Rita C. Bicho

The use of a lacertid lizard as a model for reptile ecotoxicology studies--part 1 field demographics and morphology.

At the European level, lacertid lizards have been proposed as potential model species for reptile ecotoxicology. We studied demographic and morphological aspects of natural field subpopulations of Podarcis bocagei inhabiting similar agricultural habitats which were either regularly exposed to pesticides, or not. Parameters examined in this study included population size and density, sex ratio, adult body size, fluctuating asymmetry in femoral pores and parasite prevalence. In general, we detected few statistically significant differences between the exposed and reference subpopulations. Although field situations are ecologically complex and factors other than pesticides may be acting, the absence of observable effects on field subpopulations is probably indicative that lizards are coping or compensating for this level of exposure.

The use of a lacertid lizard as a model for reptile ecotoxicology studies: Part 2 - Biomarkers of exposure and toxicity among pesticide exposed lizards

As part of a wider study examining the impacts of corn pesticides on lacertid lizards in north-western Portugal, we examined various physiological, biochemical, and histological biomarkers of exposure and effect among field populations of Podarcis bocagei. Biomarkers included body condition index, standard metabolic rate, locomotor performance, parasitization, glutathione oxidative pathways and related enzyme activity, lipid peroxidation and liver and testis histology. Few of the various biomarkers investigated provided statistically significant evidence of toxic effect. However, using a weight of evidence approach, we conclude that pesticides are affecting lizards living in the vicinity of pesticide exposed corn agriculture sites. Lizards from these locations present a profile of animals under metabolic stress with reduced condition indices, increased standard metabolic rate, lower incidence of hepatocyte vacuolation, altered iron metabolism, increased activation of GSH oxidation pathways, and even increased prevalence of hemoparasites.

Enchytraeid Reproduction Test(PLUS): hatching, growth and full life cycle test--an optional multi-endpoint test with Enchytraeus crypticus.

Soil ecotoxicity standard tests for invertebrates are usually limited to the assessment of endpoints like survival and reproduction. Adverse effects may occur at other developmental stages, e.g. embryo development, hatching or growth. The species Enchytraeus crypticus is a model organism in the standard soil ecotoxicology test, where survival and reproduction are assessed. In the present study we optimized the test method to include additional endpoints. The proposed test started with synchronized age organisms’, and included additionally hatching success, growth, maturation status and full life cycle. This allowed for the calculation of cocoon production and population growth rate. Results indicated that Cd is embryotoxic, main effect occurs on the embryo developmental stage and maturity. Further, the full life cycle test can discriminate between pre- and post-embryo formation. The increased sensitivity and full life cycle detail level makes it potentially useful for novel materials e.g. nanomaterials where the mode of action and hence effect target is unknown.Graphical Abstract

Thyroid disruption in the lizard Podarcis bocagei exposed to a mixture of herbicides: a field study

Pesticide exposure has been related with thyroid disrupting effects in different vertebrate species. However, very little is known about the effects of these compounds in reptiles. In the Mediterranean area, lacertid lizards are the most abundant vertebrate group in agroecosystems, and have been identified as potential model species for reptile ecotoxicology. The aim of this study was to understand if the herbicides applied in corn fields have thyroid disruptive effects in the lizard Podarcis bocagei. Adult male lizards were captured in north-western Portugal in corn fields treated with herbicides (exposed sites), and in organic agricultural fields (reference sites). Thyroid and male gonad morphology and functionality, and testosterone levels were investigated through histological, immunohistochemical and biochemical techniques. Lizards from exposed locations displayed thyroid follicular lumens with more reabsorption vacuoles and significantly larger follicular area than those from reference fields. Furthermore, testes of lizards from exposed locations had significantly larger seminiferous tubule diameters, significantly higher number of spermatogenic layers and displayed an up-regulation of thyroid hormone receptors when compared with lizards from reference areas. These findings strongly suggest that the complex mixture of herbicides that lizards are exposed to in agricultural areas have thyroid disrupting effects which ultimately affect the male reproductive system. Alachlor, which has demonstrated thyroid effects in mammals, may be largely responsible for the observed effects.

Effects of Ag nanomaterials (NM300K) and Ag salt (AgNO3) can be discriminated in a full life cycle long term test with Enchytraeus crypticus

Information on effects of silver nanoparticles on soil invertebrates, especially using long-term exposures, is scarce. In this study we investigated the effects of the reference Ag (NM300K) (compared to AgNO3) using the full life cycle test (FLCt) of the soil invertebrate Enchytraeus crypticus. Results showed that effects were higher compared to the standard reproduction test, which is shorter and does not cover the FLC. Both Ag forms caused a reduction on hatching success, juvenile and adult survival and reproduction with similar ECx. Differences between AgNO3 and Ag NM300K could be discriminated using the FLCt: AgNO3 decreased hatching success was shown to be a delay in the process, whereas Ag NM300K caused irreversible effects during the same time frame. These effects may have occurred during the embryo development, hatching (inhibition) or survival of hatched juveniles. Ag NM300K caused non-monotonic concentration-response effect as observed by the high effect of the lowest concentration (20mgkg-1). It is known that dispersion is higher at lower concentrations - this could explain the increased effect at low concentration. Non monotonic responses are well described in the literature, where effects of high cannot predict for low concentrations, hence special attention should be given for NMs low concentration effects.

