Exploring Alternatives for Marine Toxicity Testing: Initial Evaluation of Fish Embryo and Mysid Tests.

Current regulations require that toxicity assessments be performed using standardized toxicity testing methods, often using fish. Recent legislation in both the European Union and United States has mandated that toxicity testing alternatives implement the 3Rs of animal research (replacement, reduction, and refinement) whenever possible. There have been advances in the development of alternatives for freshwater assessments, but there is a lack of analogous developments for marine assessments. One potential alternative testing method is the fish embryo toxicity (FET) test, which uses fish embryos rather than older fish. In the present study, FET methods were applied to two marine model organisms, the sheepshead minnow and the inland silverside. Another potential alternative is the mysid shrimp survival and growth test, which uses an invertebrate model. The primary objective of the present study was to compare the sensitivity of these three potential alternative testing methods to two standardized fish-based tests using 3,4-dichloroaniline (DCA), a common reference toxicant. A secondary objective was to characterize the ontogeny of sheepshead minnows and inland silversides. This provided a temporal and visual guide that can be used to identify appropriately staged embryos for inclusion in FET tests and delineate key developmental events (e.g., somite development, eyespot formation, etc.). Comparison of the testing strategies for assessing DCA indicated that: (1) the standardized fish tests possessed comparable sensitivity to each other; (2) the mysid shrimp tests possessed comparable sensitivity to the standardized fish tests; (3) the sheepshead minnow and inland silverside FET tests were the least sensitive testing strategies employed; and (4) inclusion of sublethal endpoints (i.e., hatchability and pericardial edema) in the marine FETs increased their sensitivity. Environ Toxicol Chem 2024;43:1285-1299. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Multigenerational, Indirect Exposure to Pyrethroids Demonstrates Potential Compensatory Response and Reduced Toxicity at Higher Salinity in Estuarine Fish.

Estuarine environments are critical to fish species and serve as nurseries for developing embryos and larvae. They also undergo daily fluctuations in salinity and act as filters for pollutants. Additionally, global climate change (GCC) is altering salinity regimes within estuarine systems through changes in precipitation and sea level rise. GCC is also likely to lead to an increased use of insecticides to prevent pests from damaging agricultural crops as their habitats and mating seasons change from increased temperatures. This underscores the importance of understanding how insecticide toxicity to fish changes under different salinity conditions. In this study, larval Inland Silversides (Menidia beryllina) were exposed to bifenthrin (1.1 ng/L), cyfluthrin (0.9 ng/L), or cyhalothrin (0.7 ng/L) at either 6 or 10 practical salinity units (PSU) for 96 h during hatching, with a subset assessed for end points relevant to neurotoxicity and endocrine disruption by testing behavior, gene expression of a select suite of genes, reproduction, and growth. At both salinities, directly exposed F0 larvae were hypoactive relative to the F0 controls; however, the indirectly exposed F1 larvae were hyperactive relative to the F1 control. This could be evidence of a compensatory response to environmentally relevant concentrations of pyrethroids in fish. Effects on development, gene expression, and growth were also observed. Overall, exposure to pyrethroids at 10 PSU resulted in fewer behavioral and endocrine disruptive effects relative to those observed in organisms at 6 PSU.

Essential and non-essential elements in marine silverside Odontesthes argentinensis from southwestern Atlantic coast: Tissues bioaccumulation, biomagnification and public health risk assessment.

In order to assess the metal pollution in the southwestern Atlantic coast, levels of eleven metals: Ba, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Sr, and Zn and one metalloid: As in water and tissues of Odontesthes argentinensis from the Samborombón Bay (SB) and Partido de La Costa (PC) were examined. Additionally, bioconcentration, and bioaccumulation factors, estimated daily intake (EDI) for adults and children, and the hazard risk for fishermen and general populations were evaluated. Cadmium, Cu and Zn levels in the water samples exceeded the limits established by international guidelines for the protection of aquatic life in marine environments (CCME). The liver showed the highest concentrations of As, Cu, Fe, and Zn, while muscle showed the least levels of all elements examined. Bioaccumulation of Cd, Cu, Mn, Pb and Zn were observed in all tissues, while Ba and Sr bioaccumulated significantly in the gills. The fish δ15N values significantly varied between sites, however, no biodilution or biomagnification was observed. The Target Hazard Quotients (THQ) and the Carcinogenic Risk (CR) indices suggest that there is no human health risk to the fishermen and general populations from the O. argentinensis consumption. Furthermore, As (27%) and Cr (33%) concentrations in silversides from PC and SB, respectively, exceeded the levels recommended by local and international guidelines.

