Effects of metformin on wild fathead minnows (Pimephales promelas) using in-lake mesocosms in a boreal lake ecosystem.

Due to its widespread use for the treatment of Type-2 diabetes, metformin is routinely detected in surface waters globally. Laboratory studies have shown that environmentally relevant concentrations of metformin can adversely affect the health of adult fish, with effects observed more frequently in males. However, the potential risk to wild fish populations has yet to be fully elucidated and remains a topic of debate. To explore whether environmentally relevant metformin exposure poses a risk to wild fish populations, the present study exposed wild fathead minnows (Pimephales promelas) to 5 or 50 µg/L metformin via 2 m diameter in-lake mesocosms deployed in a natural boreal lake in Northern Ontario at the International Institute for Sustainable Development - Experimental Lakes Area (IISD-ELA). Environmental monitoring was performed at regular intervals for 8-weeks, with fish length, weight (body, liver and gonad), condition factor, gonadosomatic index, liver-somatic index, body composition (water and biomolecules) and hematocrit levels evaluated at test termination. Metabolic endpoints were also evaluated using liver, brain and muscle tissue, and gonads were evaluated histologically. Results indicate that current environmental exposure scenarios may be sufficient to adversely impact the health of wild fish populations. Adult male fish exposed to metformin had significantly reduced whole body weight and condition factor and several male fish from the high-dose metformin had oocytes in their testes. Metformin-exposed fish had altered moisture and lipid (decrease) content in their tissues. Further, brain (increase) and liver (decrease) glycogen were altered in fish exposed to high-dose metformin. To our knowledge, this study constitutes the first effort to understand metformin's effects on a wild small-bodied fish population under environmentally relevant field exposure conditions.

Effect of total dissolved gas supersaturation on the passage behavior of silver carp (Hypophthalmichthys molitrix) and ya-fish (Schizothorax prenanti) through an experimental vertical slot fishway.

Total dissolved gas (TDG) supersaturation caused by flood discharge water poses a threat to vital activities such as migration, foraging, and evasion in fish species upstream of the Yangtze River, which may impair the ability of fish to pass through fishways during the migration period, causing poor utilization of fishways. Previous studies have shown that TDG supersaturation reduces the critical and burst swimming abilities of fish, suggesting potential adverse effects on swimming performance. However, studies focusing on the impact of TDG on fish swimming behavior in experimental vertical-slot fishways remain scarce. Therefore, in this study, silver carp (Hypophthalmichthys molitrix) and ya-fish (Schizothorax prenanti) were used as the study species, and comparative passage experiments were carried out in an experimental vertical slot fishway to systematically analyze the effects of TDG supersaturation on their passage behavior. The passage success of the silver carp was 57%, 39%, 26%, and 27% at TDG levels of 100%, 110%, 120%, and 130%, respectively. Passage success of ya-fish was 73%, 37%, 31%, and 35% at TDG concentrations of 100%, 110%, 120%, and 130%, respectively. The passage time for both species increased significantly with increasing TDG levels. Furthermore, the passage routes of silver carp changed significantly compared to the control group, whereas the passage routes of ya-fish changed insignificantly. High levels of TDG supersaturation (≥120%) also contributed to a higher mortality rate of ya-fish passing through the vertical slot fishway. The research results provide valuable data on the influence of TDG supersaturation on fish movement behavior responses in experimental vertical slot fishways, offering a reference for the design of fishways and the formulation of reservoir operation schemes.

A global dataset on species occurrences and functional traits of Schizothoracinae fish.

The Schizothoracinae fish are a natural group of cyprinids widely distributed in rivers and lakes in the Qinghai-Tibetan Plateau (QTP) and adjacent regions. These fish parallelly evolved with the QTP uplift and are thus important for uncovering geological history, the paleoclimatic environment, and the mechanisms of functional adaptation to environmental change. However, a dataset including species occurrences and functional traits, which are essential for resolving the above issues and guiding relevant conservation, remains unavailable. To fill this gap, we systematically compiled a comprehensive dataset on species occurrences and functional traits of Schizothoracinae fish from our long-term field samplings and various sources (e.g., publications and online databases). The dataset includes 7,333 occurrence records and 3,204 records of 32 functional traits covering all the genera and species of Schizothoracinae fish (i.e., 12 genera and 125 species or subspecies). Sampling records spanned over 180 years. This dataset will serve as a valuable resource for future research on the evolution, historical biogeography, responses to environmental change, and conservation of the Schizothoracinae fish.

