Comparative Observation and Analysis of Preference Behavior Based on Three Types of Taxes and Locomotor Activity in the Goldfish, Carassius auratus.

Psychophysiological studies in vertebrates have focused on taxes as indicators of behavioral change. Actually, a considerable number of studies about anxiety-like and anti-anxiety-like behaviors involving geotaxis, scototaxis, and thigmotaxis have been conducted on fish. However, few analyses considering these behaviors based on taxes in fish have been conducted. Here, using goldfish, we measured the time spent in the bright or dark area of a horizontally long rectangular tank (HLRT), in the upper or lower area of a vertically long rectangular tank (VLRT), and in the central or edge area of a circular tank (CT), respectively, for the first 30 min and the last 30 min in a 3-h period after fish had been introduced to tanks. Dark, lower, and edge preference behaviors were observed for the first 30 min in all tanks. While dark and edge preference behaviors were maintained even for the last 30 min, the lower preference was lost. Swimming distance and the number of area crossings in each tank were also compared between the first 30 min and the last 30 min. Both decreased significantly or tended to decrease in the last 30 min in the HLRT and the CT, but no change was observed in the VLRT. These results suggest that, in goldfish, preference behavior is stable for a short time, and that environmental habituation may depend on the shape of the tank and the elapsed time.

Binary patch assessment by goldfish under safe and dangerous conditions.

Food resources can occur heterogeneously in space or time and differ in their abundances. A forager should be able to determine the value of a patch and choose optimally how to exploit it. However, patch choice and exploitation may be influenced by predation risk. Using the Giving up Densities (GUDs) technique, we evaluated goldfish patch assessment and choice in a binary patch choice experiment. We offered a pair of unequal food patches containing high and low food quantity. We quantified goldfish foraging behavior in the presence and absence of a predator. Goldfish groups equalized the GUDs in the two patches in safe environments but left higher GUDs in the rich patch under predation risk. The results suggest that goldfish can use both "patch assessment rule" and "fixed time rule" to exploit resource patches and trade off food and danger.

Ecological risk assessment of a contaminated stream using multi-level integrated biomarker response in Carassius auratus.

The goal of this study was to evaluate the adverse effects of wastewater effluents on freshwater crucian carp, Carassius auratus, inhabiting Sincheon stream using the integrated biomarker response (IBR) at the genotoxic (micronucleus [MN] test), oxidative stress (activity of catalase [CAT] and glutathione S-transferase [GST], and level of lipid peroxidation [LPO]), histopathological (degree of tissue changes [DTC]), and physiological (condition factor [CF] and liver somatic index [LSI]) levels. The CF and LSI were significantly (p < 0.05) enhanced in fish from downstream sites (DS1 and DS2) as compared to that of upstream (US) fish samples. Moreover, a significant increase in morphometric indices (DTC) was observed in C. auratus collected from downstream sites (p < 0.05) and histopathological responses showed the degree of pathogenicity in the order of liver > kidney > gills. The activities of CAT, GST, and LPO in fish from the DS1 and DS2 sites were notably increased in gills, liver, and kidney compared to that of fish from the US site. Additionally, the MN test level in C. auratus from the DS1 and DS2 were significantly increased (p < 0.05) when compared with that of the US site. Considering the higher bioaccumulation of Cd, Co, Cr, Mn, Ni, and Pb in gills, liver, and kidney of C. auratus collected from downstream sites compared to that of the upstream site (p < 0.05), the observed toxicity was likely attributable to metal accumulation. The multi-level IBR index was higher at the DS1 site (15.08) than at the DS2 (1.02) and the reference US (0.00) sites. Therefore, these findings demonstrated that wastewater effluent discharge induces significant DNA damage, oxidative stress, and tissue injuries in C. auratus and suggested that the multi-level IBR approach should be used to quantify these effects on fish in streams and rivers.

Thyroid disruption in male goldfish (Carassius auratus) exposed to leachate from a municipal waste treatment plant: Assessment combining chemical analysis and in vivo bioassay.

