Elevated temperature exacerbates pharmaceutical-induced oxidative stress in zebrafish (Danio rerio) larvae.

There is growing evidence that rising global temperatures resulting from climate change may exacerbate the toxic effect of pollutants and heterotherms, including fish, in which homestatic mechanisms are directly influenced by environmental temperature will be most affected. Pharmaceuticals discharged into the environment are potentially harmful to wildlife as many of their drug targets are conserved across divergent phyla. Oxidative stress (OS) is a major mechanism by which many pharmaceutical contaminants can induce toxicity but this has received little consideration in the context of effects in wildlife. Further, these mechanisms are relatively poorly understood, particularly regarding multiple stressor interactions. We used transgenic TG(EpRE:mCherry) zebrafish, developed in our laboratory for detecting OS, as our experimental model. We show that the oxidative effects of high concentrations of pharmaceuticals from three different therapeutic classes (paracetamol, diclofenac and doxorubicin) are increased at temperatures elevated by 2-5 °C above those for zebrafish standard husbandry and relevant to their current natural environment (and predicted under the IPCC 2023 scenarios for intermediate to very high greenhouse gas emissions). These OS responses were primarily seen in the pronephros, liver, and gastrointestinal tract. The increase in OS at the increased water temperature may have resulted from the elevated temperature acting as a direct additive physiological stressor to the OS imposed by the drugs and/or via the temperature increasing the chemicals oxidative effect. For paracetamol, it appeared that the elevated responses at the higher temperature of 33 °C were in part due to an increase in uptake of the drug. Our data illustrate that risk assessments for chemicals inducing OS in fish (and likely other heterotherms) should consider the influence of temperature to ensure environmental protection in future environments.

Predicting population-level impacts of projected climate heating on a temperate freshwater fish.

Climate heating has the potential to drive changes in ecosystems at multiple levels of biological organization. Temperature directly affects the inherent physiology of plants and animals, resulting in changes in rates of photosynthesis and respiration, and trophic interactions. Predicting temperature-dependent changes in physiological and trophic processes, however, is challenging because environmental conditions and ecosystem structure vary across biogeographical regions of the globe. To realistically predict the effects of projected climate heating on wildlife populations, mechanistic tools are required to incorporate the inherent physiological effects of temperature changes, as well as the associated effects on food availability within and across comparable ecosystems. Here we applied an agent-based bioenergetics model to explore the combined effects of projected temperature increases for 2100 (1.4, 2.7, and 4.4°C), and associated changes in prey availability, on three-spined stickleback (Gasterosteus aculeatus) populations representing latitudes 50, 55, and 60°N. Our results showed a decline in population density after a simulated 1.4°C temperature increase at 50°N. In all other modeled scenarios there was an increase (inflation) in population density and biomass (per unit area) with climate heating, and this inflation increased with increasing latitude. We conclude that agent-based bioenergetics models are valuable tools in discerning the impacts of climate change on wild fish populations, which play important roles in aquatic food webs.

In field use of water samples for genomic surveillance of infectious spleen and kidney necrosis virus (ISKNV) infecting tilapia fish in Lake Volta, Ghana.

Viral outbreaks are a constant threat to aquaculture, limiting production for better global food security. A lack of diagnostic testing and monitoring in resource-limited areas hinders the capacity to respond rapidly to disease outbreaks and to prevent viral pathogens becoming endemic in fisheries productive waters. Recent developments in diagnostic testing for emerging viruses, however, offers a solution for rapid in situ monitoring of viral outbreaks. Genomic epidemiology has furthermore proven highly effective in detecting viral mutations involved in pathogenesis and assisting in resolving chains of transmission. Here, we demonstrate the application of an in-field epidemiological tool kit to track viral outbreaks in aquaculture on farms with reduced access to diagnostic labs, and with non-destructive sampling. Inspired by the "lab in a suitcase" approach used for genomic surveillance of human viral pathogens and wastewater monitoring of COVID19, we evaluated the feasibility of real-time genome sequencing surveillance of the fish pathogen, Infectious spleen and kidney necrosis virus (ISKNV) in Lake Volta. Viral fractions from water samples collected from cages holding Nile tilapia (Oreochromis niloticus) with suspected ongoing ISKNV infections were concentrated and used as a template for whole genome sequencing, using a previously developed tiled PCR method for ISKNV. Mutations in ISKNV in samples collected from the water surrounding the cages matched those collected from infected caged fish, illustrating that water samples can be used for detecting predominant ISKNV variants in an ongoing outbreak. This approach allows for the detection of ISKNV and tracking of the dynamics of variant frequencies, and may thus assist in guiding control measures for the rapid isolation and quarantine of infected farms and facilities.