Identifying transcriptomic indicators of tertiary treated municipal effluent in longnose dace (Rhinichthys cataractae) caged under semi-controlled conditions in experimental raceways.

To evaluate effects of tertiary treated wastewater treatment plant effluent (MWWE) on transcriptomic responses in longnose dace (Rhinichthys cataractae; LND) we conducted a semi-controlled study in experimental raceways (Advancing Canadian Water Assets facility) imbedded in the Pine Creek treatment plant (Calgary, AB). LND collected from a reference site in the Bow River (REF) were caged in raceways containing either 5 % Pine Creek effluent (PC) or Bow River water (BR; control) over 28 d. Liver transcriptomes were analyzed in males and females sampled on days 7, 14 and 28 from BR and PC, and compared to REF fish on day 0. Concurrent with the caging, selected environmental substances of concern were analyzed in the BR and PC. Significantly different unigenes (SDUs) in females (vs males) within both BR and PC raceways increased over time and compared to REF fish. Moreover, SDUs in females and males within the same treatment (i.e., BR, PC) showed a temporal increase as well as compared to REF fish. Time was the dominant factor affecting SDUs, whereas sex and treatment had less of an impact on the transcriptome profiling. Gene Set Enrichment Analysis of BR vs PC over time revealed effects on genes involved in growth, metabolism of carbohydrates and lipids, and immune system on day 7; however, by day 28, 80-100 % of the transcripts localized to enriched biomarkers were associated with tissue immune responses in both sexes. Exposure to 5 % effluent had significant effects on female liver somatic index but no effects were observed on other phenotypic health indices in either sex. BR was used as the source of reference water, but analyses showed trace amounts of ESOCs. Analyses did not point towards definitive response patterns that could be used in field-based ecotoxicogenomic studies on the impacts of well-treated MWWE but suggested compromised adaptive immune responses.

Microplastics may induce food dilution and endocrine disrupting effects in fathead minnows (Pimephales promelas), and decrease offspring quality.

Microplastics are a complex environmental contaminant that have been reported to cause a variety of impacts, although the mechanism of these impacts remains unclear. Many studies have investigated either sub-organismal or apical endpoints, while very few have attempted to integrate and link endpoints seen at multiple levels of organization. Here, we exposed fathead minnows to microplastics for their entire lifecycle, from the egg stage through to reproduction, and raised a subset of the offspring in clean water. We show that both preconsumer and environmentally sourced microplastics impact adult growth, lipid storage, and external colouration, suggesting a potential food dilution effect. Environmentally sourced microplastics, but not preconsumer microplastics, had further endocrine disrupting impacts on the parental generation and their offspring in the low concentration treatments such that egg production began later, eggs were less viable, and the offspring had higher rates of malformation. Low dose effects are a typical dose-response for endocrine disrupting contaminants. These results suggest that microplastic exposure, at concentrations relevant to what is being found in the environment, has potential implications for forage fish populations. Our findings also highlight the importance of using an integrative approach to understanding the mechanisms behind how and why microplastics impact organisms.

Effects of Age and Exposure Duration on the Sensitivity of Early Life Stage Fathead Minnow (Pimephales promelas) to Waterborne Propranolol Exposure.

Propranolol is a heavily prescribed, nonspecific beta-adrenoceptor (bAR) antagonist frequently found in wastewater effluents, prompting concern over its potential to adversely affect exposed organisms. In the present study, the transcriptional responses of 4, 5, and 6 days postfertilization (dpf) ±1 h fathead minnow, exposed for 6, 24, or 48 h to 0.66 or 3.3 mg/L (nominal) propranolol were characterized using RNA sequencing. The number of differentially expressed genes (DEGs) was used as an estimate of sensitivity. A trend toward increased sensitivity with age was observed; fish >7 dpf at the end of exposure were particularly sensitive to propranolol. The DEGs largely overlapped among treatment groups, suggesting a highly consistent response that was independent of age. Cluster analysis was performed using normalized count data for unexposed and propranolol-exposed fish. Control fish clustered tightly by age, with fish ≥7 dpf clustering away from younger fish, reflecting developmental differences. When clustering was conducted using exposed fish, in cases where propranolol induced a minimal or no transcriptional response, the results mirrored those of the control fish and did not appreciably cluster by treatment. In treatment groups that displayed a more robust transcriptional response, the effects of propranolol were evident; however, fish <7 dpf clustered away from older fish, despite having similar numbers of DEGs. Increased sensitivity at 7 dpf coincided with developmental milestones with the potential to alter propranolol pharmacokinetics or pharmacodynamics, such as the onset of exogenous feeding and gill functionality as well as increased systemic expression of bAR. These results may have broader implications because toxicity testing often utilizes fish <4 dpf, prior to the onset of these potentially important developmental milestones, which may result in an underestimation of risk for some chemicals. Environ Toxicol Chem 2024;43:807-820. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Interaction of total dissolved gas supersaturation and suspended sediment on swimming performance of bighead carp (Hypopthalmichthys nobilis).

