Combined effect of anthracene and polyethylene microplastics on swimming speed and cytochrome P4501A monooxygenase expression of Java medaka (Oryzias javanicus).

Microplastics have been detected in a variety of aquatic ecosystems, and the combined effect of microplastics and chemical pollutants has become a matter of increasing concern. We conducted a 12-d co-exposure test of anthracene and spherical or fragmented polyethylene microplastics (size 200 µm) on Java medaka (Oryzias javanicus). The accumulation of anthracene in Java medaka muscle reached a plateau on day 5 in all anthracene exposure groups, and no significant differences were detected among the groups (ANT, 20.4 ± 5.5; ANT + SPPE-MP, 24.7 ± 2.7; ANT + FRPE-MP, 24.6 ± 4.7 µg/g). However, co-exposure to anthracene and spherical or fragmented polyethylene microplastics increased the duration of slow swimming in a swimming behavior test (control, 4.1 ± 1.4; ANT, 5.2 ± 2.8; ANT + SPPE-MP, 12.4 ± 3.7; ANT + FRPE-MP, 17.4 ± 5.1 min/30 min), and co-exposure to anthracene and fragmented polyethylene microplastics induced higher cytochrome P4501A monooxygenase (CYP1A) expression in Java medaka livers than the other anthracene exposure groups (ANT, 189 ± 74; ANT + SPPE-MP, 203 ± 75; ANT + FRPE-MP 272 ± 36% of control). Polyethylene microplastics appear to be weak vectors of anthracene at the size tested (200 µm), and the effect of shape (spherical or fragmented) on the vector effect was small. However, the presence of polyethylene microplastics could affect the swimming behavior and CYP1A expression in Java medaka.

Peek-A-Boo Test: A Simple Test for Assessing the Effect of Anxiolytics on Fish Behavior.

The potential of pharmaceuticals and personal care products to alter the behavior of aquatic organisms is a growing concern. To assess the actual effect of these substances on aquatic organisms, a simple but effective behavioral test is required. We devised a simple behavioral (Peek-A-Boo) test to assess the effect of anxiolytics on the behavior of a model fish (medaka, Oryzias latipes). In the Peek-A-Boo test, we investigated the response of medaka to an image of a predator fish (donko fish, Odontobutis obscura). The test revealed that the time taken for test medaka exposed to diazepam (0.8, 4, 20, or 100 µg/L) to approach the image was shorter by a factor of 0.22 to 0.65, and the time spent in the area close to the image was longer by a factor of 1.8 to 2.7 than in the solvent control group for all diazepam exposure groups (p < 0.05). Hence, we confirmed that the test could detect changes in medaka behavior caused by diazepam with high sensitivity. The Peek-A-Boo test we devised is a simple behavioral test with high sensitivity for fish behavioral alteration. Environ Toxicol Chem 2023;42:2358-2363. © 2023 SETAC.

Combined effect of diazepam and polystyrene microplastics on the social behavior of medaka (Oryzias latipes).

The combined effect of microplastics and pharmaceuticals on aquatic organisms is an issue of concern. In this laboratory study, we evaluated the combined effect of polystyrene microplastics (2-µm diameter) and diazepam on the social behavior of medaka (Oryzias latipes) by using the shoaling behavior test with five treatment groups: solvent control, polystyrene microplastics exposure (0.04 mg/L), low-concentration diazepam exposure (0.03 mg/L), high-concentration diazepam exposure (0.3 mg/L), and polystyrene microplastics and low-concentration diazepam co-exposure. After 7 days of exposure, the shoal-leaving behavior of the high-concentration diazepam exposure group (8.9 ± 8.3 counts/medaka) and the co-exposure group (6.8 ± 6.7 counts/medaka) was significantly greater than that in the solvent control group (1.8 ± 2.6 counts/medaka). Even after 5 days of recovery, medaka in the co-exposure group left the shoal more often (7.3 ± 5.0 counts/medaka) than those in the solvent control group (2.6 ± 2.6 counts/medaka), whereas the shoal-leaving behavior in other exposure groups, except for the high-concentration diazepam exposure group, was restored. Our findings show that the combined effects of diazepam and polystyrene microplastics suppressed medaka social behavior, suggesting that the presence of microplastics can enhance the adverse effects of pollutants on the social behavior of aquatic organisms.

