An Automated Low-Cost Swim Tunnel for Measuring Swimming Performance in Fish.

The study of swimming behavior is an important part of fish biology research and the swim tunnel is used to study swimming performance as well as metabolism of fish. In this investigation, we have developed a user-friendly, automated, modular, and low-cost swim tunnel that permits to study the performance of one or more fish separately, as well as a small group of individuals. To validate our swim tunnel, we assessed swimming activity of four different species (zebrafish, medaka, guppy, and cavefish) recording reliable data of swimming behavior and performance. Because swimming behavior has been recently used in different fields from physiology to ecotoxicology, our setup could help researchers with a low-cost solution.

Assessment of reproductive and developmental effects of graphene oxide on Japanese medaka (Oryzias latipes).

Due to its unique properties, graphene oxide (GO) has potential for biomedical and electronic applications, however environmental contamination including aquatic ecosystem is inevitable. Moreover, potential risks of GO in aquatic life are inadequately explored. Present study was designed to evaluate GO as an endocrine disrupting chemical (EDC) using the model Japanese medaka (Oryzias latipes). GO was injected intraperitoneally (25-200 µg/g) once to breeding pairs and continued pair breeding an additional 21 days. Eggs laid were analyzed for fecundity and the fertilized eggs were evaluated for developmental abnormalities including hatching. Histopathological evaluation of gonads, liver, and kidneys was made 21 days post-injection. LD50 was found to be sex-dependent. Fecundity tended to reduce in a dose-dependent manner during early post-injection days; however, the overall evaluation showed no significant difference. The hatchability of embryos was reduced significantly in the 200 µg/g group; edema (yolk and cardiovascular) and embryo-mortality remained unaltered. Histopathological assessment identified black particles, probably agglomerated GO, in the gonads of GO-treated fish. However, folliculogenesis in stromal compartments of ovary and the composition of germinal elements in testis remained almost unaltered. Moreover, granulosa and Leydig cells morphology did not indicate any significant EDC-related effects. Although liver and kidney histopathology did not show GO as an EDC, some GO-treated fish accumulated proteinaceous fluid in hepatic vessels and induced hyperplasia in interstitial lymphoid cells (HIL) located in kidneys. GO agglomerated in medaka gonads after 21-days post-injection. However, gonad histopathology including granulosa and Leydig cells alterations were associated with GO toxicity rather than EDC effects.

Toxicity assessment of pollutants sorbed on environmental microplastics collected on beaches: Part II-adverse effects on Japanese medaka early life stages.

While microplastics are present in great abundance across all seas and oceans, little is known about their effects on marine life. In the aquatic environment, they can accumulate a variety of chemicals and can be ingested by many marine organisms including fish, with chronic physical and chemical effects. The purpose of this paper is to evaluate the toxic effects of pollutants sorbed at the surface of environmental microplastics (MPs), collected on various beaches from three islands of the Pacific Ocean. Developmental toxicity of virgin MPs or artificially coated with B[a]P and environmental MPs from Easter Island, Guam and Hawaii was evaluated on embryos and prolarvae of Japanese medaka. Mortality, hatching success, biometry, malformations, EROD activity and DNA damage were analyzed after exposure to DMSO extracts. No toxicity was observed for extracts of virgin MPs whatever the endpoint considered. Extracts of virgin MPs coated with 250 µg.g-1 of B(a)P induced lethal effects with high embryo mortality (+81%) and low hatching rate (-28%) and sublethal effects including biometry and swimming behavior changes, increase of EROD activity (+94%) and DNA damage (+60%). Environmental MPs collected on the three selected islands exhibited different polymer, pollutant and toxicity patterns. The highest toxicity was detected for MPs extract from Hawaï with head/body length and swimming speed decreases and induction of EROD activity and DNA stand breaks. This study reports the possible sublethal toxicity of organic pollutants sorbed on MPs to fish early life stages.

Medaka (Oryzias latipes) Multigeneration Test with Triclosan.

