Sublethal Endpoints Research Articles

Genotoxicity of mixtures of glyphosate with 2,4-dichlorophenoxyacetic acid chemical forms towards Cnesterodon decemmaculatus (Pisces, Poeciliidae).

Acute genotoxicity of commercial glyphosate (GLY) (Credit®)-, 2,4-D-acid (2,4-D) (Dedalo Elite)-, 2,4-D-amine (2,4-D DMA) (Weedar Full®)- and 2,4-D-ester (2,4-D BE) (Herbifen Super®)-based herbicide formulations alone and their combinations were analysed in Cnesterodon decemmaculatus. Mortality was evaluated as a lethal end-point and the single cell gel electrophoresis (SCGE) bioassay was used as a sublethal end-point. LC5096h values for Dedalo Elite was 0.46mg/L and Herbifen Super® was 2.67mg/L based on 2,4-D and 2,4-D BE, respectively. Results reveal a higher toxicity exerted on C. decemmaculatus after exposure to 2,4-D- rather than 2,4-D BE-based herbicide formulations. Overall, results demonstrated an enhancement in the genetic damage index committed to an enhancement of damaged erythrocytes of C. decemmaculatus when exposed to Credit®, Dedalo Elite, Weedar Full® and Herbifen Super® at 5% and 10% of LC5096h values alone as well as in their combinations. Overall, the combination of GLY plus 2,4-D or GLY plus 2,4-D DMA showed a synergistic pattern whereas the combination of GLY plus 2,4-D BE was antagonic. Furthermore, this research is pioneer in the assessment of lethality and genotoxicity induced by 2,4-D-, 2,4-D DMA- and 2,4-D BE-based formulations when combined with GLY-based formulated herbicides in fish after they are acutely exposed.

Foaming at the mouth: Ingestion of floral foam microplastics by aquatic animals

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. Regular foam and biofoam microplastics showed similar FTIR spectra. Both types of microplastics were consumed by all six invertebrate species tested: the freshwater gastropod Physa acuta, the marine gastropod Bembicium nanum, the marine bivalve Mytilus galloprovincialis, adults and neonates of the freshwater crustacean Daphnia magna, the marine amphipod Allorchestes compressa, and nauplii of the marine crustacean Artemia sp. For all species, the occurrence of ingestion was similar for regular foam and biofoam microplastics. Biofoam microplastics leached more than twice as much phenolic compounds than regular foam microplastics. The leachates from regular foam and biofoam microplastics showed the same acute toxicity to Artemia nauplii (24-h LC50 = 27.4 mg mL−1 and 22.8 mg mL−1, respectively) and D. magna (48-h LC50 = 17.8 mg mL−1 and 15.3 mg mL−1, respectively). However, biofoam microplastic leachate was twice as toxic to embryos of the zebrafish, Danio rerio, compared with leachate from regular foam microplastic (96-h LC50 = 43.8 mg mL−1 vs 27.1 mg mL−1). Using M. galloprovincialis, we show that regular foam microplastic leachate and the physical presence of the microplastics exerted separate and cumulative effects on catalase (CAT) activity, glutathione-s-transferase (GST) activity and lipid peroxidation. Microplastic ingestion did not affect the activity of acetylcholinesterase (AChE). Taken together, these results show 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.

An imazethapyr-based herbicide formulation induces genotoxic, biochemical, and individual organizational effects in Leptodactylus latinasus tadpoles (Anura: Leptodactylidae).