Influence of Salinity on the Partitioning Behavior of Six Commonly Used Pesticides in Fish Eggs.

Salinity has been reported to impact the octanol-water partition coefficient of organic contaminants entering aquatic ecosystems. However, limited data are available on the impacts of salinity on their partitioning from the aqueous phase to adjacent organic compartments. The pesticides bifenthrin, chlorpyrifos, dicloran, myclobutanil, penconazole, and triadimefon were used to investigate the effects of salinity on their partitioning to capelin (Mallotus villosus) eggs in 5 practical salinity units (PSU) versus 25 PSU artificial seawater (ASW). The partitioning coefficient was significantly higher in 25 versus 5 PSU ASW for bifenthrin, chlorpyrifos, dicloran, penconazole, and triadimefon by 31%, 28%, 35%, 28%, and 20%, respectively, while for myclobutanil there was no significant difference. Moreover, pesticide partitioning to store-bought capelin eggs was consistent with the partitioning observed for the standard assay species, inland silversides (Menidia beryllina) eggs, after partitioning between the eggs and exposure solution had reached a state of equilibrium. The present study illustrates the importance of considering the influence of salinity on the environmental partitioning and fate of hydrophobic organic contaminants in aquatic ecosystems. Environ Toxicol Chem 2024;43:299-306. © 2023 SETAC.

Mitochondrial genome of the jack silverside, Atherinopsis californiensis (Atherinopsidae, Atheriniformes), a nearshore fish of the California Current Ecosystem.

The jack silverside (Atherinopsis californiensis), also referred to as jacksmelt, is a neotropical silverside fish that inhabits nearshore shallow waters of the California Current Ecosystem in the Northeast Pacific Ocean, ranging from the coast of Oregon, USA, in the north to as far south as Baja California, Mexico. This fish is the sole member of its genus and is a commonly taken species when hook-and-line fishing in pelagic-neritic environments including bays, estuaries, kelp forests, and along sand beaches. Here we report the first complete mitochondrial genome of jack silverside consisting of 16,519 bp nucleotides and encoding 13 protein-coding regions, 12S and 16S rRNAs, 22 tRNAs, and an 841 bp D-loop control region. Phylogenetic analysis using all protein-coding genes of the complete mitogenome confirmed the inclusion of A. californiensis within subfamily Atherinopsinae of family Atherinopsidae, order Atheriniformes. This complete mitochondrial DNA genome will be of use for biodiversity assessments in the California Current ecosystem, while also providing a foundation for future mtDNA population genetic studies on this prominently caught species in shore- and pier-based recreational sport fishing.

Brazilian silverside, Atherinella brasiliensis (Quoy & Gaimard,1825) embryos as a test-species for marine fish ecotoxicological tests.