Effects of water temperature and light intensity on the acute toxicity of herbicide thiobencarb to a green alga, Raphidocelis subcapitata.

The present study investigated how principal environmental factors such as temperature and light intensity change the toxicological properties of thiobencarb (TB) herbicide to the green alga, Raphidocelis subcapitata. At first, we investigated the inhibitory effect of TB (0, 15.6, 31.2, 62.4, and 125 µg L-1) on growth of R. subcapitata at five temperatures (10, 15, 20, 25, or 30 °C) for 144 h exposure and calculated 72- and 144-h effective concentration values (EC10, 20, and EC50) for growth rate. All EC values significantly decreased with an increasing temperature. The maximum quantum yield of photosystem II in R. subcapitata exposed to 125 µg L-1 of TB was also significantly inhibited with increased temperature. These physiological effects could explain the lower EC values at high temperatures. Then, single and interactive effects of TB, temperature, and light intensity on growth rate were investigated by three-way of analysis of variance. As a result, single and interactive effects were detected in all explanatory variables. These results suggest that temperature and light intensity change the acute toxicity parameter in R. subcapitata exposed to TB and must be considered in evaluating the risk of TB.

Short-term and persistent impacts on behaviors related to locomotion, anxiety, and startle responses of Japanese medaka (Oryzias latipes) induced by acute, sublethal exposure to chlorpyrifos.

Although most exposures to chlorpyrifos (CPF) in natural flowing waters are brief and episodic, there have been a few reports of the persistence of abnormal fish behaviors caused by such acute exposure. The present study focused on the behavioral and biochemical responses of Japanese medaka (Oryzias latipes) to acute, sublethal exposure to CPF, as well as the persistence of the effects during a 3-week recovery test in CPF-free water. The medaka became hyperactive and exhibited an elevated anxiety state after a 4-day exposure to 0.024mg/L of CPF, but they recovered from these abnormal behavioral responses within 7days of recovery treatment. In contrast, persistent impacts on some startle responses to a sudden stimulation (induced by a ball drop) were observed in medaka exposed to CPF. The reaction latency did not change immediately after the 4-day exposure, but was significantly prolonged by as much as 21days after the termination of exposure. The post-stimulus swimming distance within 5s significantly decreased on the day immediately after the 4-day exposure, but it significantly increased after 7days of recovery treatment. The activity of acetylcholinesterase (AChE) in the brains of medaka was significantly inhibited on the day immediately after the 4-day exposure, but it returned to 80% and 110% of that in control fish on days 7 and 21 of the recovery period, respectively. However, AChE activities in the eyes of exposed medaka were persistently inhibited and declined to 33%, 71%, and 72% of that in control fish on days 0 (immediately after the 4-day exposure), 7, and 21 of recovery, respectively. Correlation analysis suggested that the changes of AChE activities in the brains of medaka may underlie some of the observed acute behavioral changes, and the changes of AChE activities in the eyes may contribute to the persistence of the abnormalities in the reaction latency of the startle response. Our findings suggest that medaka need a long time to recover from acute, sublethal exposure to CPF, and the persistence of the behavioral abnormalities might affect their fitness in natural habitats.

Comparison of social behavior responses of Japanese medaka (Oryzias latipes) to lethal and sublethal chlorpyrifos concentrations at different exposure times.

Chlorpyrifos (CPF) is one of the most widely used insecticides and has been found in both urban and rural water bodies. In this study, we studied variations in the social behavioral patterns, swimming behavior, and brain acetylcholinesterase (AChE) activity of Japanese medaka exposed to lethal (0.12mg/L) and sublethal (0.012mg/L) concentrations of CPF after different exposure times. Group behavior performance (schooling, shoaling, and solitary) was determined on day 4 of lethal exposure and on days 4, 8, and 12 of sublethal exposure. Swimming speed and brain AChE activity were measured on days 4, 8, 12, 16, and 20 of sublethal CPF exposure. We observed significant decreases in social behavior and swimming speed (i.e., hypoactivity) in fish exposed to lethal CPF concentrations for 4 days. At the sublethal concentration, there was increased schooling duration and hyperactivity of fish on day 8 but not on day 4. In contrast, 12 days of sublethal CPF exposure resulted in social behavior responses similar to those after 4 days' lethal exposure, i.e., significant decreases in schooling frequency and duration with a notable increase in duration of solitary behavior. Brain AChE activity was inhibited in a time-dependent manner. Altered fish behavior in response to organophosphorus pesticides such as CPF may be mediated by more than AChE inhibition alone.