Genotoxic, biochemical, and individual organizational effects on Leptodactylus latinasus tadpoles were evaluated after exposure to an imazethapyr (IMZT)-based commercial herbicide formulation, Pivot® H (10.59% IMZT). A determination of the value of the lethal concentration (LC50) was determined as a toxicological endpoint. Alterations in animal behavior and morphological abnormalities as well as cholinesterase (ChE), catalase (CAT), and glutathione S-transferase (GST) activities were employed as individual sublethal endpoints. Micronuclei frequencies (MNs), binucleated cells (BNs), blebbed nuclei (BLs), lobed nuclei (LBs), notched nuclei (NTs), erythroplastids (EPs), and evaluation of DNA strand breaks were employed as genotoxic endpoints. All biomarkers were evaluated after 48 and 96 h of exposure to concentrations of IMZT within 0.07-4.89 mg/L. LC5096h values of 1.01 and 0.29 mg/L IMZT were obtained for Gosner stages 25 and 36, respectively. Irregular swimming, diamond body shape, and decreased frequency of keratodonts were detected at both sampling times. Results showed that IMZT increased GST activity and MN frequency at 48 and 96 h of exposure. Other nuclear abnormalities were also observed in the circulating erythrocytes of tadpoles, i.e., NT and BL values after 48 h, and LN, BL, and EP values after 96 h. Finally, results showed that IMZT within 0.07-0.22 mg/L increased the genetic damage index in tadpoles exposed for both exposure times (48 and 96 h). This study is the first to report the sublethal biochemical effects of IMZT in anurans and is also the first report using L. latinasus tadpoles as a bioindicator for ecotoxicological studies.

Evaluation of interactive effects of UV light and nano encapsulation on the toxicity of azoxystrobin on zebrafish

The use of nanotechnology to enhance pesticide formulations holds the promise of reduced pesticide use, reduced mobility in soils, and overall improvements in agricultural practices while simultaneously maintaining yields. However, the toxicity of nano-enabled pesticides, including azoxystrobin (Az), has not been well studied compared with their conventional form. This study investigates both lethal and sub-lethal endpoints in zebrafish embryos up to 120 h post-fertilization (hpf) under either laboratory light or simulated UV light. The median lethal concentration (LC50) value of nano-enabled Az (nAz) was significantly lower than the conventional form (Az). Interestingly, artificial UV light significantly increased toxicity (decreased LC50) of both Az and nAz. Malformations were not observed but the remaining yolk sac volume was significantly increased in both types of Az at both light conditions. This decreased yolk consumption is in agreement with reduced oxygen consumption and heart rate. Catalase enzyme activity was only reduced to UV light while superoxide dismutase activity was significantly reduced by co-exposure of UV light, and either type of Az at a nominal concentration of 100 μg L−1. The co-exposure of Az at 100 μg L−1 and UV light significantly upregulated sod1, sod2, and gpx1b expression and both types of Az significantly reduced gpx1a expression. Lipid peroxidation was significantly increased in nAz and Az at 100 μg L−1 under laboratory light, while UV light induced even higher level of lipid peroxidation. The results will provide important information on the toxicity of nAz under ecologically realistic conditions.

Effects of pH and nitrites on the toxicity of a cypermetrin-based pesticide to shrimps.

The Pantanal (Brazil) is a wetland region characterized by seasonal flooding. Hydrological cycles influence the water physicochemical parameters, causing seasonal variations in pH and nitrites. Thus, the objective of this study was to evaluate the effects of varying pH and nitrite concentrations on the toxicity of the cypermethrin-based pesticide Barrage®, considering both lethal (mortality) and sublethal endpoints (growth and development). Larvae of the endemic shrimp Macrobrachium pantanalense and of the estuarine Amazonian congener Macrobrachium amazonicum were exposed to cypermethrin (through Barrage®) under several pH levels (6.5, 7.5 and 8.5) or nitrite concentrations (0.1, 0.2 and 0.4 mg/L). The pH had direct effects on all the tested endpoints for both species. For M.pantanalense, the lethal effects of the cypermethrin formulation were more pronounced at low pH (96-h LC50 = 0.004 μg/L at pH 6.5, and 0.146 μg/L at pH 8.5). For M.amazonicum, an opposite response was observed, with increased toxicity of the formulation at high pH (96-h LC50 = 0.110 μg/L at pH 6.5 and 0.044 μg/L at pH 8.5). Variations in pH also seemed to modify the sublethal effects of the formulation on larval growth and development of M.pantanalense. Nitrite concentrations affected larval growth of both species, modifying also the effects of the cypermethrin formulation on the larval development of M.amazonicum. This work shows the importance of considering abiotic factors for risk assessment either due to possible direct effects on the physiology of organisms and/or due to interactions with other stressors, particularly in fragile biomes such as Pantanal.