The fish embryo test (FET) is an alternative to the classic freshwater toxicity test used to assess environmental hazards and risks to fish. This test has been standardized and adopted by the Organization for Economic and Cooperation and Development (OECD). As salinity may affect the substances' toxicity, we describe the development of an alternative euryhaline test species for embryonic ecotoxicological tests: the Brazilian silverside Atherinella brasiliensis (Quoy & Gaimard, 1825). This species is broadly distributed along the coast of South America and is able to inhabit a broad range of environmental and saline conditions. Ours is the first study on the maintenance of a native South American species for natural reproduction and the generation of embryos for tests. The embryos used are transparent and possess fluorescent cells which have only been seen in a few species and which may be used as markers, making it an alternative assessment tool for the lethal and sublethal substances in marine and estuarine environments. We provide a detailed description and analysis of embryonic development under different salinities and temperatures. The embryos and larvae developed in similar ways at different salinities, however as temperatures increased, mortality also increased. We considered the effects of the reference toxicants Zn2+ and SDS using a protocol similar to the FET that was standardized for zebrafish. Brazilian silverside embryos are as sensitive as freshwater, or euryhaline fish, to the surfactant but are more resistant to metals prior to hatching. We were able to show the advantages of the Brazilian silverside as a model for a marine fish embryo test (FETm) with high levels of reproducibility and little contaminated waste.

A comprehensive non-redundant reference transcriptome for the Atlantic silverside Menidia menidia.

The Atlantic silverside (Menidia menidia) has been the focus of extensive research efforts in ecology, evolutionary biology, and physiology over the past three decades, but lack of genomic resources has so far hindered examination of the molecular basis underlying the remarkable patterns of phenotypic variation described in this species. We here present the first reference transcriptome for M. menidia. We sought to capture a single representative sequence from as many genes as possible by first using a combination of Trinity and the CLC Genomics Workbench to de novo assemble contigs based on RNA-seq data from multiple individuals, tissue types, and life stages. To reduce redundancy, we passed the combined raw assemblies through a stringent filtering pipeline based both on sequence similarity to related species and computational predictions of transcript quality, condensing an initial set of >480,000 contigs to a final set of 20,998 representative contigs, amounting to a total length of 53.3 Mb. In this final assembly, 91% of the contigs were functionally annotated with putative gene function and gene ontology (GO) terms and/or InterProScan identifiers. The assembly contains complete or nearly complete copies of >95% of 248 highly conserved core genes present in low copy number across higher eukaryotes, and partial copies of another 3.8%, suggesting that our assembly provides relatively comprehensive coverage of the M. menidia transcriptome. The assembly provided here will be an important resource for future research.

Temporal changes in gene expression and genotype frequency of the ornithine decarboxylase gene in native silverside Basilichthys microlepidotus: Impact of wastewater reduction due to implementation of public policies.

Human activity has caused a deterioration in the health and population size of riverine species; thus, public policies have been implemented to mitigate the anthropogenic impacts of water use, watercourse transformation, and pollution. We studied the Maipo River Basin, one of the most polluted with untreated wastewater in Chile, for a period of 12 years (2007-2019). Since the implementation of new public policies, including the operation of a wastewater collector (2012), the Maipo River Basin is currently much less polluted by untreated water than before. To analyze the impact of wastewater reduction in this river basin, we studied the native silverside (Basilichthys microlepidotus), which inhabits both polluted and unpolluted areas of the river basin. Previous studies reported the overexpression of the ornithine decarboxylase (odc) gene, heterozygote deficit, and high frequency of a homozygote odc genotype in silverside populations that inhabit wastewater-polluted sites, suggesting a phenotypic change and genotypic selection in response to pollution. Here, a population affected and another population unaffected by wastewater were studied before and after implementing the wastewater collector. The physicochemical data of water samples, changes in odc expression and microsatellite variability, and odc genotype frequencies were analyzed. The results showed physicochemical changes in the affected site before and after the operation of the wastewater collector. The microsatellite loci showed no changes in either population. The odc expression in the affected site was higher before the operation of the wastewater collector. Significant changes in the genotype frequencies of the odc gene before and after the wastewater collector operation were detected only at the affected site, wherein the homozygous dominant genotype decreased from >59% to <25%. Our results suggest that public policies aimed at mitigating aquatic pollution can indirectly affect both gene expression and genotype frequencies of important functional genes.

Phenotypic/genotypic sex mismatches and temperature-dependent sex determination in a wild population of an Old World atherinid, the cobaltcap silverside Hypoatherina tsurugae.