The medaka extended one-generation reproduction test (MEOGRT) is a tier-2 study in the US Environmental Protection Agency's Endocrine Disruptor Screening Program and a level-5 study in the Organisation for Economic Co-operation and Development's conceptual framework. Integrating nonspecific apical and endocrine-specific mechanistic endpoints, results of a MEOGRT can be used, with other data, in a weight-of-evidence evaluation to establish a dose-response relationship for risk assessment and identify potential causal relationships between an endocrine mode of action and adverse effects. The MEOGRT test design was used to evaluate the multigenerational effects of the antimicrobial agent triclosan. Japanese medaka were exposed to nominal concentrations of 1.4, 2.8, 5.6, 11, and 23 µg/L triclosan and a dilution water control starting with adult medaka (F0) through hatch in the second generation (F2). No consistent or concentration-related responses occurred in the 182-d test that suggested an endocrine-mediated effect. There were no impacts on hepatic vitellogenin, secondary sex characteristics, or sex ratio that were linked to an adverse reproductive outcome. Histopathological responses were consistent with a toxic or stress effect, particularly when considered in context with observed reductions in growth. The overall population-relevant no-observed-effect concentration was 11 µg/L based on effects on growth. The results of the present study support a previously conducted weight-of-evidence evaluation concluding that triclosan does not act as an agonist or antagonist within estrogen, androgen, thyroid, or steroidogenic pathways. Environ Toxicol Chem 2019;38:1770-1783. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Summary of the development the US Environmental Protection Agency's Medaka Extended One Generation Reproduction Test (MEOGRT) using data from 9 multigenerational medaka tests.

In response to various legislative mandates, the US Environmental Protection Agency (USEPA) formed its Endocrine Disruptor Screening Program (EDSP), which in turn, formed the basis of a tiered testing strategy to determine the potential of pesticides, commercial chemicals, and environmental contaminants to disrupt the endocrine system. The first tier of tests is intended to detect the potential for endocrine disruption mediated through estrogen, androgen, or thyroid pathways, whereas the second tier is intended to further characterize the effects on these pathways and to establish a dose-response relationship for adverse effects. One of these tier 2 tests, the Medaka Extended One Generation Reproduction Test (MEOGRT), was developed by the USEPA for the EDSP and, in collaboration with the Japanese Ministry of the Environment, for the Guidelines for the Testing of Chemicals of the Organisation for Economic Co-operation and Development (OECD). The MEOGRT protocol was iteratively modified based on knowledge gained after the successful completion of 9 tests with variations in test protocols. The present study describes both the final MEOGRT protocol that has been published by the USEPA and the OECD, and the iterations that provided valuable insights into nuances of the protocol. The various tests include exposure to 17ß-estradiol, 4-t-octylphenol, o,p'- dichlorodiphenyltrichloroethane, 4-chloro-3-methylphenol, tamoxifen, 17ß-trenbolone, vinclozolin, and prochloraz. Environ Toxicol Chem 2017;36:3387-3403. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Assessment of in vitro antiovulatory activities of nonsteroidal anti-inflammatory drugs and comparison with in vivo reproductive toxicities of medaka (Oryzias latipes).

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used therapeutic agents; however, their pharmacological actions raise concerns about potential risks to the reproductive health of aquatic vertebrates. In the present study, a medaka ovulation assay was applied as an in vitro model to evaluate NSAID-induced antiovulatory activity. We first tested five NSAIDs, including diclofenac sodium (DCF), ketoprofen (KP), salicylic acid (SA), mefenamic acid (MA), and acetylsalicylic acid (ASA) for their antiovulatory activities toward the follicles isolated from the ovaries of spawning females. Of all the chemicals tested, DCF had the highest antiovulatory activity, with the concentration that caused 50% inhibition (IC50) (101 µM). MA was the second most potent inhibitor following DCF, but KP, SA, or ASA had little inhibitory effect on the ovulation of the follicles. The in vitro antiovulatory activity of five NSAIDs showed good correlation with data published on the inhibitory activity on human COX-2. Second, we selected DCF and SA as the most and least potent NSAIDs, respectively, and examined the effects on reproduction of intact fish in order to evaluate whether the ovulation assay was a reasonable predictor of potential reproductive effects in fish. Females exposed to DCF showed a concentration-dependent decrease in the number of spawned eggs and an increment in the gonadosomatic index (GSI), possibly due to an anovulation in the females. In contrast, neither fecundity nor the GSI of females decreased at up to 20 mg/L of SA, at which acute lethality to medaka was induced. In conclusion, the medaka ovulation assay reflected the potency of NSAID-induced antiovulatory activity and may thus serve as an in vitro model for the prediction of NSAID-induced reproductive toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1710-1719, 2016.

Workshop report: The medaka model for comparative assessment of human disease mechanisms.

Results of recent studies showing the utility of medaka as a model of various human disease states were presented at the 7th Aquatic Models of Human Disease Conference (December 13-18, 2014, Austin, TX). This conference brought together many of the most highly regarded national and international scientists that employ the medaka model in their investigations. To take advantage of this opportunity, a cohort of established medaka researchers were asked to stay an extra day and represent the medaka scientific community in a workshop entitled "The Medaka Model for Comparative Assessment of Human Disease Mechanisms." The central purpose of this medaka workshop was to assess current use and project the future resource needs of the American medaka research community. The workshop sought to spur discussions of issues that would promote more informative comparative disease model studies. Finally, workshop attendees met together to propose, discuss, and agree on recommendations regarding the most effective research resources needed to enable US scientists to perform experiments leading to impacting experimental results that directly translate to human disease. Consistent with this central purpose, the workshop was divided into two sessions of invited speakers having expertise and experience in the session topics. The workshop hosted 20 scientific participants (Appendices 1 and 2), and of these, nine scientists presented formal talks. Here, we present a summary report stemming from workshop presentations and subsequent round table discussions and forward recommendations from this group that we believe represent views of the overall medaka research community.