Sound production in Japanese medaka (Oryzias latipes) and its alteration by exposure to aldicarb and copper sulfate.

This study is the first to report sound production in Japanese medaka (Oryzias latipes). Sound production was affected by exposure to the carbamate insecticide (aldicarb) and heavy-metal compound (copper sulfate). Medaka were exposed at four concentrations (aldicarb: 0, 0.25, 0.5, and 1 mg L-1; copper sulfate: 0, 0.5, 1, and 2 mg L-1), and sound characteristics were monitored for 5 h after exposure. We observed constant average interpulse intervals (approx 0.2 s) in all test groups before exposure, and in the control groups throughout the experiment. The average interpulse interval became significantly longer during the recording periods after 50 min of exposure to aldicarb, and reached a length of more than 0.3 s during the recording periods after 120 min exposure. Most medaka fish stopped to produce sound after 50 min of exposure to copper sulfate at 1 and 2 mg L-1, resulting in significantly declined number of sound pulses and pulse groups. Relative shortened interpulse intervals of sound were occasionally observed in medaka fish exposed to 0.5 mg L-1 copper sulfate. These alternations in sound characteristics due to toxicants exposure suggested that they might impair acoustic communication of medaka fish, which may be important for their reproduction and survival. Our results suggested that using acoustic changes of medaka has potential to monitor precipitate water pollutions, such as intentional poisoning or accidental leakage of industrial waste.

Effects of Calcium and Magnesium Ions on Acute Copper Toxicity to Glochidia and Early Juveniles of the Chinese Pond Mussel Anodonta woodiana.

We evaluated the effects of calcium (Ca(2+)) and magnesium (Mg(2+)) ions on copper (Cu) toxicity to glochidia and newly-transformed juvenile mussels (age 1-2 days) of the Chinese pond mussel (Anodonta woodiana). Acute Cu toxicity tests were performed with glochidia for 24 h and juveniles for 96 h with measured Ca(2+) concentrations of 1.1, 14, 26, 51, and 99 mg L(-1), or measured Mg(2+) concentrations of 2.6, 11, 21, and 39 mg L(-1). The Ca(2+) and Mg(2+) cations provided no statistically significant protection against Cu toxicity to glochidia or juveniles. The 24-h EC50 value for glochidia was 82 µg L(-1) Cu, and contrastly, 96-h EC50 value for newly-transformed juvenile mussels was as low as 12 µg L(-1) Cu, implying that the juveniles of A. woodiana are more vulnerable to Cu contamination at concentrations close to currently-accepted levels.

Elevated water temperature reduces the acute toxicity of the widely used herbicide diuron to a green alga, Pseudokirchneriella subcapitata.

In the actual environment, temperatures fluctuate drastically through season or global warming and are thought to affects risk of pollutants for aquatic biota; however, there is no report about the effect of water temperature on toxicity of widely used herbicide diuron to fresh water microalgae. The present research investigated inhibitory effect of diuron on growth and photosynthetic activity of a green alga Pseudokirchneriella subcapitata at five different temperatures (10, 15, 20, 25, and 30 °C) for 144 h of exposure. As a result, effective diuron concentrations at which a 50% decrease in algal growth occurred was increased with increasing water temperature ranging from 9.2 to 20.1 µg L(-1) for 72 h and 9.4-28.5 µg L(-1) for 144 h. The photochemical efficiency of photosystem II (F v/F m ratio) was significantly reduced at all temperatures by diuron exposure at 32 µg L(-1) after 72 h. Inhibition rates was significantly increased with decreased water temperature (P < 0.01). Intracellular H2O2 levels as an indicator of oxidative stress were also decreased with increasing temperature in both control and diuron treatment groups and were about 2.5 times higher in diuron treatment groups than that of controls (P < 0.01). Our results suggest water temperatures may affect the toxicokinetics of diuron in freshwater and should therefore be considered in environmental risk assessment.

Alterations in social behavior of Japanese medaka (Oryzias latipes) in response to sublethal chlorpyrifos exposure.

