Effect of 17beta-estradiol on the reproduction of Japanese medaka (Oryzias latipes)

Estrogenic compounds such as 17beta-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis-ova. Males treated with E2 concentrations = 55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis-ova in male medaka exposed to environmentally relevant concentrations of E2.     

Multi-generational effects of four selected environmental oestrogens on Daphnia magna

The objective of this study was to determine whether vertebrate-type oestrogens have ecotoxicological effects on a crustacean species. The effects of 17beta-oestradiol (E2), diethylstilbestrol (DES), bisphenol A (BPA) and 4-nonylphenol (4-NP) on the freshwater invertebrate Daphnia magna were assessed over first and second generations. The acute EC(50) 48 h, based on immobilisation, for E2, DES, BPA and 4-NP were 2.87 mg/l, 1.55 mg/l, 7.75 mg/l and 0.13 mg/l, respectively. The impact of the test chemicals on moulting frequency was also assessed. The EC(50) 48 h, based on the inhibition of moult number for E2, DES and 4-NP were 2.04 mg/l, 1.87 mg/l and 0.14 mg/l, respectively. BPA was not observed to impact the moulting frequency of D. magna at concentrations tested. In a series of separate studies, the effects of the four selected test compounds on the survival, moulting frequency and reproduction of first and second generational D. magna were assessed over a period of 21 d. Exposure of D. magna to 4-NP decreased the number of offspring produced in both first and second generation testing. DES proved to have no significant (p0.05) inhibition of fecundity in first generation but when second generation daphnids were exposed to DES, a significant (p0.05) reduction in the number of offspring was recorded. When D. magna were exposed to E2 or BPA, no statistically significant (p0.05) inhibition in the number of moults or offspring produced was observed.     

Effects of pharmaceuticals on Daphnia survival, growth, and reproduction

Pharmaceuticals have been globally detected in surface waters, and the ecological impacts of these biologically-active, ubiquitous chemicals are largely unknown. To evaluate the aquatic toxicity of individual pharmaceuticals and mixtures, we performed single species laboratory toxicity tests with Daphnia magna, a common freshwater zooplankton. We conducted acute (6-day) and chronic (30-day) exposure pharmaceutical bioassays and evaluated survivorship and morphology of adults and neonates, adult length, resting egg production, brood size (fecundity), and the proportion of male broods produced (sex ratio). In general, exposure to a single pharmaceutical in the 1-100 microg/l range yielded no apparent effects on the normal life processes of Daphnia. However, chronic fluoxetine exposure (36 microg/l) significantly increased Daphnia fecundity, and acute clofibric acid exposure (10 microg/l) significantly increased sex ratio. A mixture of fluoxetine (36 microg/l) and clofibric acid (100 microg/l) caused significant mortality; the same fluoxetine concentration mixed with 10 microg/l clofibric acid resulted in significant deformities, including malformed carapaces and swimming setae. Mixtures of three to five antibiotics (total antibiotic concentration 30-500 microg/l) elicited changes in Daphnia sex ratio. We conclude: (1) individual and mixtures of pharmaceuticals affect normal development and reproduction of Daphnia magna, (2) aquatic toxicity of pharmaceutical mixtures can be unpredictable and complex compared to individual pharmaceutical effects, and (3) timing and duration of pharmaceutical exposure influence aquatic toxicity.     

Acute and chronic toxicities of tributyltin to various life stages of the marine polychaete Hydroides elegans

Experiments were performed to determine the acute and chronic toxicities of tributyltin (TBT) (nominal concentrations) to polychaete Hydroides elegans. Firstly, static tests were performed to examine the lethal effect of TBT on five life stages of H. elegans. The 48-h LC(50) (95% CI) for the eggs, 2-cells, trochophores, juveniles and adults was 0.18 (0.15-0.20), 0.97 (0.77-1.23), 2.36 (2.11-2.65), 2.86 (2.63-3.12) and 4.36 (4.04-4.71) microg TBT l(-1), respectively. Secondly, a 17-d static renewal chronic test was conducted to study sensitivity of the early development (egg to juvenile) to TBT. The survivorship, percent settlement and time to reach settlement were significantly reduced at 0.01 microg TBT l(-1). Effects of TBT on the juvenile growth and maturation were also studied through a 44-d chronic test. Lower survivorship and reduced tube growth were observed only at >or= 1 microg TBT l(-1), however, only H. elegans exposed to or= 0.1 microg TBT l(-1). Our results indicate that the early development of H. elegans is highly sensitive to TBT and this polychaete can be routinely employed as a test organism for both acute and chronic ecotoxicity bioassays in tropical and subtropical regions such as southern China.     