Biomarkers of exposure and effect in a lacertid lizard (Podarcis bocagei Seoane) exposed to chlorpyrifos

In Europe, reptiles have been recently included in environmental risk-assessment processes for registration of plant-protection products. However, data on toxicity effects of most compounds are lacking. Chlorpyrifos is the most commonly used organophosphorus insecticide worldwide. In the present study, the authors exposed a lacertid lizard, Podarcis bocagei, to sublethal concentrations of chlorpyrifos. Individuals were exposed through spiked food for a period of 20 d (low dose 0.12 mg/kg/d, high dose 1.57 mg/kg/d). After exposure, various biomarkers of exposure and effect were evaluated, including the activities of glutathione S-transferase and enzymes involved in the glutathione redox cycle, glutathione concentrations, activities of esterases, liver and testes histopathologies, as well as locomotory and predatory behavior. The results indicate that sublethal, subchronic exposure to chlorpyrifos can affect P. bocagei in a dose-dependent manner. Adverse effects occurred at both the subindividual and individual levels, including inhibition of carboxylesterases and cholinesterases (ChEs), liver histopathological changes, and altered predatory behaviors. Animals exposed to chlorpyrifos took more time to capture and subdue prey items. The results suggest a link between effects at subindividual levels of organization with those observed at the whole individual level after exposure to environmentally realistic dosages of chlorpyrifos.

Non-avoidance behaviour in enchytraeids to boric acid is related to the GABAergic mechanism

Soil invertebrates, e.g. enchytraeids, are known to be able to avoid unfavourable conditions, which gives them an important ecological advantage. These organisms possess chemoreceptors that can detect stressors, which in turn activate responses such as avoidance behaviour. We studied the avoidance behaviour in response to boric acid (BA) using enchytraeids. Results showed not only no avoidance, but that increasing concentrations seemed to have an “attraction” effect. To study the underlying mechanism, a selection of genes targeting for neurotransmission pathways (acetylcholinesterase (AChE) and gamma-aminobutyric acid receptor (GABAr)) were quantified via quantitative real-time polymerase chain reaction (qPCR). Evidences were that BA is neurotoxic via the GABAergic system mechanism where it acts as a GABA-associated protein receptor (GABAAR) antagonist possibly causing anaesthetic effects. This is the first time that (non)avoidance behaviour in invertebrates was studied in relation with the GABAergic system. We strongly recommend the combination of such gene and/or functional assay studies with the avoidance behaviour test as it can bring many advantages and important interpretation lines for ecotoxicity with minor effort.

Development of an embryotoxicity test for Enchytraeus crypticus – The effect of Cd

The existing standard enchytraeid reproduction test (ERT) concerns the assessment of effects on survival and reproduction. In the present study we optimized and propose an embryotoxicity test using Enchytraeus crypticus. Cadmium (Cd) was used as a test substance. Endpoints evaluated were embryo development, number of embryonic structures, Calcium (Ca) channels quantification and hatching success with macroscopic monitoring, histological and immunohistochemistry analysis. Results showed that Cd is embryotoxic for this species, causing a decrease in the hatching success (EC50=3.1mg/kg), a delay or disruption in formation of embryonic structures depending on concentrations (<5mg Cd/kg or ⩾16mg Cd/kg). Results from immunohistochemistry suggest a competitive binding between Cd and Ca for Ca channels, resulting in changes in Ca homeostasis. The use of the E. crypticus embryotoxicity test with the combination of histological and immunohistological tools provided a good option towards mechanistic information enhancing the importance of these tests to evaluate the hazard of chemicals and possible use in risk assessment.

Multigenerational effects of copper nanomaterials (CuONMs) are different of those of CuCl 2 : exposure in the soil invertebrate Enchytraeus crypticus

Nanomaterials (NMs) are recommended to be tested in longer term exposures. Multigenerational (MG) studies are scarce and particularly important because effects can be transferred to the next generation. The current risk assessment framework does not include MG effects and this is a caveat for persistent materials. Here, the effects of copper NMs (CuONMs) and copper salt (CuCl2) were assessed in a MG exposure (4 generations in spiked soil + 2 generations in clean soil, F1 to F7 generations in total), with the standard soil model Enchytraeus crypticus, using relevant reproduction test effect concentrations (EC10, EC50), monitoring survival and reproduction. This represented ca. 1 year continuous exposure tests. MG effects varied with effect concentration and test materials: CuONMs caused increased toxicity for EC10 exposed organisms (EC50 did not change), and transfer to clean media reset effects, whereas CuCl2 reduced toxicity for EC10 and EC50, but the transfer to clean media “revived” the initial effects, i.e. close to EC50 levels in F7. Clearly CuONMs and CuCl2 cause different mechanisms of toxicity or response in the long term, not predictable based on short term or one generation studies. The present contributes for the improvement of risk assessment, adding important information for the long term exposure and effects.

Effects of europium polyoxometalate encapsulated in silica nanoparticles (nanocarriers) in soil invertebrates

Polyoxometalates (POMs) are metal oxo clusters that have been investigated for several applications in material sciences, catalysis, and biomedicine; these gained increasing interest in the field of nanotechnology as nanocarriers for drug delivery. Associated to the increasing applications, there is the need for information regarding the effects on the environment of these compounds, which is completely absent in the literature. In the present study, the effects of europium polyoxometalates encapsulated into silica nanoparticles (Eu-POM/SiO2 NPs) were assessed on the soil representative Enchytraeus crypticus. The individual materials were also assessed (Eu-POMs and SiO2 NPs). Toxicity was evaluated in various test media with increasing complexity: water, soil/water extracts, and soil. Toxicity was only observed for Eu-POM/SiO2 NPs and in the presence of soil components. Despite the fact that effects were observed for concentrations higher than current predicted environmental concentration (PEC), attention should be given to the growing use of these compounds. The present study shows the importance of assessing the effects in soil media, also compared to water. Moreover, results of “no effect” are critically needed and often unpublished. The present study can contribute to the improvement of the OECD guidelines for safety of manufactured nanomaterials on environmental toxicity in the soil compartment providing an improved test alternative.