Exposure Effects of Environmentally Relevant Concentrations of the Tricyclic Antidepressant Amitriptyline in Early Life Stage Zebrafish.

Antidepressants are one of the most globally prescribed classes of pharmaceuticals, and drug target conservation across phyla means that nontarget organisms may be at risk from the effects of exposure. Here, we address the knowledge gap for the effects of chronic exposure (28 days) to the tricyclic antidepressant amitriptyline (AMI) on fish, including for concentrations with environmental relevance, using zebrafish (Danio rerio) as our experimental model. AMI was found to bioconcentrate in zebrafish, was readily transformed to its major active metabolite nortriptyline, and induced a pharmacological effect (downregulation of the gene encoding the serotonin transporter; slc6a4a) at environmentally relevant concentrations (0.03 µg/L and above). Exposures to AMI at higher concentrations accelerated the hatch rate and reduced locomotor activity, the latter of which was abolished after a 14 day period of depuration. The lack of any response on the features of physiology and behavior we measured at concentrations found in the environment would indicate that AMI poses a relatively low level of risk to fish populations. The pseudopersistence and likely presence of multiple drugs acting via the same mechanism of action, however, together with a global trend for increased prescription rates, mean that this risk may be underestimated using current ecotoxicological assessment paradigms.

Effectiveness of eDNA for monitoring riverine macroinvertebrates.

Environmental DNA (eDNA) is a technique increasingly used for monitoring organisms in the natural environment including riverine macroinvertebrates. However, the effectiveness of eDNA for monitoring riverine macroinvertebrates compared with the more traditional method of sampling the organisms directly and identifying them via morphological analysis, has not been well established. Furthermore, the ability of the various gene markers and PCR primer sets to detect the full range of riverine invertebrate taxa has not been quantified. Here we conducted a meta-analysis of the available literature, to assess the effectiveness of eDNA sampling for detecting riverine macroinvertebrates compared with sampling for the organisms directly and applying morphological analysis. We found, on average, eDNA sampling, irrespective of the gene marker used, detected fewer riverine invertebrates than morphological sampling. The most effective PCR primer set for identifying taxa was mlCOIintF/jgHCO2198, (mlCOIintF- forward primer, jgHCO2198, - reverse primer). Regardless of the gene marker or primer sets used, however, many taxa were not detected by eDNA metabarcoding that were detected by sampling directly for these invertebrates, including over 100 members of Arthropoda. eDNA sampling failed to detect any species belonging to Nematoda, Platyhelminthes, Cnidaria or Nematomorpha and these markers applied for eDNA sampling in terrestrial systems also do not detect members of Nematoda. In addition to these issues, uncertainties relating to false positives from upstream DNA sources, the stability of DNA from different species, differences in the propensity for DNA release into the environment for different organisms, and lack of available sequence information for numerous taxa illustrates the use of eDNA is not yet applicable as a robust stand-alone method for the monitoring of riverine invertebrates. As a primary consideration, further methodological developments are needed to ensure eDNA captures some of the key freshwater taxa, notably taxa belonging to the phyla Arthropoda, Nematoda, Platyhelminthes, Cnidaria and Nematomorpha.

Factors Influencing Disease Dynamics in Small-Scale Carp Polyculture in Bangladesh.

Small-scale carp polyculture plays a key role in food supply in Bangladesh. However, factors including water pollution, limited infrastructure, and inadequate disease management hinder its sustainability. This paper reports on a survey of 231 farmers across the six major carp producing regions in Bangladesh, analyzing factors including farmers' social aspects, farm characteristics, information on disease and approaches adopted to combat them, and biosecurity practices. Almost half (46.8%) of the farms surveyed experienced disease in carp species, with clear regional variations. Eighty-four percent of farms reported carp mortalities during disease outbreaks, with an average mortality level of 10.23 ± 11.81%. Clinical signs during outbreaks lasted between a week and a month, with a peak in disease outbreaks occurring in two seasonal periods between June and July and October and December. Disease incidence was related to a range of factors including the farmer's experience, ponds/farm type, stocked species, and biosecurity practice. A combination of disinfecting measures during pond preparation and measures during stocking, including discarding fingerling transport water away from the farm, fingerling disinfection, and checking the health of fingerlings before stocking, significantly reduced disease occurrence. Treatments involving antibiotics, ciprofloxacin, erythromycin, and azithromycin were reported as ineffective, raising concerns about their non-prudent use, inadequate dosing (perhaps without appropriate veterinary guidance), and the potential for driving antimicrobial resistance in the environment. The research unveils a concerning pattern of high disease incidence across small-scale carp farms in Bangladesh, and the significant potential for disease spread highlights the need for responsible disposal practices. The study emphasizes the need for improving training and awareness programs for addressing biosecurity and disease management challenges, ensuring sustainable aquaculture and community well-being.