Effects of iron oxide nanoparticles (Fe3O4) on life history and metabolism of the Neotropical cladoceran Ceriodaphnia silvestrii

Nanoparticles (NPs) production is increasing worldwide. These products are likely to end up in aquatic environments. However, few studies evaluated the chronic toxicity of iron-based NPs (Fe-NPs) to cladocerans and their potential ecotoxicological hazards. In this study we aimed to investigate the effects of iron oxide nanoparticles (Fe3O4-NPs) to Ceriodaphnia silvestrii Daday, 1902, assessing acute (48 h) and chronic toxicity (up to 14 d). Besides traditional endpoints (immobility and lethality), we also evaluated physiological responses (respiration rates) in a 48 h-exposure. No immobility was observed (EC50 > 100 mg L−1) after 48 h, whereas respiration rates at the highest concentration were 400% of that in control, indicating that this endpoint was more sensitive during acute toxicity. In chronic assays, Fe3O4-NPs affected body length (8.24% growth inhibition in 7 d-exposure) and number of eggs (7-d IC10: 3.53, IC20: 6.69 mg Fe L−1) and neonates (7-d IC10: 1.25, IC20: 3.75 mg Fe L−1). Based on species sensitivity distribution (SSD), C. silvestrii was a sensitive organism, suggesting Fe-NPs as a possible threat for this species. Our results also indicate that the NPs caused a physical barrier, impairing food absorption, since we observed NPs agglomerations into cladocerans’ gut. We demonstrate that Fe3O4-NPs affects C. silvestrii metabolism and reproduction and our results support the use of sublethal endpoints to assess environmental safety. The release of these NPs into freshwater environments should be carefully evaluated, since disturbances on cladoceran population dynamics could cause strong impacts on the entire food web structure and ultimately on ecosystem functioning.

Multi-Laboratory Hazard Assessment of Contaminated Microplastic Particles by Means of Enhanced Fish Embryo Test With the Zebrafish (Danio rerio)

As wide-spread pollutants in the marine environment, microplastics (MPs) have raised public concern about potential toxic effects in aquatic organisms, and, among others, MPs were suspected to act as a vector for organic pollutants to biota. The purpose of the present study was to investigate effects by three model pollutants, oxybenzone (BP3), benzo[a]pyrene (BaP) and perfluorooctane sulfonate (PFOS) adsorbed to polyethylene MPs on the basis of a standard assay, the acute fish embryo toxicity test (FET; OECD TG 236) with zebrafish (Danio rerio) supplemented by additional endpoints such as induction of ethoxyresorufin-O-deethylase (EROD) activity, modification of cyp1a gene transcription and changes in larval swimming behavior. FET assays were performed in three laboratories using slightly different husbandry and exposure conditions, which, however, were all fully compatible with the limits defined by OECD TG 236. This allowed for testing of potential changes in the FET assay due to protocol variations. The standard endpoints of the FET (acute embryotoxicity) did not reveal any acute toxicity for both virgin MPs and MPs spiked with BP3, BaP and PFOS. With respect to sublethal endpoints, EROD activity was increased after exposure to MPs spiked with BP3 (3 h pulse) and MPs spiked with BaP (96 h continuous exposure). Cyp1a transcription was increased upon exposure to MPs spiked with BP3 or BaP. For the selected combination of MPs particles and contaminants, the basic FET proved not sensitive enough to reveal effects of (virgin and spiked) MPs. However, given that the FET can easily be supplemented by a broad variety of more subtle and sensitive endpoints, an enhanced FET protocol may provide a relevant approach with developmental stages of a vertebrate animal model, which is not protected by current EU animal welfare legislation (Directive EU 2010/63).