The behavioral and biochemical responses of Japanese medaka (Oryzias latipes) to acute and subacute (sublethal) levels of chlorpyrifos were studied. In the acute exposure test, medaka were exposed to 0.018, 0.055, 0.166, or 0.500 mg L(-1) chlorpyrifos for 4 d. As a result, fish showed hypoactivity compared to the control (at 0.018, 0.055, and 0.166 mg L(-1), swimming speeds were 55.6%, 39.0%, and 27.3% those of the control), Brain acetylcholinesterase activity and swimming speed were significantly correlated. In the subacute toxicity test, medaka were exposed to 0.012 mg L(-1) chlorpyrifos (10% of LC(50)) for 8 d. On day 4, there were no significant differences in behavioral and biochemical endpoints in exposed fish as compared to the control. On day 8, exposed fish became hyperactive, and the swimming speed of the social group increased to 2 times that of the control, whereas acetylcholinesterase activity was decreased to 68% that of the control. In addition, fish exhibited significant alterations in social behavior (schooling duration increased to 2.6 times and solitary duration decreased to 28% that of the control). Our findings clearly demonstrate a subacute effect of chlorpyrifos on the social behavior of medaka, which may pose a risk at population level because of the disturbance of social behavior. In addition, the recorded behavioral alterations may provide a useful tool for assessing the toxicity of organophosphorous pesticides to aquatic organisms.

Acute effects of triclosan, diclofenac and carbamazepine on feeding performance of Japanese medaka fish (Oryzias latipes).

The toxicity of three pharmaceutical and personal care products (PPCPs)--carbamazepine (CBMZ), diclofenac (DCF), and triclosan (TCS)--was examined by measuring their effects on feeding behavior and swimming speed of adult Japanese medaka fish (Oryzias latipes). Medaka were exposed to 6.15 mg L(-1) CBMZ, 1.0 mg L(-1) DCF, 0.17 mg L(-1) TCS, or no PPCP (control) for 9 d. Fish behaviors were monitored during days 5-9 of the exposure period. Feeding behavior (time to eat midge larvae, TE) and swimming speed (SS) of individual exposed and control fish were tracked in two dimensions, using an automated system with a digital charge-coupled device camera. As a result, feeding behavior was affected by exposure to CBMZ and DCF, while SS was altered by exposure to CBMZ and TCS. Thus, TCS, DCF and CBMZ appear to affect fish behaviors through different mechanisms. Overall, the results suggest that behavioral changes may provide a sensitive indicator for assessing the toxicity of PPCPs to aquatic organisms.

The application of entropy for detecting behavioral responses in Japanese medaka (Oryzias latipes) exposed to different toxicants.

Growing concern over environmental pollution by chemicals that may directly or indirectly impact human and ecosystem health make it increasingly necessary to establish a biological early warning system to minimize these impacts. To achieve this, simple and reliable approaches and criteria for environmental risk assessment must be developed. We propose a new approach for detecting behavioral responses to toxicants in flowing water. We conducted a series of tests to determine toxic responses of Japanese medaka (Oryzias latipes) to lethal concentrations of potassium cyanide (KCN; 10 mg L(-1)) and phenol (25 mg L(-1)). Fish movements were tracked by three-dimensional (3D) biomonitoring under flow-through conditions. The 3D data were used to evaluate behavioral responses in terms of swimming speed, vertical position in the water, and entropies of these parameters. Fish exposed to KCN surfaced more frequently than unexposed fish, whereas fish exposed to phenol stayed near the bottom. Maximum swimming speed significantly increased after exposure to both test chemicals, whereas median swimming speed significantly decreased after exposure. Despite large variations in swimming behavior between individual fish, toxic behavioral responses could be identified by the significant decrease in the entropy of vertical position after 10-30 min of exposure to the test chemicals. We conclude that the use of entropy can contribute to the development of a biological early warning system for detecting toxicants at early stages of pollution. To use the present approach to detect lower concentrations of toxicants, further studies should be conducted using long-term monitoring of fish exposed to sublethal concentrations.

In ovo nanoinjection of triclosan, diclofenac and carbamazepine affects embryonic development of medaka fish (Oryzias latipes).