Influence of host phylogeny and water physicochemistry on microbial assemblages of the fish skin microbiome.

The skin of fish contains a diverse microbiota that has symbiotic functions with the host, facilitating pathogen exclusion, immune system priming, and nutrient degradation. The composition of fish skin microbiomes varies across species and in response to a variety of stressors, however, there has been no systematic analysis across these studies to evaluate how these factors shape fish skin microbiomes. Here, we examined 1922 fish skin microbiomes from 36 studies that included 98 species and nine rearing conditions to investigate associations between fish skin microbiome, fish species, and water physiochemical factors. Proteobacteria, particularly the class Gammaproteobacteria, were present in all marine and freshwater fish skin microbiomes. Acinetobacter, Aeromonas, Ralstonia, Sphingomonas and Flavobacterium were the most abundant genera within freshwater fish skin microbiomes, and Alteromonas, Photobacterium, Pseudoalteromonas, Psychrobacter and Vibrio were the most abundant in saltwater fish. Our results show that different culturing (rearing) environments have a small but significant effect on the skin bacterial community compositions. Water temperature, pH, dissolved oxygen concentration, and salinity significantly correlated with differences in beta-diversity but not necessarily alpha-diversity. To improve study comparability on fish skin microbiomes, we provide recommendations for approaches to the analyses of sequencing data and improve study reproducibility.

Beyond compliance: harmonising research and husbandry practices to improve experimental reproducibility using fish models.

Reproducibility in animal research is impacted by the environment, by husbandry practices in the laboratory and by the animals' provenance. These factors, however, are often not adequately considered by researchers. A disconnect between researchers and animal care staff can result in inappropriate housing and husbandry decisions for scientific studies with those animals. This is especially the case for the research in neuro-behaviour, epigenetics, and the impact of climate change, as heritable phenotypic, behavioural or physiological changes are known to result from the animals' environmental housing, husbandry, provenance and prior experience. This can lead to greater variation (even major differences) in data outcomes among studies, driving scientific uncertainties. Herein, we illustrate some of the endpoints measured in fish studies known to be intrinsically linked to the environment and husbandry conditions and assess the significance of housing and husbandry practice decisions for research adopting these endpoints for different fish species. We highlight the different priorities and challenges faced by researchers and animal care staff and how harmonising their activities and building greater understanding of how husbandry practices affect the fish will improve reproducibility in research outcomes. We furthermore illustrate how improving engagement between stakeholders, including regulatory bodies, can better underpin fish husbandry decisions and where researchers could help to drive best husbandry practices through their own research with fish models.

Assessment of the impacts of GABA and AChE targeting pesticides on freshwater invertebrate family richness in English Rivers.

Globally, riverine system biodiversity is threatened by a range of stressors, spanning pollution, sedimentation, alterations to water flow, and climate change. Pesticides have been associated with population level impacts on freshwater invertebrates for acute high-level exposures, but far less is known about the chronic impact of episodic exposure to specific classes of pesticides or their mixtures. Here we employed the use of the UK Environment Agency's monitoring datasets over 40 years (covering years 1980 to 2019) to assess the impacts of AChE (acetylcholinesterase) and GABA (gamma-aminobutyric acid) receptor targeting pesticides on invertebrate family richness at English river sites. Concentrations of AChE and GABA pesticides toxic to freshwater invertebrates occurred (measured) across 18 of the 66 river sites assessed. For one of the three river sites (all found in the Midlands region of England) where data recorded over the past 40 years were sufficient for robust modelling studies, both AChE and GABA pesticides associated with invertebrate family richness. Here, where AChE total pesticide concentrations were classified as high, 46 of 64 invertebrate families were absent, and where GABA total pesticide concentration were classified as high, 16 of 64 invertebrate families were absent. Using a combination of field evidence and laboratory toxicity thresholds for population relevant endpoints we identify families of invertebrates most at risk in the selected English rivers to AChE and GABA pesticides. We, furthermore, provide strong evidence that the absence of the invertebrate family Polycentropodidae (caddisfly) from one field site is due to exposure effects to AChE pesticides.