Lethal and sublethal toxicity assessment of Bacillus thuringiensis var. israelensis and Beauveria bassiana based bioinsecticides to the aquatic insect Chironomus riparius

Despite being considered environmentally safe, a deeper environmental risk assessment is needed for microbial insecticides; special attention should be devoted to their sublethal toxicity to non-target species.This study evaluated effects of VectoBac® 12AS - VB (based on the bacterium Bacillus thurigiensis var. israelensis) and Naturalis®-L - NL (based on the fungus Beauveria bassiana) on the aquatic insect Chironomus riparius life-history and biochemical responses.Acute tests estimated a 48 h-LC50 (median lethal concentration) of 1.85 μg/L (VB) and 34.7 mg/L (NL). Under sublethal exposure, VB decreased adults' emergence (LOEC – lowest observed effect concentration of 80 ng/L) while NL impaired larval growth (LOEC of 0.32 mg/L) and delayed emergence (LOEC of 2 mg/L for males and 0.8 mg/L for females). Despite not being monotonic, phenoloxidase activity increased (LOEC of 20 ng/L (VB) and 2 mg/L (NL)), suggesting activation of the immune system. There were no indications of oxidative damage nor neurotoxicity. Catalase activity was stimulated with all VB treatments, possibly associated with detoxification of immune response products. Under NL exposure, glutathione-S-transferase activity increased but did not show a dose-dependent response and, total glutathione decreased in the highest concentration. Exposure to both formulations caused the increase in protein content, while carbohydrate and lipids were not altered.This study revealed the susceptibility of C. riparius to VB and NL at concentrations below the ones recommended for field application, with potential population-level effects. These results add important information for the risk assessment of these microbial insecticides in aquatic ecosystems, considering relevant sublethal endpoints and raising concern about the adverse effects on non-target aquatic organisms.

Acute and chronic effects of perfluoroalkyl substance mixtures on larval American bullfrogs (Rana catesbeiana)

Discovery of elevated concentrations of perfluoroalkyl substances (PFAS) in ground and surface waters globally has heightened concern over their potential adverse health effects. The effects of PFAS are known largely from acute toxicity studies of single PFAS compounds in model organisms, while little is understood concerning effects of mixtures on wildlife. To address this gap, we examined the acute and chronic effects of two of the most common PFAS (perfluorooctanesulfonic acid [PFOS] and perfluorooctanoic acid [PFOA]) and their mixtures on survival, growth, and development of American bullfrog (Rana catesbeiana) tadpoles. In 96 h acute toxicity tests, PFOS was 10X more toxic than PFOA and effects of the two chemicals in combination appeared additive. The effects of PFOS, PFOA, and their interaction varied by the sublethal endpoint under consideration in a 72 d exposure. Effects of PFAS on tadpole mass and developmental stage were largely driven by PFOS and there was no evidence of interactions suggesting deviations from additivity. However, for snout-vent length, reductions in length in mixture treatments were greater than expected based on the effects of the two chemicals independently (i.e. non-additivity). Further, effects on snout-vent length in single chemical exposures were only observed with PFOA. Our results highlight the importance of assessing combined effects of PFAS co-occurring in the environment and suggest caution in extrapolating the effects of acute toxicity studies to more environmentally relevant exposures. Future studies examining effects of environmentally relevant mixtures on wildlife will be essential for effective environmental risk assessment and management.

Environmental levels of neonicotinoids reduce prey consumption, mobility and emergence of the damselfly Ischnura elegans

Abstract Freshwaters are among the most endangered ecosystems in the world as a result of anthropogenic interference such as pollution. Pollution in the form of neonicotinoids has been intensively studied, but data thus far is often conflicted by contrasting responses between laboratory and field experiments. In addition, toxicity data are scarce and contradictory for insects such as Odonates (dragonflies and damselflies) and a potential risk to them may therefore be overlooked. We investigate the potential risk of neonicotinoids to Odonates by exposing nymphs of the blue‐tailed damselfly Ischnura elegans to environmentally relevant concentrations of the neonicotinoid thiacloprid. We consider I. elegans as an indicator species for other Odonates as it is an abundant, widespread and eurytopic species. We analyse the effects of thiacloprid on multiple endpoints (survival, consumption, growth, molting, mobility and emergence), using cage‐experiments as well as controlled field observations in naturally colonized experimental ditches. In addition, we assess sensitivity by either feeding the damselfly nymphs with lab‐cultured prey or by letting them feed freely on natural aquatic invertebrates. All sublethal endpoints of I. elegans are affected to some degree, and strongly depend on the food offered; free‐feeding nymphs are more sensitive than culture‐fed nymphs. Environmental relevant concentrations of thiacloprid strongly reduce the emergence of I. elegans and this effect is more substantial in the natural populations compared to the caged damselflies. This is likely explained by exclusion of additional biotic pressures such as predation in the caged experiment. Policy implications. Literature reports that one out of seven Odonates is threatened and 24% of the species have declining populations. Our observations show that current risks of neonicotinoids to Odonates are underestimated in laboratory experiments as the toxicity is governed by multiple biotic factors such as food quantity/quality and predation. Given the widespread abundance of blue‐tailed damselfly Ischnura elegans, the observed sensitivity to neonicotinoids and current population trends of this species, these results indicate neonicotinoids play a central role in the Odonate decline in general.