Several New World atheriniforms have been recognized as temperature-dependent sex determined (TSD) and yet possess a genotypic sex determinant (amhy) which is primarily functional at mid-range temperatures. In contrast, little is known about the sex determination in Old World atheriniforms, even though such knowledge is crucial to understand the evolution of sex determination mechanisms in fishes and to model the effects of global warming and climate change on their populations. This study examined the effects of water temperature on sex determination of an Old World atheriniform, the cobaltcap silverside Hypoatherina tsurugae, in which we recently described an amhy homologue. We first assessed the occurrence of phenotypic/genotypic sex mismatches in wild specimens from Tokyo Bay for three years (2014-2016) and used otolith analysis to estimate their birth dates and approximate thermal history during the presumptive period of sex determination. Phenotypic sex ratios became progressively biased towards males (47.3%-78.2%) during the period and were associated with year-to-year increases in the frequency of XX-males (7.3%-52.0%) and decreases in XY/YY-females (14.5%-0%). The breeding season had similar length but was delayed by about 1 month per year between 2014 and 2016, causing larvae to experience higher temperatures during the period of sex determination from year to year. Larval rearing experiments confirmed increased likelihood of feminization and masculinization at low and high temperatures, respectively. The results suggest that cobaltcap silverside has TSD, or more specifically the coexistence of genotypic and environmental sex determinants, and that it affects sex ratios in wild populations.

Estuarine fish assemblages present a species-specific difference in the multixenobiotics resistance activity.

We investigated the activity of the multixenobiotic resistance (MXR) phenotype, a biological defense system in aquatic organisms, in the fish assemblages of two tropical estuaries with different degrees of environmental impacts, the Paraiba River and Mamanguape River Estuaries. The aim of this work was to compare the activity of the MXR phenotype of different fishes to test the hypothesis that each species has an inherent activity level and to use this activity as a bioindicator of aquatic contamination. We assessed the MXR activity of the gills, using rhodamine B (RB) accumulation assay. The results demonstrated a species-specific difference in the MXR activity of fishes caught in the same estuarine zone. Also, the pelagic species Eucinostomus melanopterus, Eucinostomus argenteus, and Lutjanus jocu had higher RB accumulation, while the demersal species Sphoeroides testudineus and Sphoeroides greeleyi had the lowest RB accumulation, suggesting that the ecological characteristic of fish in the water column exerts an influence on MXR activity. Besides, we demonstrated the potential of using the gill MXR activity of the key estuarine species, the Brazilian silversides Atherinella brasiliensis, as a tool for biomonitoring estuaries.

Occurrence of organochlorine compounds in fish from freshwater environments of the central Andes, Argentina.

The Central Andes region is considered an area of high environmental relevance in South America and it is rich in glaciers, which are the main fresh water sources and reservoirs of the region. The presence of organochlorine compounds (OCs) is an environmental threat due to their persistence and adverse effects on wildlife and humans. The levels of OCs, such as chlordane compounds (CHLs), dichlorodiphenyltrichloroethane and metabolites (DDTs), hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), and polychlorinated biphenyls (PCBs), were determined in muscle and liver tissues of fish and in sediments collected from different sites of the central Andes: the Mendoza River and the Yaucha River (rainbow trout: Oncorhynchus mykiss) and Carrizal Dam (silverside: Odontesthes bonariensis). Regardless of studied site and fish tissue, the general OC trend was: DDTs > PCBs > HCHs > CHLs > HCB. The results showed clear geographic distribution patterns for DDTs. Among the sampling sites, silverside fish showed the highest average load of DDTs (614 and 867 ng g-1 lw in muscle and liver tissues, respectively). Based on the biota-sediment bioaccumulation factor (BSAF), both rainbow trout and silverside fish showed comparable capability to accumulate p,p'-DDT in their tissues. Intraspecific correlational analysis showed that CB-138 in muscle and p,p'-DDT in liver were clearly correlated with the lipid content in silverside specimens, which highlights the relevance of taking into account the fat content and the type of target tissues.

Environmental regulation of sex determination in fishes: Insights from Atheriniformes.