Effects of chlorpyrifos on reproductive performances of guppy (Poecilia reticulata)

Guppy (Poecilia reticulata) was selected to investigate the effects of chlorpyrifos on reproductive performances. Male and female guppy with proven fertility were selected from our own colony and the groups of fish (n=72/group) were exposed to pre-determined chlorpyrifos concentrations (0.002 microg/l, 2 microg/l) based on the 96-h LC50 for guppy. Mating behavior of males was recorded on the 2nd day of exposure. Offspring were counted and survival recorded on the 14th day. Gonopodial thrusts (8/15 min) in 0.002 microg/l and (4/15 min) in 2 microg/l were significantly different from the control group (11/15 min). Similarly, live birth reduced significantly to 8/female in 2 microg/l compared to 27/female in the control group. Survival of offspring after 14 days was reduced to 47% in the 2 microg/l group compared to 94% of survival in the control. Our findings demonstrate that low soluble concentrations of chlorpyrifos affect mating behavior, number of offspring and offspring survival of guppy.     

Nitrophenols in precipitation

From 1995 to 1998 the concentrations of 4-nitrophenol, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenol, dinitro-ortho-cresol (DNOC) and 2,4-di-nitrophenol were measured monthly by HPLC in precipitation at eight different locations in Bavaria (Germany). Samples were collected by purpose-constructed computerised rainwater samplers which record electronically various sensor data each hour and adjust the sample temperature to 4 degrees C. The highest nitrophenol (NP) concentrations were measured for 4-NP. The median at all locations is higher than 1 microg/l. The median of the other NPs ranges between 0.2 and 0.8 microg/l. Considering the rain amounts the highest depositions were calculated for the regions Spessart, Bayerischer Wald and Chiemgauer Alpen. The median of 4-NP depositions extents to 200 microg/(month m2). The highest medians of the other NP depositions reach approximately 50 microg/(month m2).     

Bisphenol A induces yolk-sac oedema and other adverse effects in landlocked salmon (Salmo salar m. sebago) yolk-sac fry

Accumulation and toxicity of waterborne bisphenol A were studied in landlocked salmon (Salmo salar m. sebago) yolk-sac fry. In a short-term (96 h) exposure to five bisphenol A concentrations yolk-sac fry had higher accumulation rates and bioconcentration factors (BCF96) than earlier studies have shown for salmon eggs. Furthermore, the conditional uptake rate constant tended to decrease as exposure concentration increased. Fry were also exposed to bisphenol A for 42 days at three concentrations (10, 100 and 1000 microg/l), and changes in behaviour, morphology and histological structure were observed. After 6 days of exposure, the highest concentration (1000 microg/l) of bisphenol A caused fluid accumulation (oedema) in the yolk sac and haemorrhages in the front part of the yolk sac and in the head around the gill arches. Later on, the fry at 1000 microg/l showed phlegmatic behaviour and had darker skin coloration than the fry in the other treatments. At the two highest concentrations (100 and 1000 microg/l) histological changes were seen in liver cell nuclei, where strongly stained fragments were observed. In the control fry and the fry exposed to 10 microg/l the nucleolus was clearly visible and spherical in shape and no strongly stained fragments were present. This study shows that high concentrations of bisphenol A may have both morphological and histological effects on salmon yolk-sac fry.     

Life stage- dependent bioconcentration of a nonylphenol isomer in Daphnia magna

Bioaccumulation is an important aspect for the fate and effects of xenobiotics in the environment. In this study we used a radiolabeled nonylphenol isomer to investigate the bioconcentration in Daphnia magna at different ages. Apart from the total radioactivity we measured the metabolism of p353-NP in D. magna, to calculate the amount of p353-NP compared to total radioactivity found within the daphnids. Bioconcentration factors, based on wet weight, calculated from the rate constants for total radioactivity in neonates and adults were 4271 kg/l and 760 kg/l respectively, leading to a 5.6 deviance in bioconcentration. This deviance was even more pronounced, nearly one order of magnitude, for the p353-NP concentration with bioconcentration factors of 302 kg/l for neonates and 31 kg/l for adults. We were able to describe the bioconcentration for all daphnids by a weight-dependent one- compartment model. These results pointed out that it is not possible to compare bioconcentration experiments conducted with different substances and different sized daphnids. Additionally it was shown that it is not possible to describe the bioconcentration by measuring the total radioactivity. Metabolism of nonylphenol occurs at a very fast rate and bioconcentration is not triggered by the partition between two phases, but by metabolism. Discrimination between the two mechanisms was achieved using radiolabeled substances and a pseudo two-compartment model to describe metabolism and elimination by two rate constants which afterwards can be compared between different substances.     