When dams discharge floodwaters, the river downstream often becomes supersaturated in total dissolved gases (TDG) and contains high volumes of suspended sediments (SS). Supersaturated TDG and high SS concentrations in water may affect fish physiologically in ways that manifest in swimming performance. Despite increasing awareness of the separate effects of TDG supersaturation and SS, knowledge about their synergistic effects remains unknown. To explore the interactive effects of TDG and SS on the swimming performance of bighead carp, the juveniles were exposed to 100, 110, 115, 120, 125, 130, 135, and 140% of TDG-supersaturated water with SS concentrations of 0, 50, 100, and 150 mg/L, respectively, and the critical swimming ability speed (Ucrit ) and burst swimming ability speed (Uburst ) were measured. The results indicated that the swimming ability (Ucrit and Uburst ) decreased when TDG levels and SS concentrations increased. TDG and SS did not interact significantly to decrease both Ucrit and Uburst . In contrast, exposure to TDG alone significantly decreased both Ucrit and Uburst , whereas exposure to SS alone decreased only Uburst . In addition, our results suggested that there was a negative linear relationship between TDG and fatigue time. Swimming ability can decline significantly due to high TDG levels (>130%). Therefore, high TDG levels (>130%) should be restricted during reservoir operation to prevent the stress caused by TDG.

Multi-omic responses of fish exposed to complex chemical mixtures in the Shenandoah River watershed.

To evaluate relationships between different anthropogenic impacts, contaminant occurrence, and fish health, we conducted in situ fish exposures across the Shenandoah River watershed at five sites with different land use. Exposure water was analyzed for over 500 chemical constituents, and organismal, metabolomic, and transcriptomic endpoints were measured in fathead minnows. Adverse reproductive outcomes were observed in fish exposed in the upper watershed at both wastewater treatment plant (WWTP) effluent- and agriculture-impacted sites, including decreased gonadosomatic index and altered secondary sex characteristics. This was accompanied with increased mortality at the site most impacted by agricultural activities. Molecular biomarkers of estrogen exposure were unchanged and consistent with low or non-detectable concentrations of common estrogens, indicating that alternative mechanisms were involved in organismal adverse outcomes. Hepatic metabolomic and transcriptomic profiles were altered in a site-specific manner, consistent with variation in land use and contaminant profiles. Integrated biomarker response data were useful for evaluating mechanistic linkages between contaminants and adverse outcomes, suggesting that reproductive endocrine disruption, altered lipid processes, and immunosuppression may have been involved in these organismal impacts. This study demonstrated linkages between human-impact, contaminant occurrence, and exposure effects in the Shenandoah River watershed and showed increased risk of adverse outcomes in fathead minnows exposed to complex mixtures at sites impacted by municipal wastewater discharges and agricultural practices.

From behaviour to complex communities: Resilience to anthropogenic noise in a fish-induced trophic cascade.