Sex determination is the first step toward the establishment of phenotypic sex in most vertebrates. Aquatic poikilotherms such as teleost fishes exhibit a high diversity of sex-determination mechanisms and gonadal phenotypes that are remarkably plastic and responsive to a variety of environmental factors (e.g., water temperature, pH, salinity, photoperiod, population density). This chapter reviews current knowledge of genotypic and environmental sex determination systems in fishes with special reference to Atheriniformes-one of the best-characterized taxa in this field-and offers perspectives to guide and stimulate further research.

Sensitivities of an endemic, endangered California smelt and two non-native fishes to serial increases in temperature and salinity: implications for shifting community structure with climate change.

In many aquatic systems, native fishes are in decline and the factors responsible are often elusive. In the San Francisco Estuary (SFE) in California, interactions among native and non-native species are key factors contributing to the decline in abundance of endemic, endangered Delta Smelt (Hypomesus transpacificus). Climate change and drought-related stressors are further exacerbating declines. To assess how multiple environmental changes affect the physiology of native Delta Smelt and non-native Mississippi Silverside (Menidia beryllina) and Largemouth Bass (Micropterus salmoides), fishes were exposed to serial exposures of a single stressor (elevated temperature or salinity) followed by two stressors (elevated temperature and salinity) to determine how a single stressor affects the capacity to cope with the addition of a second stressor. Critical thermal maximum (CTMax; a measure of upper temperature tolerance) was determined after 0, 2, 4 and 7 days following single and multiple stressors of elevated temperature (16°C vs. 20°C) and salinity (2.4 vs. 8-12 ppt, depending on species). Under control conditions, non-native fishes had significantly higher CTMax than the native Delta Smelt. An initial temperature or salinity stressor did not negatively affect the ability of any species to tolerate a subsequent multiple stressor. While elevated salinity had little effect on CTMax, a 4°C increase in temperature increased CTMax. Bass experienced an additive effect of increased temperature and salinity on CTMax, such that CTMax further increased under multiple stressors. In addition, Bass demonstrated physiological sensitivity to multiple stressors demonstrated by changes in hematocrit and plasma osmolality, whereas the physiology of Silversides remained unaffected. Non-native Bass and Mississippi Silversides showed consistently higher thermal tolerance limits than the native Delta Smelt, supporting their abundance in warmer SFE habitats. Continued increases in SFE water temperatures predicted with climate change may further impact endangered Delta Smelt populations directly if habitat temperatures exceed thermal limits.

Robust quantification of fish early life CO2 sensitivities via serial experimentation.

Despite the remarkable expansion of laboratory studies, robust estimates of single species CO2 sensitivities remain largely elusive. We conducted a meta-analysis of 20 CO2 exposure experiments conducted over 6 years on offspring of wild Atlantic silversides (Menidia menidia) to robustly constrain CO2 effects on early life survival and growth. We conclude that early stages of this species are generally tolerant to CO2 levels of approximately 2000 µatm, likely because they already experience these conditions on diel to seasonal timescales. Still, high CO2 conditions measurably reduced fitness in this species by significantly decreasing average embryo survival (-9%) and embryo+larval survival (-13%). Survival traits had much larger coefficients of variation (greater than 30%) than larval length or growth (3-11%). CO2 sensitivities varied seasonally and were highest at the beginning and end of the species' spawning season (April-July), likely due to the combined effects of transgenerational plasticity and maternal provisioning. Our analyses suggest that serial experimentation is a powerful, yet underused tool for robustly estimating small but true CO2 effects in fish early life stages.

Heavy Metal Content in Chilean Fish Related to Habitat Use, Tissue Type and River of Origin.