Exposure to single and binary mixtures of fullerenes and triclosan: Reproductive and behavioral effects in the freshwater snail Radix balthica

Emerging pollutants occur in complex mixtures in rivers and have the potential to interact with freshwater organisms. The chronic effects of nominal exposure to 3 μg/L of fullerenes (C60) and 1 μg/L of triclosan (TCS) alone and in a binary mixture, were evaluated using the freshwater snail Radix balthica. Pollutants accumulation, reproductive output and feeding behavior were selected as sublethal endpoints. After 21 days of exposure, we did not observe interactive effects between TCS and C60 on the studied endpoints, except for the accumulation of C60 in R. balthica in TCS + C60 treatment, which was lower than when the fullerenes were alone. Neither TCS nor C60 caused significant effects on reproduction, expressed as number of eggs per individual, but an increase in the clutch size was observed in treatments with TCS at the third week of exposure, independently of the presence of C60 (16.15 ± 1.67 and 18.9 ± 4.01 eggs/egg mass in TCS and TCS + C60 treatments, respectively, vs. 13.17 ± 4.01 in control). The presence of C60 significantly enhanced the grazing activity of R. balthica during the first seven days (4.95 ± 1.35 and 3.91 ± 0.59% of the area grazed per individual in C60 and TCS + C60 treatments, respectively, vs 2.6 ± 0.39% in control). The accumulation of TCS was quite similar in treatments where this pollutant was present (BAF ≈ 1007 L/kg d.w.); however, the accumulation of C60 was higher when the nanoparticles were alone (BAF = 254.88 L/kg d.w.) than when it was in the binary mixture (BAF = 7.79 L/kg d.w). Overall, although TCS has been listed as an endocrine disrupter compound, no significant effects on reproduction were observed in the assayed conditions. Regarding C60, the limited effects on feeding activity and the low BAF obtained in this experiment indicate that fullerenes do not have ecological consequences of relevance at the studied environmental concentrations in freshwater snails.

How glyphosate and its associated acidity affect early development in zebrafish (Danio rerio).

BackgroundGlyphosate is among the most extensively used pesticides worldwide. Following the ongoing highly controversial debate on this compound, its potential impact on non-target organisms is a fundamental scientific issue. In its pure compound form, glyphosate is known for its acidic properties.MethodsWe exposed zebrafish (Danio rerio) embryos to concentrations between 10 μM and 10 mM glyphosate in an unbuffered aqueous medium, as well as at pH 7, for 96 hours post fertilization (hpf). Furthermore, we investigated the effects of aqueous media in the range of pH 3 to 8, in comparison with 1 mM glyphosate treatment at the respective pH levels. Additionally, we exposed zebrafish to 7-deoxy-sedoheptulose (7dSh), another substance that interferes with the shikimate pathway by a mechanism analogous to that of glyphosate, at a concentration of one mM. The observed endpoints included mortality, the hatching rate, developmental delays at 24 hpf, the heart rate at 48 hpf and the malformation rate at 96 hpf. LC10/50, EC10 and, if reasonable, EC50 values were determined for unbuffered glyphosate.ResultsThe results revealed high mortalities in all treatments associated with low pH, including high concentrations of unbuffered glyphosate (>500 μM), low pH controls and glyphosate treatments with pH < 3.4. Sublethal endpoints like developmental delays and malformations occurred mainly at higher concentrations of unbuffered glyphosate. In contrast, effects on the hatching rate became particularly prominent in treatments at pH 7, showing that glyphosate significantly accelerates hatching compared with the control and 7dSh, even at the lowest tested concentration. Glyphosate also affected the heart rate, resulting in alterations both at pH 7 and, even more pronounced, in the unbuffered system. In higher concentrations, glyphosate tended to accelerate the heart rate in zebrafish embryos, again, when not masked by the decelerating influence of its low pH. At pH > 4, no mortality occurred, neither in the control nor in glyphosate treatments. At 1 mM, 7dSh did not induce any mortality, developmental delays or malformations; only slightly accelerated hatching and a decelerated heart rate were observed. Our results demonstrate that lethal impacts in zebrafish embryos can be attributed mainly to low pH, but we could also show a pH-independent effect of glyphosate on the development of zebrafish embryos on a sublethal level.