Zinc oxide nanoparticles disrupt development and function of the olfactory sensory system impairing olfaction-mediated behaviour in zebrafish.

Zinc (Zn) is an essential metal present in numerous enzymes throughout the body, playing a vital role in animal and human health. However, the increasing use of zinc oxide nanomaterials (ZnONPs) in a diverse range of products has raised concerns regarding their potential impacts on health and the environment. Despite these concerns, the toxicity of ZnONP exposure on animal health remain poorly understood. To help address this knowledge gap, we have developed a highly sensitive oxidative stress (OS) biosensor zebrafish capable of detecting cell/tissue-specific OS responses to low doses of various oxidative stressors, including Zn, in a live fish embryo. Using live-imaging analysis with this biosensor zebrafish embryo, we discovered that the olfactory sensory neurons in the brain are especially sensitive to ZnOP exposure. Furthermore, through studies monitoring neutrophil migration and neuronal activation in the embryonic brain and via behaviour analysis, we have found that sub-lethal doses of ZnONPs (ranging from 0.033 to 1 mg/L nominal concentrations), which had no visible effect on embryo growth or morphology, cause significant localised inflammation, disrupting the neurophysiology of olfactory brain tissues and ultimately impaired olfaction-mediated behaviour. Collectively, these findings establish a potent and important effect mechanism for ZnONP toxicity, indicating the olfactory sensory system as the primary target for ZnONPs as an environmental toxicant in aquatic environments. Our result also highlights that even low doses of ZnONPs can have detrimental effects on the olfactory sensory system, surpassing previous expectations. The importance of olfaction in environment sensing, sex behaviours and overall fitness across species raises concerns about the potential impact of ZnONPs on olfaction-mediated brain function and behaviour in animals and humans. Our study emphasises the need for greater consideration of the potential risks associated with these nanomaterials.

Challenges and Recommendations in Assessing Potential Endocrine-Disrupting Properties of Metals in Aquatic Organisms.

New tools and refined frameworks for identifying and regulating endocrine-disrupting chemicals (EDCs) are being developed as our scientific understanding of how they work advances. Although focus has largely been on organic chemicals, the potential for metals to act as EDCs in aquatic systems is receiving increasing attention. Metal interactions with the endocrine system are complicated because some metals are essential to physiological systems, including the endocrine system, and nonessential metals can have similar physiochemical attributes that allow substitution into or interference with these systems. Consequently, elevated metal exposure could potentially cause endocrine disruption (ED) but can also cause indirect effects on the endocrine system via multiple pathways or elicit physiologically appropriate compensatory endocrine-mediated responses (endocrine modulation). These latter two effects can be confused with, but are clearly not, ED. In the present study, we provide several case studies that exemplify the challenges encountered in evaluating the endocrine-disrupting (ED) potential of metals, followed by recommendations on how to meet them. Given that metals have multiple modes of action (MOAs), we recommend that assessments use metal-specific adverse outcome pathway networks to ensure that accurate causal links are made between MOAs and effects on the endocrine system. We recommend more focus on establishing molecular initiating events for chronic metal toxicity because these are poorly understood and would reduce uncertainty regarding the potential for metals to be EDCs. Finally, more generalized MOAs such as oxidative stress could be involved in metal interactions with the endocrine system, and we suggest it may be experimentally efficient to evaluate these MOAs when ED is inferred. These experiments, however, must provide explicit linkage to the ED endpoints of interest. Environ Toxicol Chem 2023;42:2564-2579. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Comparative analysis of gonadal transcriptomes between turtle and alligator identifies common molecular cues activated during the temperature-sensitive period for sex determination.