A new spiked sediment assay using embryos of the Japanese medaka specifically designed for a reliable toxicity assessment of hydrophobic chemicals.

Despite their low water solubility, hydrophobic pollutants are widespread in the aquatic environment and could represent a threat for living organisms. EU regulations on chemicals require accurate and reliable data on chemical toxicity. Current normalised fish toxicity assays, in particular those advocated by OECD guidelines, do not allow reliable toxicity assessment of hydrophobic compounds due to their low water solubility. In order to accurately evaluate the toxicity of this kind of compounds, a new spiked sediment assay using embryos of the Japanese medaka was developed. It consists of directly exposing fertilised eggs, during their entire embryonic development, onto the reference sediment spiked with the test compound. A large set of lethal or sublethal effects in embryos and newly hatched larvae, including non-invasive endpoints is analysed in order to maximise the sensitivity of the test. The approach was validated using four model pollutants with different modes of action: DMBA, PCB126, PCB153 and 4-nonylphenol (NP). All compounds, except PCB153, induced a dose-dependent increase in toxic effects. In fact, lethal effects only occurred at the highest tested concentration. In contrast, sub-lethal effects including skeletal deformations, cardiac activity modulation, body length reduction and hatching delay were observed at low to moderate concentrations of DMBA and PCB126. NP induced subtle effects in embryos, altering cardiac activity and hatching success but only at high concentrations. Although a few more improvements would make it a fully standardised assay, this spiked sediment assay using medaka embryos proves to be sensitive enough to measure hydrophobic chemical toxicity using an environmentally realistic mode of exposure.

Full life-cycle assessment of gene flow consistent with fitness differences in transgenic and wild-type Japanese medaka fish (Oryzias latipes).

Transgenic fish in development for aquaculture could escape from farms and interbreed with wild relatives in the nearby environment. Predicting whether escapes would result in transgene introgression is a major challenge in assessing environmental risks of transgenic fish. Previous studies have simulated gene flow from transgenic fish using mathematical modeling of fitness traits to predict the relative selective value of transgenic genotypes. Here, we present the first study of gene flow over the full life cycle in openly-breeding populations of transgenic animals, along with measurement of fitness traits. We conducted two invasion experiments in which we released two lines of growth-enhanced transgenic fish (T67 and T400), Japanese medaka (Oryzias latipes), into populations of wild-type (W) medaka in structured mesocosms. After several generations, the frequency of transgenic fish varied across replicates in the first invasion experiment (6 months), but the frequency of transgenic fish decreased in the second experiment (19 months). We also measured selected fitness traits in transgenic and wild-type medaka because these traits could be used to predict the relative selective value of a genotype. We found that: T400 males were more fertile than W males; offspring of W females lived longer than those with transgenic mothers; and W and T67 females reached sexual maturity sooner than T400 females. In contrast with other research that reported larger transgenic males had a mating advantage, we found that W males obtained more matings with females than T males; genetic background effects may account for our differing results as we compared W and T fish derived from different strains. The decreasing frequency of transgenic fish in the second invasion experiment suggests that transgenic fish had a selective disadvantage in the experimental environment. Our finding of transgenic advantage of some fitness traits and wild-type advantage in others is consistent with our invasion experiment results.

Hazard assessment of commonly used agricultural antibiotics on aquatic ecosystems.

In this study, eleven commonly used antibiotics including sulfonamides, tetracyclines, aminoglycosides, fluoroquinolones, and beta-lactams were evaluated for their acute and chronic aquatic toxicities using standard test organisms e.g., Vibrio fischeri, Daphnia magna, Moina macrocopa, and Oryzias latipes. Among the antibiotics tested for acute toxicity, neomycin was most toxic followed by trimethoprim, sulfamethoxazole and enrofloxacin. Sulfamethazine, oxytetracycline, chlortetracycline, sulfadimethoxine and sulfathiazole were of intermediate toxicity, while ampicillin and amoxicillin were least toxic to the test organisms. There were no trends in sensitivity among test organisms or among different classes of the antibiotics. Only the beta-lactam class was the least toxic. In chronic toxicity test, neomycin affected reproduction and adult survival of D. magna and M. macrocopa with low mg/l levels exposure. Predicted no effect concentrations (PNECs) were derived from the acute and chronic toxicity information gleaned from this study and from literature. When the PNECs were compared with measured environmental concentrations (MECs) reported elsewhere for the test compounds, hazard quotients for sulfamethoxazole, sulfathiazole, chlortetracycline, oxytetracycline, and amoxicillin exceeded unity, which suggests potential ecological implication. Therefore, further studies including monitoring and detailed toxicological studies are required to assess potential ecological risk of these frequently used veterinary antibiotics.