Host-parasite interaction as a toxicity test endpoint using asymmetrical exposures

Interspecific relationships frequently determine the effect a pollutant can have on an organism, and this is especially true in closely interacting species such as hosts and parasites. The high spatial and temporal variability of contaminant concentrations combined with the movement of aquatic biota can further influence the consequences that are associated with contamination. We used a full factorial design for the exposed and unexposed partners of the relationship between the parasitic larvae (glochidia) of the European freshwater mussel (Anodonta anatina) and its host fish (Squalius cephalus) to identify the sources of variation in the sublethal endpoints of species interaction (the intensity of parasite attachment, the spatial position of glochidia on the host body, and encapsulation success). We used the water-borne human pharmaceutical compounds methamphetamine (a central nervous system stimulant) and tramadol (an opioid) at environmentally relevant concentrations (˜ 6.7 and 3.8 nmol L−1 of methamphetamine and tramadol, respectively) as a proxy for contaminant exposure because these compounds are emerging aquatic stressors that are known for high spatial and temporal variability in their detected concentration levels. The relationship between the bivalve and the fish species was influenced by the preceding contact with both methamphetamine and tramadol, but this effect was highly asymmetric. Our experimental design enabled us to identify the specific changes in the relationship outcome that are elicited by the exposure of individual partners, such as the significant increase in glochidia infection success rate from 59.6 ± 3.9% to 78.7 ± 2.8% (means ± s.e.) that was associated with host exposure to methamphetamine. Additionally, the significant interaction effect of the exposure was demonstrated by the lowered proportion of glochidia attached to gills after the coexposure of both partners to tramadol. The impact of pharmaceuticals on wild aquatic host–parasite relationships provides an example of the risks that are associated with the unintentional discharge of biologically active compounds into freshwater habitats. Given the increasing evidence showing the ecological impact of waste pharmaceuticals, the use of multitrophic interaction endpoints after joint and unilateral exposures provides an important step towards the realistic risk assessment of these compounds.

A novel screening method for transition metal-based anticancer compounds using zebrafish embryo-larval assay and inductively coupled plasma-mass spectrometry analysis.

As novel metallodrugs continue to emerge, they are evaluated using models, including zebrafish, that offer unique sublethal endpoints. Testing metal-based anticancer compounds with high-throughput zebrafish toxicological assays requires analytical methods with the sensitivity to detect these sublethal tissue doses in very small sample masses (e.g., egg mass 100μg). A robust bioanalytical model, zebrafish embryos coupled with inductively coupled plasma-mass spectrometry (ICPMS) for measurement of delivered dose, creates a very effective means for screening metal-based chemotherapeutic agents. In this study, we used ICPMS quantitation with the zebrafish embryo assays to detect metal equivalents at multiple response endpoints for two compounds, the chemotherapeutic agent cisplatin and ruthenium (Ru)-based prospective metallodrug, PMC79. We hypothesized that cisplatin and PMC79 have different mechanisms for inducing apoptosis and result in similar lesions but different potencies following water-borne exposure. An ICPMS method was developed to detect the metal in waterborne solution and tissue (detection limit: 5 parts per trillion for Ru or platinum [Pt]). The Ru-based compound was more potent (LC50 : 7.8μm) than cisplatin (LC50 : 158μm) and induced disparate lesions. Lethality from cisplatin exposure exhibited a threshold (values >15mg/L) while no threshold was observed for delayed hatching (lowest observed adverse effect level 3.75mg/L cisplatin; 8.7 Pt (ng)/organism). The Ru organometallic did not have a threshold for lethality. Cisplatin-induced delayed hatching was investigated further by larval-Pt distribution and preferentially distributed to the chorion. We propose that zebrafish embryo-larval assays coupled with ICPMS serve as a powerful platform to evaluate relative potency and toxic effects of metallodrug candidates.