We examined the toxicity of three pharmaceuticals and personal care products (PPCPs) - triclosan (TCS), diclofenac (DCF), and carbamazepine (CBMZ) - on embryonic development of Japanese medaka (Oryzias latipes) using in ovo nanoinjection. Medaka eggs (8h post-fertilization; late blastula stage) were injected with 0.5nL of triolein (vehicle control) or 0.5nL of PPCPs, using different doses of TCS (1, 5, or 9ng), DCF (1, 5, or 12ng), or CBMZ (1, 5, or 12ng) per egg in triolein, in addition to uninjected control. Following injection, we recorded survival, embryonic lesions, delay in embryonic development (eye, embryonic body and internal organs), heart beat rate, hatchability, and hatching time of embryos and upward swimming of larvae. As a result, injected PPCPs caused toxic responses to medaka embryos during embryonic development and around the day of hatching. Based on estimated EC(50) values of PPCPs doses on survival of injected embryos at hatching, TCS (at a dose of 4.2ngegg(-1)) was generally more toxic to medaka embryos, followed by DCF (6.0ngegg(-1)), and CBMZ (13.1ngegg(-1)). We conclude that the nanoinjection medaka embryos model is a valuable tool for analyzing the effects of chemicals on the development of fish embryos and feasibility of nanoinjecting PPCPs into small fish eggs perhaps mimicking early exposure resulting from oocyte uptake of contaminants from maternal extra gonadal tissues.

The effects of methyltestosterone on the sexual development and reproduction of adult medaka (Oryzias latipes).

We examined the effects of methyltestosterone (MT) on the reproduction of medaka (Oryzias latipes). Six mating pairs of medaka were exposed for 3 weeks to MT at the measured concentrations of 22.5, 46.8, 88.1, 188, and 380 ng/L. The fecundity and fertility of paired medaka were significantly decreased in the 46.8, 88.1, 188, and 380 ng/L MT groups compared with those of controls. The gonadosomatic indices of female fish exposed to >or=46.8 ng/L were increased significantly. Gonadal histology showed degeneration of oocytes in the ovaries of fish in all MT treatment groups. Hepatic vitellogenin concentrations were significantly decreased in female fish treated with MT at 188 and 380 ng/L, and the hatchability and survival rate of the offspring were decreased in all MT treatment groups. These results clearly demonstrate that MT at >or=46.8 ng/L inhibited gonadal development and adversely affected the reproduction of medaka.

Association of glutathione S-transferase gene polymorphisms (GSTM1 and GSTT1) of vitiligo in Korean population.

Vitiligo is an acquired pigmentary disorder of the skin involving melanocyte dysfunction. It has been reported that melanocyte impairment could be related to increased oxidative stress. The glutathione S-transferases (GSTs) are group of polymorphic enzymes that are important in protection against oxidative stress. To find the relationship between GSTM1 and GSTT1 polymorphisms with vitiligo susceptibility, GSTM1 and GSTT1 (homozygous deletion vs. non-deleted) polymorphisms between vitiligo patients (n=310) and healthy controls (n=549) were analyzed. We observed significant association in null alleles of the GSTM1 (P<0.001, OR=2.048, 95% CI=1.529-2.743). GSTM1 null type was also statistically different between two vitiligo subtypes and controls (Focal P<0.001, OR=2.224, 95% CI=1.499-3.298; Generalized P=0.001, OR=1.974, 95% CI=1.342-2.904). However, no significant association in GSTT1 (P=0.869, OR=1.024, 95% CI=0.775-1.353) was observed with vitiligo. In combined analysis of GSTM1 and GSTT1, both null type and GSTM1/GSTT1 (null/present) group showed significant differences between controls and vitiligo patients. These results suggest that GSTM1 null type might be associated with vitiligo susceptibility in Korean population.

In situ on-line toxicity biomonitoring in water: recent developments.

In situ on-line biomonitoring is an emerging branch of aquatic biomonitoring. On-line biomonitoring systems use behavioral and/or physiological stress responses of caged test organisms exposed in situ either in a bypass system or directly in-stream. Sudden pollution waves are detected by several existing single-species on-line biomonitors, which until now have been placed mostly in streamside laboratories. However, recent achievements have been multispecies biomonitors, mobile biomonitors for direct in-stream use, development of new instruments, new methods for data analysis and alarm generation, biomonitors for use in soil and sediment, and scientific research supporting responses as seen in on-line biomonitors by linking them to other biological and ecological effects. Mobile on-line monitoring platforms containing an array of biomonitors, biosensors, and chemical monitoring equipment might be the future trend, especially in monitoring transboundary rivers at country borders as well as in coastal zones.

Quantification of para-phenylenediamine and heavy metals in henna dye.