Sound emissions from human activities represent a pervasive environmental stressor. Individual responses in terms of behaviour, physiology or anatomy are well documented but whether they propagate through nested ecological interactions to alter complex communities needs to be better understood. This is even more relevant for freshwater ecosystems that harbour a disproportionate fraction of biodiversity but receive less attention than marine and terrestrial systems. We conducted a mesocosm investigation to study the effect of chronic exposure to motorboat noise on the dynamics of a freshwater community including phytoplankton, zooplankton, and roach as a planktivorous fish. In addition, we performed a microcosm investigation to test whether roach's feeding behaviour was influenced by the noise condition they experienced in the mesocosms. Indeed, compared to other freshwater fish, the response of roach to motorboat noise apparently does not weaken with repeated exposure, suggesting the absence of habituation. As expected under the trophic cascade hypothesis, predation by roach induced structural changes in the planktonic communities with a decrease in the main grazing zooplankton that slightly benefited green algae. Surprisingly, although the microcosm investigation revealed persistent alterations in the feeding behaviour of the roach exposed to chronic noise, the dynamics of the roach-dominated planktonic communities did not differ between the noisy and noiseless mesocosms. It might be that roach's individual response to noise was not strong enough to cascade or that the biological cues coming from the conspecifics and the many planktonic organisms have diverted each fish's attention from noise. Our work suggests that the top-down structuring influence of roach on planktonic communities might be resilient to noise and highlights how extrapolating impacts from individual responses to complex communities can be tricky.

Chronic Exposure to Environmentally Relevant Concentrations of Imidacloprid Impact Survival and Ecologically Relevant Behaviors of Fathead Minnow Larvae.

Imidacloprid (IM) has emerged as a contaminant of concern in several areas within the United States due to its frequent detection in aquatic ecosystems and its pseudo-persistence, which pose potential risks to nontarget species. We evaluated the sublethal toxicity of IM to fathead minnow larvae following chronic exposure beginning just after fertilization. Our in silico analysis and in vivo bioassays suggest that IM has a low binding affinity for the vertebrate nicotinate acetylcholine receptor (nAChR), as expected. However, chronic exposure to ≥0.16 µg IM/L reduced survival by 10%, and exposure to ≥18 µg IM/L reduced survival by approximately 20%-40%. Surviving fish exposed to ≥0.16 µg IM/L showed reduced growth, altered embryonic motor activity, and premature hatching. Furthermore, a significant proportion of fish exposed to ≥0.16 µg IM/L were slower to respond to vibrational stimuli and slower to swim away, indicating that chronic exposure to IM has the potential to impair the ability of larvae to escape predation. The adverse health effects we observed indicate that chronic exposure to environmentally relevant concentrations of IM may elicit sublethal responses that culminate in a significant increase in mortality during early life stages, ultimately translating to reduced recruitment in wild fish populations. Environ Toxicol Chem 2023;42:2184-2192. © 2023 SETAC.

Fish muscle mercury concentration and bioaccumulation fluctuate year-round - Insights from cyprinid and percid fishes in a humic boreal lake.

Boreal lakes demonstrate pronounced seasonality, where the warm open-water season and subsequent cold and ice-covered season dominate natural cycles. While fish muscle total mercury concentration (mg/kg) [THg] is well documented in open-water summer months, there is limited knowledge on the ice-covered winter and spring mercury dynamics in fish from various foraging and thermal guilds. This year-round study tested how seasonality influences [THg] and its bioaccumulation in three percids, perch (Perca fluviatilis), pikeperch (Sander lucioperca), ruffe (Gymnocephalus cernua), and three cyprinids, roach (Rutilus rutilus), bleak (Alburnus alburnus), and bream (Abramis brama) in deep boreal mesotrophic Lake Pääjärvi, southern Finland. Fish were sampled and [THg] was quantified in the dorsal muscle during four seasons in this humic lake. Bioaccumulation regression slopes (mean ± STD, 0.039 ± 0.030, range 0.013-0.114) between [THg] and fish length were steepest during and after spawning and shallowest during autumn and winter for all species. Fish [THg] was significantly higher in the winter-spring than summer-autumn in all percids, however, not in cyprinids. The lowest [THg] was observed in summer and autumn, likely due to recovery from spring spawning, somatic growth and lipid accumulation. Fish [THg] was best described by multiple regression models (R2adj: 52-76%) which included total length and varying combinations of seasonally changing environmental (water temperature, total carbon, total nitrogen, and oxygen saturation) and biotic factors (gonadosomatic index, and sex) in all species. The seasonal variation in [THg] and bioaccumulation slopes across multiple species suggests a need for standardized sampling seasons in long-term monitoring to avoid any seasonality bias. From the fisheries and fish consumption perspective in seasonally ice-covered lakes, monitoring of both winter-spring and summer-autumn would improve knowledge of [THg] variation in fish muscle.

Chronic Toxicity of Iron to Aquatic Organisms under Variable pH, Hardness, and Dissolved Organic Carbon Conditions.