Predicting the toxic effects of Cu and Cd on Chlamydomonas reinhardtii with a DEBtox model

Predicting the sublethal effects of pollutants to aquatic organism is essential in realistic chemical risk assessment. However, only a few dynamics models for sublethal endpoints are available. Here, we investigated the toxic effects of the essential metal Cu and the nonessential metal Cd on Chlamydomonas reinhardtiiunder both single and combined exposure, compared the effectiveness of different effect endpoints as toxic effect factors, and developed a Dynamic Energy Budget toxicology (DEBtox) model to predict the sublethal effects of Cu and Cd on C. reinhardtii. The results showed that the chlorophyll fluorescence parameter is a better toxic effect indicator than others for short-term exposure (<24 h), while algal cell growth is preferred for long-term exposure (2–6 days). The developed DEBtox model could successfully predict single metal toxicity to C. reinhardtii, while the combined metal DEBtox model slightly overestimates the joint toxicity of Cu-Cd due to the antagonistic effect of Cu-Cd on C. reinhardtii. This study is helpful to understanding and better predictions of metal sublethal toxic effects on aquatic organisms.

Viability of the marine amphipod Parhyale hawaiensis growth rate as an endpoint for chronic ecotoxicological tests

There is a lack of suitable marine species for ecotoxicological tests. Parhyale hawaiensis is a marine amphipod with circumtropical distribution. Recently, acute ecotoxicological procedures were standardized for these model organisms. However, its potential for chronic toxicity assessment still unclear. Sediment quality assessments using sublethal responses of benthic organisms, such as growth and reproduction, have been used to successfully evaluate moderately contaminated areas. The aim of this work was to evaluate the viability of growth rate as an sublethal endpoint for chronic toxicity tests.

Varying the exposure period and duration of neuroactive pharmaceuticals and their metabolites modulates effects on the visual motor response in zebrafish (Danio rerio) larvae

Pharmaceuticals and personal care products are emerging contaminants that are increasingly detected in surface waters around the world. Despite the rise in environmental detections, measured concentrations are still typically low, raising the importance of environmental risk assessments that focus on ecologically relevant sublethal endpoints, such as altered behavior. Neuroactive pharmaceuticals, like mental health medications, pain killers, etc., may be particularly potent in this regard as they are specifically designed to cause behavioral changes without causing physiologic impairment in mammalian systems. We screened 15 different popular neuroactive pharmaceuticals, ranging from antidepressants (including 3 major antidepressant metabolites), anxiety medications, and pain killers, under three different exposure scenarios (repeated, late acute and early transient exposure) to look for behavioral effects in larval zebrafish using the visual motor response (VMR). Drugs were screened at 0, 1, 10, and 100 μg/L in the repeated exposure scenario, and at 0 and 100 μg/L in the late acute and early transient exposure scenarios. Eight of the 15 compounds tested, specifically the antidepressants amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, sertraline, nor-sertraline, venlafaxine, and the antipsychotic drug haloperidol decreased swimming activity by 25% to 40% under repeated exposure conditions. Five of the compounds (amitriptyline, fluoxetine, nor-fluoxetine, paroxetine, and sertraline) also significantly decreased activity by 17% to 31% in the late acute exposure paradigm. Three compounds (fluoxetine, paroxetine and venlafaxine) significantly altered swimming activity with early transient exposure, however creating a hyperactive response and increasing activity from 24% to 28%, while haloperidol significantly decreased activity by 31%. This paper is, to our knowledge, the first to screen so many neuroactive pharmaceuticals, including major metabolites, in parallel under multiple exposure conditions. We show that antidepressants most consistently alter VMR swimming activity. Additionally, we show that major antidepressant metabolites can potentially alter behavior as much as their parent compounds. Furthermore, we show that the magnitude and direction of behavioral effect is dependent on the exposure duration and period, indicating that a more diverse experimental approach might be needed to more accurately assess the risk these compounds pose to the environment.