Several classes of thyroid-disrupting chemicals (TDCs) have been found in refuse leachate, but the potential impacts of leachate on the thyroid cascade of aquatic organisms are yet not known. In this study, we chemically analyzed frequently reported TDCs, as well as conducted a bioassay, to evaluate the potential thyroid-disrupting effects of leachate. We used radioimmunoassay to determine the effects of leachate exposure on plasma 3,3',5-triiodo-l-thyronine (T3), 3,3',5,5'-l-thyroxine (T4), and thyroid-stimulating hormone (TSH) levels in adult male goldfish (Carassius auratus). We also investigated the impacts of leachate treatment on hepatic and gonadal deiodinases [types I (D1), II (D2), and III (D3)] and gonadal thyroid receptor (TRα-1 and TRß) mRNA expressions by using real-time polymerase chain reaction. The results indicated the presence of five TDCs (bisphenol A, 4-t-octylphenol, di-n-butyl phthalate, di-n-octyl phthalate, and diethylhexyl phthalate); their mean concentrations in the leachate were 18.11, 2.76, 4.86, 0.21, and 9.16 µg/L, respectively. Leachate exposure induced plasma T3 and TSH levels in male fish, without influencing the plasma T4 levels. The highly elevated D2 mRNA levels in the liver were speculated to be the primary reason for the induction of plasma T3 levels. Disruption of thyroid functions by leachate was also suggested by the up-regulation of D1 and D2 as well as TRα-1 mRNA levels in the gonads. Prominent thyroid disruptions despite the very low TDC concentrations in the exposure media used in the bioassay strongly indicated the existence of unidentified TDCs in the leachate. Our study indicated the necessity of conducting in vivo bioassays to detect thyroid dysfunctions caused by leachate.

Physiological and condition-related traits in the gynogenetic-sexual Carassius auratus complex: different investments promoting the coexistence of two reproductive forms?

BACKGROUND: Carassius auratus complex is an extraordinary species complex including the diploid and polyploid forms exhibiting asexual and sexual reproduction modes. The coexistence of both forms in the same habitats is currently reported. The stable coexistence of asexual and sexual forms assumes some disadvantages for asexuals that balance the costs of sex. In our study, we hypothesized and tested the differences in physiological (including heamatological and immunological), growth-related, condition-related, and fitness-related traits between gynogenetic females and sexuals. RESULTS: Our results revealed similar growth performance in gynogenetic females and sexuals measured by body size and weight, or expressed by condition factor. The energy allocation in reproduction measured by the relative size of gonads revealed no difference between gynogenetic and sexual females; in addition, both females in spawning expressed the same estradiol levels in blood plasma. We found a gender specific trade-off between investment in reproduction and immunocompetence (measured by the spleen-somatic index). Higher aerobic performance expressed by the heart index and higher oxygen-carrying capacity were found in sexual males, with increasing values before and during spawning. Our study evidenced significantly lower aerobic performance but higher oxygen-carrying capacity per erythrocyte in gynogenetic females when compared to sexuals. IgM production differed between gynogens and sexuals of C. auratus complex. CONCLUSIONS: Our study indicates that a similar amount of energy is invested by both gynogenetic and sexual females of C. auratus complex in reproductive behaviour. We suggest that lower aerobic performance in gynogens may represent their physiological disadvantage balancing the cost of sexual reproduction. A trade-off between the number of erythrocytes and the oxygen-carrying capacity per erythrocyte in sexual males and gynogenetic females may contribute to the coexistence of gynogenetic and sexual forms. In addition, the differences in specific immunity between gynogens and sexuals may also reduce the evolutionary disadvantage of sexual reproduction. In conclusion, we propose that several mechanisms contribute to the coexistence of the gynogenetic-sexual C. auratus complex.

Antagonistic joint toxicity assessment of two current-use phthalates with waterborne copper in liver of Carassius auratus using biochemical biomarkers.

Di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) are two kinds of widely-used phthalates, whereas Cu (II) is a common valence state of copper. They have been ubiquitously detected in the aquatic environment, but information on their joint toxicity on aquatic organisms is scarce. In this study, we evaluated the combined effects of copper and these two phthalates to the goldfish (Carassius auratus) by detecting the antioxidant responses in liver after exposure for 7 and 21 days. The exposure concentrations were in a range relevant to their levels in the natural aquatic environment. The results indicated that DBP, DEHP and Cu (II) can affect the antioxidant status in fish liver, evidenced by the significant alterations of antioxidant defenses (superoxide dismutase, catalase, glutathione) and malondialdehyde. Antagonistic effects were found in the joint toxicity of Cu (II) and DBP or DEHP using the integrated biomarker response (IBR) index. These findings have important implications in the risk assessments of phthalates mixed with some heavy metals in the aquatic environment.

A multi-biomarker assessment of single and combined effects of norfloxacin and sulfamethoxazole on male goldfish (Carassius auratus).

In the present study, the sublethal effects of norfloxacin alone and in combination with sulfamethoxazole in goldfish (Carassius auratus) were investigated, the biomarkers including acetylcholinesterase (AChE) in brain, 7-ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST), and superoxides dismutase (SOD) activities in liver, vitellogenin (Vtg) in serum and DNA damage in gonad were determined after 1, 2, 4 and 7 days of exposure. Brain AChE activity was significantly inhibited by norfloxacin (≥0.4 mg/L) after 4 and 7 days and the mixtures with sulfamethoxazole (≥0.24 mg/L) after 4 days of exposure, and significant concentration-response relationships were obtained. Liver EROD, GST and SOD activities were significantly increased by the individual and mixed pharmaceuticals in most cases and exhibited analogously bell-shaped concentration-response curves. Serum Vtg was increased by the highest concentration of norfloxacin and two higher concentrations of the mixtures. Higher concentrations of the test antibiotics induced significant DNA damage in a concentration- and time-dependent manner. The results indicated that selected antibiotics possesses cytotoxic and genotoxic potential against the non-target organism C. auratus.