A series of chronic toxicity tests was conducted exposing three aquatic species to iron (Fe) in laboratory freshwaters. The test organisms included the green algae Raphidocelis subcapitata, the cladoceran Ceriodaphnia dubia, and the fathead minnow Pimephales promelas. They were exposed to Fe (as Fe (III) sulfate) in waters under varying pH (5.9-8.5), hardness (10.3-255 mg/L CaCO3 ), and dissolved organic carbon (DOC; 0.3-10.9 mg/L) conditions. Measured total Fe was used for calculations of biological effect concentrations because dissolved Fe was only a fraction of nominal and did not consistently increase as total Fe increased. This was indicative of the high concentrations of Fe required to elicit a biological response and that Fe species that did not pass through a 0.20- or 0.45-µm filter (dissolved fraction) contributed to Fe toxicity. The concentrations frequently exceeded the solubility limits of Fe(III) under circumneutral pH conditions relevant to most natural surface waters. Chronic toxicity endpoints (10% effect concentrations [EC10s]) ranged from 442 to 9607 µg total Fe/L for R. subcapitata growth, from 383 to 15 947 µg total Fe/L for C. dubia reproduction, and from 192 to 58,308 µg total Fe/L for P. promelas growth. Toxicity to R. subcapitata was variably influenced by all three water quality parameters, but especially DOC. Toxicity to C. dubia was influenced by DOC, less so by hardness, but not by pH. Toxicity to P. promelas was variable, but greatest under low hardness, low pH, and low DOC conditions. These data were used to develop an Fe-specific, bioavailability-based multiple linear regression model as part of a companion publication. Environ Toxicol Chem 2023;42:1371-1385. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of developmental exposure to neurotoxic algal metabolites on predator-prey interactions in larval Pimephales promelas.

Harmful algal blooms are a growing environmental concern in aquatic systems. Although it is known that some of the secondary metabolites produced by cyanobacteria can alter predator-prey dynamics in aquatic communities by reducing foraging and/or predator evasion success, the mechanisms underpinning such responses are largely unknown. In this study, we examined the effects of a potent algal neurotoxin, ß-N-methylamino-L-alanine (BMAA), on the development and behavior of larval Fathead Minnows, Pimephales promelas, during predator-prey interactions. We exposed eggs and larvae to environmentally relevant concentrations of BMAA for 21 days, then tested subjects in prey-capture and predator-evasion assays designed to isolate the effects of exposure at sequential points of the stimulus-response pathway. Exposure was associated with changes in the ability of larvae to detect and respond to environmental stimuli (i.e., a live prey item and a simulated vibrational predator), as well as changes in behavior and locomotor performance during the response. Our findings suggest that chronic exposure to neurodegenerative cyanotoxins could alter the outcomes of predator-prey interactions in natural systems by impairing an animal's ability to perceive, process, and respond to relevant biotic stimuli.

The influence of flow velocity on the response of rheophilic fish to visual cues.

The strong association with visual cues exhibited by fish that prefer to inhabit flowing water (rheophilic species) may help reduce the energetic costs of maintaining position due to the provision of spatial points of reference. If this "Station Holding Hypothesis" is true, a positive relationship between the association with visual cues and flow velocity is expected. This hypothesis was tested experimentally by quantifying the response of common minnow (Phoxinus phoxinus) and brown trout (Salmo trutta) to visual cues under three flow velocities. In contradiction to the prediction, there was no evidence that the association with strong visual cues was positively related to flow velocity when fish were presented with vertical black stripes in an open channel flume, although interspecific variation in response was observed. The association with visual cues was relatively weak in trout, compared to minnow that spent 660% more time associated with the zone in which visual cues were present during the treatment, than the control when visual cues were absent. Trout tended to be more exploratory and made short visits to the area where visual cues were present, whereas minnow associated with the cues for longer. The strong association with visual cues independent of flow velocity exhibited by minnow and the weak association across all velocities by trout suggest that this behaviour is unlikely to reflect a strategy to minimise the energetic cost of maintaining position in flowing water. Minnow may have used the visual cues as a proxy indicator of physical structure that provides alternative benefits, such as refuge from predators. Trout may have employed alternative cues (e.g. mechanosensory) to seek more energetically favourable regions of the experimental area, reducing the importance of stationary visual stimuli.