Sub-lethal Responses of the Polychaete Armandia agilis in Whole-sediment Toxicity Testing.

The present study assessed biochemical responses as sublethal endpoints in the polychaete Armandia agilis exposed to contaminated sediments to in order to assess its potential use as a test organism. Sediment samples from several locations at a dredging site were obtained and used in whole-sediment exposures. Samples were tested with A. agilis to determine the 10-day toxicity of the 100% sample and the enzymatic activity of catalase (CAT), glutathione-S-transferase (GST) and acetylcholinesterase (AChE) biochemical measurements made in whole-body homogenates of a subset of the surviving organisms. Biochemical responses reported in A. agilis were not statistically different from the reference site sediment, however, the integrated analysis demonstrated that contaminants bound to sediment samples influenced the sublethal effects.

Toxicity study on Clinacanthus nutans leaf hexane fraction using Danio rerio embryos.

Clinacanthus nutans, an herbal shrub belonging to the Acanthaceae family, is traditionally used as a functional food to treat various ailments in Malaysia and Indonesia. Although the polar fraction of this plant shows non-toxic effect, the toxicity of the non-polar extract is not reported so far. The present study aimed to assess the toxic effect and determine the lethal concentration of this non-polar fraction using zebrafish embryos. The n-hexane fraction was partitioned from the crude extract of C. nutans obtained using 80% methanolic solution. After spawning of the adult male and female zebrafish, the eggs were collected, transferred into a 96-well plate and incubated with the n-hexane fraction at concentrations of 15.63 μg/ml, 31.25 μg/ml, 62.5 μg/ml, 125 μg/ml, 250 μg/ml and 500 μg/ml in 2% DMSO. The survival and sublethal endpoint were assessed, the mortality and hatchability rates were calculated based on microscopic observation, while the heartbeat rate was measured using DanioScope software. The median lethal concentration (LC50) of the C. nutans n-hexane fraction, which was determined using probit analysis, was calculated to be 75.49 μg/mL, which is harmful. Moreover, gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of palmitic acid, phytol, hexadecanoic acid, 1-monopalmitin, stigmast-5-ene, pentadecanoic acid, heptadecanoic acid, 1-linolenoylglycerol and stigmasterol in the n-hexane fraction.

Identifying sublethal endpoints for evaluating neurotoxic compounds utilizing the fish embryo toxicity test

Fish embryos are increasingly being utilized in aquatic toxicity testing, as evidenced by the Organisation for Economic Co-operation and Development's approval of the fish embryo acute toxicity (FET) test. However, the FET test only allows for the estimation of acute toxicity, whereas other test methods such as the larval growth and survival (LGS) test allow for the estimation of both acute and chronic toxicity. Additionally, it has been demonstrated that the FET test is less sensitive than other test methods for some neurotoxic compounds. To address these limitations, efforts to identify sublethal endpoints that increase FET test sensitivity and allow for the prediction of sublethal adverse effects have begun. As such, the objectives of the current study were 1) to compare estimated LC50 values from the FET and LGS test for three known neurotoxicants: fluoride (F), nickel (Ni), and cadmium (Cd) and 2) to evaluate the responsiveness of potential sublethal endpoints for the FET test related to growth (i.e., wet weight and snout-vent length), neurological development (i.e., spontaneous contraction frequency and eye size), and cardiovascular function (i.e., heart rate and pericardial area). The calculated LC50 values from the F and Cd FET test were significantly higher than those from the LGS test, demonstrating that the FET test is less sensitive than the LGS test for neurotoxic compounds. Only Cd exposure resulted in alterations in any of the sublethal endpoints investigated. Embryos/eleutheroembryos exposed to Cd displayed alterations in length, eye size, and pericardial area at concentrations five-fold less than the estimated LC50 value, suggesting that for Cd the inclusion of these sublethal endpoints would improve the sensitivity of the FET test. Overall, these results provide evidence that for some neurotoxicants, the inclusion of sublehtal endpoints may improve the utility of the FET test; however, further research utilizing a broader range of neurotoxicants with differing mechanisms of action, is needed to fully establish such endpoints in the context of routine FET test.