Assessment of in vivo estrogenic response and identification of environmental estrogens in influent and effluent from a sewage treatment plant.

The in vivo estrogenic response and estrogenic contents of the influent and effluent collected from a sewage treatment plant located in Jiaozuo were assessed. The bioassay showed significant serum vitellogenin (VTG) induction in all the treated male goldfish (Carassius auratus) and significant gonad atrophies were only observed in the fish induced the most VTG expressions. Six target estrogens (estrone, 17ß-estradiol, 17α-ethynylestradiol, 4-n-octylphenol, 4-n-nonylphenol and bisphenol A) were detected in different polar fractions, with the exception of the 25 % and 50 % methanol fractions extracted from the influent and the 25 %, 50 %, 95 % and 100 % methanol fractions extracted from the effluent. For both the influent and effluent, natural and synthetic steroidal estrogens were detected in those extracted fractions induced the most abundant VTG expressions.

Ecotoxicological effects of waterborne PFOS exposure on swimming performance and energy expenditure in juvenile goldfish (Carassius auratus).

The potential risks of perfluorooctane sulfonate (PFOS) are of increasing ecological concern. Swimming performance is linked to the fitness and health of fish. However, the impacts of PFOS on swimming performance remain largely unknown. We investigated the ecotoxicological effects of acute exposure to PFOS on the swimming performance and energy expenditure of juvenile goldfish (Carassius auratus). The fish were exposed to a range of PFOS concentrations (0, 0.5, 2, 8 and 32 mg/L) for 48 hr. The spontaneous swimming activity, fast-start swimming performance, critical swimming speed (U(crit)) and active metabolic rate (AMR) of the goldfish were examined after exposure to PFOS. PFOS exposure resulted in remarkable effects on spontaneous activity. Motion distance was reduced, and the proportion of motionless time increased with increasing concentrations of PFOS. However, no significant alterations in the fast-start performance-related kinematic parameters, such as latency time, maximum linear velocity, maximum linear acceleration or escape distance during the first 120 msec after stimulus, were observed after PFOS exposure. Unexpectedly, although PFOS exposure had marked influences on the swimming oxygen consumption rates and AMR of goldfish, the U(crit) of the goldfish was not significantly affected by PFOS. This may result in a noteworthy increase in the energetic cost of transport. The overall results indicate that, in contrast to spontaneous activity, underlying swimming capabilities are maintained in goldfish after short-term exposure to PFOS, but energy expenditure during the process of swimming is dramatically aggravated.

Assessment of the health status of wild fish from the Wheatley Harbour Area of Concern, Ontario, Canada.

The overall health and endocrine function of wild brown bullhead (Ameiurus nebulosus) and goldfish (Carassius auratus) from the Wheatley Harbour Area of Concern (Lake Erie, Ontario, Canada) was assessed using a suite of physiological and biochemical endpoints. Smaller gonads were detected in female brown bullhead and goldfish from Wheatley Harbour compared with Hillman Marsh (Ontario, Canada) reference fish. Female brown bullhead exhibited decreased in vitro synthesis of 17ß-estradiol. Female goldfish had decreased plasma vitellogenin concentrations. Plasma testosterone and 11-ketotestosterone were significantly depressed in males of both species. Perturbations in the thyroid status were detected, but varied between sexes and species. Observed differences included lower plasma concentrations of thyroid hormones and/or elevated liver deiodinase activity. Histological evaluation of the thyroid tissue indicated that in the case of female goldfish, those perturbations stimulated the thyroid (as indicated by increased thyroid epithelial cell height) and partially depleted the thyroxine reserves, as indicated by decreased colloid and elevated thyroid activation index. Increased mixed-function oxygenase activity in brown bullhead from Wheatley Harbour was consistent with exposure to planar aromatic contaminants. A principal component analysis of selected variables showed the separation of fish by collection site. The endpoints most strongly associated with the separation were generally those exhibiting significant differences between sites. The results of the present study indicate that the health of fish populations within Wheatley Harbour warrants continued attention.

Toxicity assessment on three direct dyes (D-BLL, D-GLN, D-3RNL) using oxidative stress bioassay and quantum parameter calculation.