Henna (Lawsonia inermis, family Lythraceae) is a shrub cultivated in India, Sri Lanka and North Africa and contains the active dye lawsone (2-hydroxy-1,4-naphthoquinone). Henna dye is obtained from the dried leaves, which are powdered and mixed with oil or water and are used to prepare hair and body dyes. Temporary henna tattoos are readily available worldwide, last on the skin for several weeks and offer a self-limited, convenient alternative to a permanent tattoo. The addition of para-phenylenediamine (PPD), which is widely recognised as a sensitizer, increases the risk of allergic contact dermatitis from henna tattoo mixtures, and a number of cases have been reported. We examined 15 henna samples available in Korea for the presence of PPD and heavy metals such as nickel, cobalt, chromium, lead and mercury using high-performance liquid chromatography (HPLC), atomic absorption spectroscopy (AAS), mercury analyser and inductively coupled plasma emission spectroscopy. PPD, nickel and cobalt were detected in 3, 11 and 4 samples, respectively.

Effect of tributyltin on reproduction in Japanese whiting, Sillago japonica.

We examined the effect of tributyltin (TBT) on reproduction in the Japanese whiting, Sillago japonica. Mature fish were placed in indoor 500-L polyethylene tanks (five males and three females per tank) with a flow-through system and received dietary exposure to tributyltin oxide at concentrations of 2, 20, or 200 microg/g for 30 days during the active spawning period. Eggs spawned from the fish were collected daily, and the floating egg rate, larval deformity, hatchability, and viable hatch were monitored. TBT concentration in eggs of 200-microg/g group ranged from 85.0 to 159.6 ng/g in the evaluation period (days 5-30). In this period, the floating egg rate (83.2%), viable hatchability (82.2%), and total number of viable larvae (422,000 larvae per 100 g of female) were all significantly decreased in the 200-microg/g group compared with the control group (93.0%, 91.9%, and 709,000 larvae, respectively). The rate of deformity (2.6%) in the 200-microg/g group was about three times that in the control group (0.8%), although this difference was not significant. From these results, the lowest observed effect concentration of TBT in eggs on reproduction in Japanese whiting was estimated to be less than 159.6 ng/g-eggs.

Anti-androgen flutamide affects gonadal development and reproduction in medaka (Oryzias latipes).

The effects of an anti-androgenic chemical on the reproduction of medaka (Oryzias latipes) were examined. Six mating pairs of medaka were exposed to flutamide (FLU) at measured concentrations of 0.101, 0.202, 0.397, 0.787, or 1.56 mg/L for 3 weeks. During the exposure period, one female died in the 1.56-mg/L treatment group, but no lesions or pathological abnormalities were observed. The fecundity and fertility of paired medaka were significantly decreased at 1.56 mg/L compared with those of the controls. Histological examination showed intersex gonads (testis-ova) in males exposed to FLU at 0.202, 0.397, and 0.787 mg/L. However, concentrations of hepatic vitellogenin (Vtg) in both sexes were not statistically different from those in the controls. These results demonstrate that FLU affected gonadal development in male medaka and reproductive capacity in paired medaka. This is the first report of testis-ova in male medaka exposed to FLU.

Fertilization success and sexual behavior in male medaka, Oryzias latipes, exposed to tributyltin.

We evaluated the effects of tributyltin (TBT), polychlorinated biphenyls (PCBs), and a mixture of TBT and PCBs on reproduction, gonadal histology, and sexual behavior in Japanese medaka (Oryzias latipes). We administered TBT (1 microg g(-1) body weight [b.w.] daily), PCBs (1 microg g(-1) b.w. daily), or both to medaka for 3 weeks. We assessed reproductive success during week 3 and the sexual behavior of male medaka after the exposure period. Fertilization success was significantly decreased in groups that received TBT. The profiles of fertility developed a bimodal distribution in the TBT and TBT+PCBs groups, and the numbers of males that performed "following" and "dancing" were significantly decreased in the TBT+PCBs group. The frequency of "dancing" also decreased after treatment with TBT, whereas fish that received PCBs only showed no difference in sexual behavior. Testes that had oocytes showed normal spermatogenesis, even though two of the five males treated with TBT+PCBs had some indication of testis-ova, which did not occur in any other group. Our results indicate that TBT, but not PCBs, affects sexual behavior and reproduction in medaka.