Short-term responses of Oryzias latipes (Pisces: Adrianichthyidae) and Macrobrachium nipponense (Crustacea: Palaemonidae) to municipal and pharmaceutical waste water in Beijing, China: survival, behaviour, biochemical biomarkers.

Whole effluent toxicity was assessed for the fish Oryzias latipes and the prawn Macrobrachium nipponense for 18 h in a dilution series (0-66%) of the inflow and effluent of a municipal waste water treatment plant as well as waste water from a teramycin producing pharmaceutical industry, before, during and after a pilot laboratory purification process. The waste waters caused acute toxicity as measured by inhibition of light emission in the luminiscent bacterium Vibrio qingaiensis sp. nov. (Q67). EROD and aryl hydrocarbon hydroxylase activity in in vitro carp liver-cells showed a dose-dependent toxic response to the municipal waste water. Behavioural responses and time-to-death of fish and prawn, recorded online with the "Multispecies Freshwater Biomonitor" proved to be concentration- and time-dependent sensitive toxicity indicators in both types of waste water. Behaviour changed stepwise from normal activity to (increased or decreased) activity to more time spent on ventilation and finally to increased morbidity at higher concentration and time of exposure. The municipal waste water treatment plant managed to reduce toxicity to bacteria (Q67), prawn and fish. The pharmaceutical waste water treatment process still has to be improved, in order to reduce toxicity for fish and prawn.

Assessment of thyroid function in adult medaka (Oryzias latipes) and juvenile rainbow trout (Oncorhynchus mykiss) using immunostaining methods.

The effect of injected bovine TSH on the pattern of anti-T(4) and anti-T(3) immunostaining of the thyroid tissue was examined in rainbow trout (Oncorhynchus mykiss) and medaka (Oryzias latipes) to determine if the previously reported immunostaining of the cytoplasm of thyrocytes is due to the presence of colloid pinocytotic vesicles and is thus indicative of thyroid hormone release. We hypothesized that the number of immunostained thyrocytes should increase following a TSH challenge, and this should parallel other indicators of increased thyroid activity. In medaka, immunostained thyrocytes were only found following the TSH challenge, and were most marked after 24 to 72 hours; the immunostaining was associated with large colloid-filled cytoplasmic vesicles. In trout, the number and staining intensity of immunostained thyrocytes were increased after the TSH challenge; the immunostaining was present throughout the cytoplasm of the thyrocytes. These findings support the working hypothesis that the immunostaining of the thyrocytes is associated with the pinocytosis of thyroglobulin by the thyrocytes in parallel with an increase in release of thyroid hormone, and that this investigational approach provides a reliable indicator of thyroid hormone release activity.

Environmental complex mixture toxicity assessment.

Trichloroethylene (TCE) was found as a contaminant in the well supplying water to an aquatic testing laboratory. The groundwater was routinely screened by a commercial laboratory for volatile and semivolatile compounds, metals, herbicides, pesticides, and polychlorinated biphenyls using U.S. Environmental Protection Agency methods. Although TCE was the only reportable peak on the gas chromatograph, with average concentrations of 0.200 mg/l, other small peaks were also present, indicating the possibility that the contamination was not limited to TCE alone. A chronic 6-month carcinogenicity assay was conducted on-site in a biomonitoring trailer, using the Japanese medaka fish (Oryzias latipes) in an initiation-promotion protocol, with diethylnitrosamine (DEN) as the initiator and the TCE-contaminated groundwater as a promoter. Study results indicated no evidence of carcinogenic potential of the groundwater without initiation. There was, however, a tumor-promotional effect of the groundwater after DEN initiation. A follow-up laboratory study was conducted using reagent grade TCE added to carbon-filtered groundwater to simulate TCE concentrations comparable to those found in the contaminated groundwater. Study results indicated no promotional effects of TCE. These studies emphasize the necessity for on-site bioassays to assess potential environmental hazards. In this instance, chemical analysis of the groundwater identified TCE as the only reportable contaminant, but other compounds present below reportable limits were noted and may have had a synergistic effect on tumor promotion observed with the groundwater exposure. Laboratory toxicity testing of single compounds can produce toxicity data specific to that compound for that species but cannot take into account the possible toxic effects of mixtures of compounds.