Textile dyes and dye industrial effluents are widely known for esthetic and toxicity problems. The toxicity of three direct dyes, Direct Blending Rebine (D-BLL), Direct Blending Scarlet (D-GLN), and Direct Blending Yellow (D-3RNL), were examined by the antioxidase and lipid peroxide index. Fish (Carassius auratus) were exposed to 100mg/L test compounds or injected with 200µg/kg corresponding dyes, and then samples of liver were collected at different times (0.5, 1, 3, 5, 7, 10, 13, 17 and 22d ) for analysis of superoxide dismutase (SOD), catalase (CAT), and contents of malondialdehyde (MDA). There is an obvious difference between two poisoning conditions and results indicated injection pattern have a more sensitive response. Besides, SOD, CAT and MDA levels displayed different variation trend following the prolonged duration, implying that dye metabolism generated less toxic or more active substance. The comparison among their intensity of enzyme inhibition showed that the toxicity order is D-BLL>D-GLN>D-3RNL. Additionally, three direct dye molecules were optimized based on the quantum mechanical charge density of a solute molecule interacting with a continuum description of the solvent (SMD) of Self-consistent Reaction Field Theory (SCRF) on B3LYP/LAN2BM level and the stable configurations were obtained. Wiberg bond orders were analyzed and atom in molecule (AIM) 2000 program was employed to estimate the interaction between atoms. The possible degradation pathways and toxicities were speculated based on the computations. The calculation is consistent with the experimental results and analysis.

An integrated assessment of estrogenic contamination and feminization risk in fish in Taihu Lake, China.

An integrated approach, combining biological and chemical methods, was used to assess potential exposure to exogenous estrogens and their possible interference with the endocrine system of male goldfish (Carassius auratus) in Taihu Lake. A suite of biomarkers in caged fish after in situ exposure for 28 days, coupled with six selected exogenous estrogens in water, were determined at eight biomonitoring stations. The compounds estrone, 17ß-estradiol (E2), estriol, 17α-ethinylestradiol (EE2), bisphenol-A and diethylstilbestrol were detected in most of the samples and the concentrations of total estradiol equivalent (EEQ) ranged between 5.69 and 17.8 ng/L. E2 and EE2 were thought to be the major causal agents responsible for the estrogenic activities. Elevated serum vitellogenin and E2, gonadal DNA damage and reduced gonadosomatic index were observed in fish collected from most stations. The integrated biomarker response (IBR) index showed good agreement with the observed total EEQ levels in water, and feminization risk in fish may be present in northern Taihu Lake, especially in Meiliang Bay and Zhushan Bay.

Assessment of lateral line function: a potential technique for studies in ototoxicity.

In zebrafish neuromast hair cells, the process of programmed cell death ototoxic damage is strikingly similar to that of degenerating hair cells of the mammalian organ of Corti. Therefore, in vivo zebrafish assays involving the lateral line have been developed for drug ototoxicity screening. This is accomplished by examination of canal neuromast morphology in treated larvae using fluorescent dyes. To-date however, physiological confirmation of lateral line dysfunction resulting from such ototoxins has not been reported in the scientific literature--neither for larval nor adult zebrafish. Here we describe a rapid, non-invasive far-field electrophysiological method for assessing lateral line function. We suggest that ototoxic and otoprotective agents identified in larval studies may be assessed using this tool in adult fish. In this way, potential drug candidates can be further screened en route to testing in mammalian models, before potential clinical trials begin.

Assessment of estrogenic endocrine-disrupting chemical actions in the brain using in vivo somatic gene transfer.

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element-thymidine kinase-luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17ss-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 microg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Toxicity of boron to rainbow trout: a weight-of-the-evidence assessment.

From the large data set available on the toxicity of boron to aquatic organisms, the toxicity of boron to the early life stages of rainbow trout (Oncorhyncus mykiss) is the seminal issue relative to setting water quality criteria and effluent standards. Issues associated with the early life stage studies are the flat concentration-response curve, the low threshold of toxicity, and teratogenic effects observed. Recent laboratory and field studies offer new experimental data that make a weight-of-the-evidence assessment timely. In a re-examination of the effect of boron on the embryo-larval stage in rainbow trout and zebrafish, adverse effects due to boron deficiency are observed which decrease with increasing dose. It was found that low concentrations of boron stimulate embryonic growth in rainbow trout and increase the viability and survival of embryonic zebrafish. As boron concentration is further increased, the dose-response curve becomes flat as homeostatic processes are active; this is followed at higher doses by a new adverse response that increases with increasing dose. As a result, the dose-response relationship is U shaped, consistent with the characteristic shape of an essential micronutrient. Thus, effects originally reported to be toxicity at low exposures rather may be due to boron deficiency. Water analyses in trout hatcheries and field studies in wild trout streams add additional information on the toxicity of boron to trout. Of particular note is a controlled field study carried out in the Firehole River in Yellowstone Park (WY, USA), where trout populations survive and reproduction successfully occurs in natural water containing boron concentrations up to and in some cases greater than 1.0 mg B/L. Teratogenic effects due to boron exposure were not observed in any of these more recent studies.