Cost-Effective Pharmaceutical Implants in Fish: Validating the Performance of Slow-Release Implants for the Antidepressant Fluoxetine.

Internal, slow-release implants can be an effective way to manipulate animal physiology or deliver a chemical exposure over long periods of time without the need for an exogenous exposure route. Slow-release implants involve dissolving a compound in a lipid-based carrier, which is inserted into the body of an organism. However, the release kinetics of the compound from the implant to body tissues also requires careful validation. We tested and validated a slow-release implant methodology for exposing fish to a pharmaceutical pollutant, fluoxetine. We tested two lipid-based carriers (coconut oil or vegetable shortening) in the common roach (Rutilus rutilus). The implants contained either a high (50 µg/g), low (25 µg/g), or control (0 µg/g) concentration of fluoxetine, and we measured tissue uptake in the brain, muscle, and plasma of implanted fish over 25 days. The two carriers released fluoxetine differently over time: coconut oil released fluoxetine in an accelerating manner (tissue uptake displayed a positive quadratic curvature), whereas vegetable shortening released fluoxetine in a decelerating manner (a negative quadratic curvature). For both carrier types, fluoxetine was measured at the highest concentration in the brain, followed by muscle and plasma. By comparing the implant exposures with waterborne exposures in the published literature, we showed that the implants delivered an internal exposure that would be similar if fish were exposed in surface waters containing effluents. Overall, we showed that slow-release internal implants are an effective method for delivering chronic exposures of fluoxetine over at least 1-month time scales. Internal exposures can be an especially powerful experimental tool when coupled with field-based study designs to assess the impacts of pharmaceutical pollutants in complex natural environments. Environ Toxicol Chem 2023;42:1326-1336. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of hypoxia and reoxygenation on oxidative stress, histological structure, and apoptosis in a new hypoxia-tolerant variety of blunt snout bream (Megalobrama amblycephala).

A new hypoxia-tolerant variety of blunt snout bream was obtained by successive breeding of the wild population, which markedly improved hypoxia tolerance. In this study, the hypoxia-tolerant variety was exposed to hypoxia (2.0 mg O2·L-1) for 4, 7 days. The contents of blood biochemical indicators including the number of red blood cells (RBC), total cholesterol (T-CHO), total protein (TP), triglyceride (TG), glucose (GLU), and lactic acid (LD) increased significantly (P < 0.05) under hypoxia. The glycogen content in the liver and muscle decreased significantly (P < 0.05) and the LD content in the brain, muscle and liver increased significantly (P < 0.05) under hypoxia. The levels of oxidative stress-related indicators i.e., superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and total antioxidant capacity (T-AOC) also changed significantly (P < 0.05) in the heart, liver, and intestine of the new variety under hypoxia. Additionally, hypoxia has caused injuries to the heart, liver, and intestine, but it shows amazing repair ability during reoxygenation. The apoptotic cells and apoptosis rate in the heart, liver, and intestine increased under hypoxia. Under hypoxia, the expression of the B-cell lymphomas 2 (Bcl-2) gene in the heart, liver, and intestine was significantly (P < 0.05) down-regulated, while the expression of the BCL2-associated agonist of cell death (Bad) gene was significantly (P < 0.05) up-regulated. These results are of great significance for enriching the basic data of blunt snout bream new variety in response to hypoxia and promoting the healthy development of its culture industry.

Case Study in 21st-Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Reproductive Outcomes in Fish In Vivo.