In this study, we analyze the concentration of ten metals in two freshwater fish-the benthic catfish Trichomycterus areolatus and the limnetic silverside Basilichthys microlepidotus-in order to detect possible accumulation differences related to fish habitat (benthic or pelagic), tissue type (gill, liver and muscle), and the river of origin (four different rivers) in central Chile. The MANOVA performed with all variables and metals, revealed independent effects of fish, tissue and river. In the case of the fish factor, Cu, Cr, Mo and Zn showed statistically higher concentrations in catfish compared with silverside for all tissues and in all rivers (p < 0.05). In the case of the tissue factor, Al, Cr, Fe and Mn had statistically higher concentrations in liver and gills than in muscle (p < 0.05). For the river effect, the analysis showed higher concentrations of Cr, Mn and Pb in the Cogoti river and the lower concentrations in the Recoleta river. These results suggest that not all metals have the same pattern of accumulation; however, some metals tend to accumulate more in readily catfish, probably due to their benthic habit, and in liver and gill tissue, probably as a result of accumulation from food sources and respiration.

Presence of trace elements in the silverside Odontesthes argentinensis.

The silverside Odontesthes argentinensis is an economically significant resource for commercial fisheries in South America. We evaluated the presence of trace elements in the stomach content and fish tissues (muscle and otoliths) of O. argentinensis. In addition, we assessed the presence of trace elements in its prey (zooplankton) and in seawater in a coastal temperate area. The most abundant trace elements found in the water, zooplankton, stomach content, and fish tissues (muscle and otoliths) constituted of Ba, Mn, Sr and Zn, while Cd, Cu and Pb were observed in lower concentrations. We concluded that O. argentinensis specimens captured from the environment, within the length range analyzed for muscle samples (total length: <21cm), are deemed fit for human consumption because the concentrations of trace elements mostly meet the standards established in the Argentine Food Code. The information obtained in this study is vital for O. argentinensis farming in closed systems.

Review of the Odontesthes perugiae species group from Río de La Plata drainage, with the description of a new species (Atherinomorpha: Atherinopsidae).

Specimens presently identified as belonging to Odontesthes perugiae species group from rio Paraná, rio Uruguay and rio Negro in Argentina, Brazil and Uruguay are revised. Two species are recognized: Odontesthes perugiae, type species of the genus with Odontesthes orientalis as a junior synonym, and a new species described herein. Odontesthes perugiae is redescribed and its distribution restricted to the lower rio Paraná, lower rio Uruguay and rio Negro basins. The new species is endemic from the upper rio Uruguay above Salto do Yucumã (Brazil) or Saltos del Moconá (Argentina) falls. Both species share with the remaining taxa included in the O. perugiae species-group (O. bicudo, O. ledae, O. mirinensis, and O. piquava) the possession of teeth of the outer row on dentary and premaxilla shorter than those in the inner row, being distinguished from them by the number of gill rakers, body measurements and some osteological characters.

Visual sensitivity and spatial resolution of the planktivorous fish, Atherinomorus forskalii (Atherinidae; Rüppell, 1838), to a polarized grating.

Polarized light detection has been documented in only a small number of fish species. The benefit of polarization vision for fish is not fully understood, nor is the transduction mechanism that underlies it. Past studies proposed that one possible advantage of polarization vision is that it enhances the contrast of zooplankton targets by breaking their transparency. Here, we used an optomotor apparatus to test the responses of the planktivorous Hardyhead silverside fish Atherinomorus forskalii (Atherinidae) to vertical unpolarized (intensity) and polarized gratings. We also tested and compared the spatial and temporal resolutions of A. forskalii in the intensity and polarization domains. A. forskalii responded to the polarization pattern, but only under illumination that included ultraviolet-blue (λ>380nm) wavelengths. The spatial resolution of A. forskalii was measured as a minimum separable angle of 0.57° (a 1-mm prey viewed from 100-mm distance). The temporal resolution to unpolarized vs. polarized gratings was constant, at 33 and 10Hz respectively at most of the stripe widths tested. At the smallest stripe width tested (1mm=the minimal separable angle), which correlates with the size of prey typically consumed by these fish, the temporal resolution to the polarized grating increased to 42Hz. We conclude that A. forskalii is polarization sensitive, may use polarization vision to improve detection of its planktonic prey, and that polarization may be perceived by the fish via a separate visual pathway than intensity.