoints like survival, locomotion, head regeneration and DNA damage. In addition, a feeding assay based on planarian's predatory behaviour was performed. Results indicated that TBT is toxic to planarians with LC50's of 1.87 µg L(-1) Sn and 1.31 µg L(-1) Sn at 48 h and 96 h of exposure respectively. Sub-lethal exposures to TBT significantly reduced locomotion and feeding, delayed head regeneration and caused DNA damage in planarians. The behavioural endpoints (feeding and locomotion) and head regeneration were the most sensitive parameters followed by DNA damage. Similar to other aquatic model organisms, S. mediterranea showed high sensitivity towards TBT exposure. Based on our results, and though further research is required concerning their sensitivity to other pollutants, the use of freshwater planarians as a model species in ecotoxicology is discussed.