The mode of sex determination in vertebrates can be categorized as genotypic or environmental. In the case of genotypic sex determination (GSD), the sexual fate of an organism is determined by the chromosome composition with some having dominant genes, named sex-determining genes, that drive the sex phenotypes. By contrast, many reptiles exhibit environmental sex determination (ESD), whereby environmental stimuli drive sex determination, and most notably temperature. To date, temperature-dependent sex determination (TSD) has been found in most turtles, some lizards, and all crocodylians, but commonalities in the controlling processes are not well established. Recent innovative sequencing technology has enabled investigations into gonadal transcriptomic profiles during temperature-sensitive periods (TSP) in various TSD species which can help elucidate the controlling mechanisms. In this study, we conducted a time-course analysis of the gonadal transcriptome during the male-producing temperature (26℃) of the Reeve's turtle (Chinese three-keeled pond turtle) Mauremys reevesii. We then compared the transcriptome profiles for this turtle species during the TSP with that for the American alligator Alligator mississippiensis to identify conserved reptilian TSD-related genes. Our transcriptome-based findings provide an opportunity to retrieve the candidate molecular cues that are activated during TSP and compare these target responses between TSD and GSD turtle species, and between TSD species.

Pesticide pollution associations with riverine invertebrate communities in England.

Globally freshwater biodiversity has experienced major decline and chemical pollutants are believed to have played a significant role in this decline, but this has not been well quantified for most riverine invertebrate populations. Here we applied a biogeographically independent trait-based bioindicator, SPEARpesticides across sites across five regions (Northern, Midlands and Western, Anglian, Southeast, and Southwest) in England to investigate for associations specifically between pesticide use/pollution and riverine invertebrate communities over a 55-year period (1965-2019). Both spatially and temporally post-1990, the Anglian and Thames regions consistently showed the lowest SPEARpesticides scores, illustrating the presence of fewer pesticide sensitive species. The Anglian region had the highest pesticide use compared to all other regions from 1990 to 2018 and there were negative relationships between the level of pesticide/insecticide use and the regional SPEARpesticides score. Biochemical Oxygen Demand and ammonia, as measures of general water quality, were also negatively correlated with the SPEARpesticides scores across the regions, but these factors were not the driver for the lower SPEARpesticides scores seen in the Anglian region. Based on SPEARpesticides scores, riverine invertebrate communities in England have been most impacted in the Anglian region and we evidence chronic insecticide exposure is likely a significant factor in shaping the status of those invertebrate communities.

Endocrine Disruption Is Reduced but Still Widespread in Wild Roach (Rutilus rutilus) Living in English Rivers.

Endocrine disruption of wild fish, primarily resulting in the feminization of males, has been reported in English river sites for several decades. Estrogenic activity emanating from wastewater treatment works (WwTW) has been conclusively demonstrated to be the main driver of these feminized phenotypes. Here, we revisit 10 English river sites previously surveyed in the late 1990s and early 2000s to assess how the frequency and severity of feminization now compare with the historical surveys. In the contemporary assessment, 60% of the sites revisited still showed endocrine disruption at the tissue organization level (oocytes present in otherwise male gonads; intersex) and 90% of sites had average male plasma vitellogenin concentrations (female-specific yolk protein; a sensitive biomarker of estrogen exposure) above natural baseline levels. In contrast to the historic surveys, none of the males sampled in the contemporary survey had ovarian cavities. At one of the larger WwTW, improvements to treatment technology may have driven a significant reduction in intersex induction, whereas at several of the smaller WwTW sites, the frequencies of feminization did not differ from those observed in the late 1990s. In conclusion, we show that although the severity of feminization is now reduced at many of the revisited sites, endocrine-disrupting chemicals are still impacting wild fish living downstream of WwTW in England.

Microbiomes in the context of developing sustainable intensified aquaculture.

With an ever-growing human population, the need for sustainable production of nutritional food sources has never been greater. Aquaculture is a key industry engaged in active development to increase production in line with this need while remaining sustainable in terms of environmental impact and promoting good welfare and health in farmed species. Microbiomes fundamentally underpin animal health, being a key part of their digestive, metabolic and defense systems, in the latter case protecting against opportunistic pathogens in the environment. The potential to manipulate the microbiome to the advantage of enhancing health, welfare and production is an intriguing prospect that has gained considerable traction in recent years. In this review we first set out what is known about the role of the microbiome in aquaculture production systems across the phylogenetic spectrum of cultured animals, from invertebrates to finfish. With a view to reducing environmental footprint and tightening biological and physical control, investment in "closed" aquaculture systems is on the rise, but little is known about how the microbial systems of these closed systems affect the health of cultured organisms. Through comparisons of the microbiomes and their dynamics across phylogenetically distinct animals and different aquaculture systems, we focus on microbial communities in terms of their functionality in order to identify what features within these microbiomes need to be harnessed for optimizing healthy intensified production in support of a sustainable future for aquaculture.