To reduce the use of intact animals for chemical safety testing, while ensuring protection of ecosystems and human health, there is a demand for new approach methodologies (NAMs) that provide relevant scientific information at a quality equivalent to or better than traditional approaches. The present case study examined whether bioactivity and associated potency measured in an in vitro screening assay for aromatase inhibition could be used together with an adverse outcome pathway (AOP) and mechanistically based computational models to predict previously uncharacterized in vivo effects. Model simulations were used to inform designs of 60-h and 10-21-day in vivo exposures of adult fathead minnows (Pimephales promelas) to three or four test concentrations of the in vitro aromatase inhibitor imazalil ranging from 0.12 to 260 µg/L water. Consistent with an AOP linking aromatase inhibition to reproductive impairment in fish, exposure to the fungicide resulted in significant reductions in ex vivo production of 17ß-estradiol (E2) by ovary tissue (≥165 µg imazalil/L), plasma E2 concentrations (≥74 µg imazalil/L), vitellogenin (Vtg) messenger RNA expression (≥165 µg imazalil/L), Vtg plasma concentrations (≥74 µg imazalil/L), uptake of Vtg into oocytes (≥260 µg imazalil/L), and overall reproductive output in terms of cumulative fecundity, number of spawning events, and eggs per spawning event (≥24 µg imazalil/L). Despite many potential sources of uncertainty in potency and efficacy estimates based on model simulations, observed magnitudes of apical effects were quite consistent with model predictions, and in vivo potency was within an order of magnitude of that predicted based on in vitro relative potency. Overall, our study suggests that NAMs and AOP-based approaches can support meaningful reduction and refinement of animal testing. Environ Toxicol Chem 2023;42:100-116. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Chromium supplemented carbohydrate diets for Cirrhinus mrigala: effects on body composition, gut enzyme activity and hematological parameters / Dietas com carboidratos suplementados com cromo para Cirrhinus mrigala: efeitos sobre a composição corporal, atividade enzimática intestinal e parâmetros hematológicos

A ninety days nutritional trial was directed to explore the effects of dietary chromium on body composition, gut enzyme activity and physiological status of Cirrhinus mrigala by using G & NG corn. Six experimental diets were prepared by using different levels of chromium chloride hexahydrate (0, 0.2, 0.4 mg/kg, each with G & NG corn). For this experimental trial, 480 fingerlings, irrespective of sex were distributed in six aquariums each with replicate. Results revealed that gelatinized corn along with increasing level of Cr2Cl3.6H2O have a positive impact upon body composition of fish. Hematology was positively correlated with chromium chloride hexahydrate supplementation in gelatinized corn. Amylase gut enzyme also showed significant (P<0.05) increase in group fed with chromium chloride hexahydrate supplemented diet (G corn). However, corn with chromium chloride hexahydrate supplementation did not revealed any significant impact on gut protease enzyme activity. From these results it can be concluded that both chromium chloride hexahydrate and gelatinized corn in fish feed are very beneficial to improve body composition, enzymes activity and physiological health status of fish.

Um ensaio nutricional de 90 dias foi dirigido para explorar os efeitos do cromo dietético na composição corporal, atividade enzimática intestinal e estado fisiológico de Cirrhinus mrigala usando milho G & NG. Seis dietas experimentais foram preparadas usando diferentes níveis de cloreto de cromo hexa-hidratado (0, 0,2, 0,4 mg/kg, cada um com milho G e NG). Para este ensaio experimental, 480 alevinos, independentemente do sexo, foram distribuídos em seis aquários, cada um com réplicas. Os resultados revelaram que o milho gelatinizado, juntamente com o aumento do nível de Cr2Cl3.6H2O, tem um impacto positivo na composição corporal dos peixes. A hematologia foi positivamente correlacionada com a suplementação de cloreto de cromo hexa-hidratado em milho gelatinizado. A enzima amilase intestinal também apresentou aumento significativo (P <0,05) no grupo alimentado com dieta suplementada com cloreto de cromo hexa-hidratado (milho G). No entanto, o milho com suplementação de hexahidrato de cloreto de cromo não revelou nenhum impacto significativo na atividade da enzima protease intestinal. Desses resultados, pode-se concluir que tanto o cloreto de cromo hexa-hidratado quanto o milho gelatinizado na alimentação de peixes são muito benéficos para melhorar a composição corporal, a atividade de enzimas e o estado fisiológico de saúde dos peixes.

Avoidance of copper by fathead minnows (Pimephales promelas) requires an intact olfactory system.

Fish can detect and respond to a wide variety of cations in their environment, including copper. Most often fish will avoid copper during behavioural trials; however, fish may also show no response or an attraction response, depending on the concentration(s) used. While it may seem intuitive that the response to copper requires olfaction, there is little direct evidence to support this, and what evidence there is remains incomplete. In order to test if olfaction is required for avoidance of copper by fathead minnows (Pimephales promelas) copper-induced movement was compared between fish with an intact olfactory system and fish with induced anosmia. Fish in a control group or a mock-anosmic group avoided copper (approximately 10 µg/L or 62.7 nM copper sulphate) while anosmic fish did not. The evidence demonstrates that an intact olfactory system is required for copper sensing in fish.