Correction: Alathari et al. A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia. Viruses 2023, 15, 965.

In the original publication [...].

A review of non-destructive biomonitoring techniques to assess the impacts of pollution on reproductive health in frogs and toads.

In anuran amphibians (frogs and toads), evidence linking pollution to population declines is limited, in particular through impaired reproduction. Here we review the evidence for pollutant-induced alterations on reproductive endpoints in wild anurans with a particular focus on the application of non-destructive endpoints including on sex ratios, male reproductive phenotypes (data are too scarce for females) and reproductive outputs (reflective of mating success). Data evidencing alterations in sex ratio in wild anurans are scarce, however, both feminisation and masculinisation in response to pollution have been reported (seven studies). Male nuptial pad morphology and calling behaviour display high sensitivity to pollutant-exposure and are important features determining male breeding success, however there is considerable variation in these endpoints and inconsistencies in the responses of them to pollution are reported in wild anurans. Data for clutch size are insufficient to assess sensitivity to pollutants (five studies only). However, hatch success and offspring fitness (tadpole survival/development) are sensitive to pollution, with clear linkages to population stability. In conclusion, there are a wide range of non destructive measures with good potential for application to assess/monitor reproductive health in wild anurans, however, a greater understanding of pollutant effects on these endpoints is needed. There measures deserve wider application as they are relatively simple and inexpensive to implement, and as they can be applied non-destructively are widely applicable to our declining anuran populations.

Factors Determining the Susceptibility of Fish to Effects of Human Pharmaceuticals.

The increasing levels and frequencies at which active pharmaceutical ingredients (APIs) are being detected in the environment are of significant concern, especially considering the potential adverse effects they may have on nontarget species such as fish. With many pharmaceuticals lacking environmental risk assessments, there is a need to better define and understand the potential risks that APIs and their biotransformation products pose to fish, while still minimizing the use of experimental animals. There are both extrinsic (environment- and drug-related) and intrinsic (fish-related) factors that make fish potentially vulnerable to the effects of human drugs, but which are not necessarily captured in nonfish tests. This critical review explores these factors, particularly focusing on the distinctive physiological processes in fish that underlie drug absorption, distribution, metabolism, excretion and toxicity (ADMET). Focal points include the impact of fish life stage and species on drug absorption (A) via multiple routes; the potential implications of fish's unique blood pH and plasma composition on the distribution (D) of drug molecules throughout the body; how fish's endothermic nature and the varied expression and activity of drug-metabolizing enzymes in their tissues may affect drug metabolism (M); and how their distinctive physiologies may impact the relative contribution of different excretory organs to the excretion (E) of APIs and metabolites. These discussions give insight into where existing data on drug properties, pharmacokinetics and pharmacodynamics from mammalian and clinical studies may or may not help to inform on environmental risks of APIs in fish.

Oxidative stress, genotoxic effects, and other damages caused by chronic exposure to silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs), and their mixtures in zebrafish (Danio rerio).

This study assessed the oxidative stress impacts of Ag NPs and ZnO NPs and their mixtures in zebrafish (Danio rerio). Zebrafish were exposed to sublethal concentrations of each NP and a mixture for 28 days followed by a 28-day recovery period (without NP exposure) and measurements made on hepatic levels of antioxidant enzymes (CAT, SOD, and GPx), MDA levels, expression of the genes for the Hsp70 and Hsp90, and MT, blood biochemical parameters (total protein, globulin, albumin, AST, ALT, ALP, and LDH), and genotoxicity in erythrocytes (via measurement of micronuclei (MN) and nuclear (NA) abnormalities). There was a tendency for an increase in the variation in the responses of antioxidant defense systems and there were higher MDA levels with increasing exposure concentration of Ag NPs and with increasing exposure time. Total protein, globulin, and albumin decreased during the exposure period, especially on the days of 28. Moreover, levels of AST and LDH increased significantly in the NPs co-exposure treatments, while levels of ALT and ALP significantly decreased. The highest expression levels for these genes occurred on day 14 and in the NPs co-exposure treatments. For exposure to both NPs individually and as a mixture, the frequency of MN and other NA were significantly increased (p < 0.05). During the recovery periods, most of the effects seen were reduced, most notably in the individual NPs treatments. The overall results suggest that the toxic effects of Ag NPs and ZnO NPs in combination significantly increase their toxicity in zebrafish.