Avoidance by olfaction in a fish, medaka (Oryzias latipes), to aquatic contaminants

Behavioral tests using a fish, medaka (Oryzias latipes), from which the olfactory organs were resected confirmed that fish avoid aquatic contaminants such as surfactants and pesticides mainly by olfaction. Control medaka (non-resected) exhibited significant avoidance to 10, 20 and 30 microg liter(-1) of LAS (sodium linear laurylbenzene sulfonate), 90 and 100 microg liter(-1) of fenitrothion (dimethyl 4-nitro-m-toryl phosphorothionate), and 500 microg liter(-1) of POE-ether (polyoxyethylene lauryl ether). By contrast, medaka with bilateral nose resections (BNRM) exhibited no avoidance to 10-50 microg liter(-1) of LAS, 90 and 100 microg liter(-1) of fenitrothion, and 500 microg liter(-1) of POE-ether. Although medaka with unilateral nose resection (UNRM) avoided some concentrations of these toxicants, the UNRM exhibited no avoidance to 10 and 40 microg liter(-1) of LAS and 90 microg liter(-1) of fenitrothion. That is, the avoidance response of the UNRM was less distinct than that of the controls. This difference may have been caused by the deterioration of detecting ability and the paralyzation or adaptation of olfaction.     

Morphological effects of Bisphenol-A on the early life stages of medaka (Oryzias latipes).

Bisphenol-A (BPA), a widely used polycarbonate plasticizer, has been of concern because it has been shown to leach out of plastics and other epoxy products. Primary sources of environmental releases are expected to be from BPA and epoxy manufacturing facilities. Although environmental concentrations may be limited, little is known about the effects of this compound on fish, particularly during their most sensitive early life stages. A pilot study exposing newly hatched medaka to 200 microg BPA/l for 96 h showed to be non-lethal. In addition, medaka embryos were exposed beginning at 5 h post-fertilization, for 9 days at 25 degrees C, to concentrations of 20 or 200 microg BPA/l (24 h static renewal). Embryos were monitored daily for development and gross abnormalities. Embryos exposed to 200 microg BPA/l did not exhibit abnormalities until after day 4. However, between days 4 and 8 the severity index (SI) score of embryos was significantly greater than those exposed to lesser concentrations. By day 9, SI scores were not statistically different among treatments. BPA caused transient embryonic deformities in medaka at environmentally relevant concentrations.     

Bioconcentration factor of relatively low concentrations of chlorophenols in Japanese medaka

Bioconcentration factors (BCF) for pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP) in Japanese medaka (Oryzias latipes) were determined at five different concentrations of the chemicals, between 0.1 and 10 microg/l (PCP), 0.3 and 30 microg/l (2,4-DCP), in the ambient water. Medaka were exposed to each chemicals in a continuous-flow system during the embryonic development period and 60 days after hatching from eggs collected in the laboratory. Both the exposure time and the aqueous concentrations are much more realistic and closer to natural aquatic environments than those used in conventional BCF studies. The BCF values of PCP were from (4.9+/-2.8)x10(3) at the aqueous concentration of 0.074+/-0.028 microg/l to (2.1+/-1.4)x10(3) at 9.70+/-0.56 microg/l. The BCF value of 2,4-DCP were from (3.4+/-3.0)x10(2) at 0.235+/-0.060 microg/l to 92+/-27 at 27.3+/-1.6 microg/l. Generally, BCF values increased as the aqueous concentrations of PCP or 2,4-DCP decreased. This finding suggests that a relatively low and realistic aqueous concentration of these compounds is necessary to more accurately determine their BCF values in natural aquatic environments. Conventional BCF experiments at higher aqueous concentrations may underestimate the BCF values.     

Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol

Mating pairs of Japanese medaka (Oryzias latipes) received 17beta-estradiol (E2; dose, 3 and 30 microg/g body weight daily) in their diet and E2-free diet (control) for two weeks. The effect on reproductive success was assessed in the mating pairs, and sexual and nonsexual behaviors in the male were quantified. The fecundities of the E2-treated groups were reduced significantly (p < 0.05) compared with those of control, and the sexual behaviors (following, dancing, floating, and crossing) of male fish in response to prostaglandin F2alpha (PG)-injected E2-unexposed females were suppressed dramatically. While the fertility of spawned eggs and the frequencies of the nonsexual behaviors of resting and swimming in a straight line performed by male were unchanged in E2-exposed groups. Therefore, sexual behavior in male may be linked with reproductive success in mating pair of madaka exposed to E2. This assay for sexual behavior using PG may provide a useful tool for assessing the effect of endocrine-disrupting chemicals.     

Styrene dimers and trimers affect reproduction of daphnid (Ceriodaphnia dubia)

The endocrine disruptor activity of styrene in humans and other vertebrates appears to be negligible. However, offspring numbers were reduced in Ceriodaphnia dubia bred in polystyrene cups. Styrene dimers and trimers were found to be eluted from the polystyrene cups by hexane and methanol with gas chromatography-mass spectrometry. Styrene dimers and trimers at concentrations of 0.04-1.7 microg/l affected C. dubia fertility (25% reduction after seven days), suggesting that styrenes have the potential to impair crustacean populations in the aquatic environment.     

Aerobic thermophilic treatment of sewage sludge contaminated with 4-nonylphenol

4-Nonylphenol (4-NP) occurs in sewage sludge as a result of the breakdown of detergents which contains nonylphenol ethoxylates (NPEs). 4-NP is of environmental concern because of its toxicity to biological systems. The present paper reports results of aerobic treatment under thermophilic conditions of sewage sludge artificially contaminated with 4-NP. Experiments were carried out using three parallel laboratory-scale batch reactors operating with blank, 50 and 100 mg/l of 4-NP concentration. For the two studied concentrations up to 66% 4-NP reduction was achieved at a specific air flow rate of 16 l/(l.h) and a thermophilic temperature of 60 degrees C, within 10 days of operation. The presence of 4-NP has minor effect on the rate of sludge oxidation and the nitrogen and phosphorous content in the sludge.     

Effect of certain chemicals on the reproduction of medaka (Oryzias latipes)

In order to understand the effects of estrogenic chemicals on fish reproduction, we exposed male medaka (Oryzias latipes) to a natural estrogen [17 beta-estradiol (17 beta-E2)] and three estrogenic chemicals [bisphenol-A, nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP)]. After two weeks' exposure, one male medaka was kept together with two female medaka for spawning, and the number of eggs and hatchings were compared to those of a negative control group. The results indicated that exposure to 17 beta-E2 caused a significant decrease in the number of eggs and hatchings as compared to the negative control group at and above 3 nmol/l. Also, the highest concentrations of bisphenol-A and NP caused a decrease in the number of hatchings, but no decrease in hatchings was observed in DEHP treatments. In the treatment using these chemicals the decrease in egg numbers was not so much as in hatching numbers. When compared to other in vitro studies, concentrations observed to have adverse effects on reproduction in this study are generally lower. In addition, it was suggested that physical alterations, such as an induction of plasma vitellogenin, were caused at much lower concentrations than those at which a decline in reproductivity was actually induced.     

Uptake and depuration of the anti-depressant fluoxetine by the Japanese medaka (Oryzias latipes)

The selective serotonin reuptake inhibitor (SSRI) class of anti-depressants is among the most widely prescribed groups of pharmaceuticals. Consequently, aquatic ecosystems impacted by municipal wastewater discharges are predicted to receive substantial annual loadings of these compounds. Although SSRIs have been detected in fish tissues, little is known of their uptake and depuration in freshwater fish species. In this study, Japanese medaka (Oryzias latipes) were exposed to fluoxetine at a nominal concentration of 0.64 microg L(-1) for 7d and subsequently allowed to depurate in clean water over a 21d period. Fluoxetine uptake by medaka was observed within the first 5h of exposure and the biologically active metabolite, norfluoxetine, was also detected in medaka tissues during this timeframe. A maximum fluoxetine concentration was measured in medaka by the third day of the uptake phase, yielding an uptake rate constant (k(1)) of 5.9+/-0.5 (d(-1)). During the depuration phase of the experiment, a half life of 9.4+/-1.1d was determined for fluoxetine. Using these data, bioconcentration factor (BCF) values of 74 and 80 were estimated for fluoxetine and a pseudo-BCF (the ratio of the concentration of norfluoxetine in medaka and the aqueous fluoxetine concentration) of 117 was calculated for norfluoxetine. These results indicate longer persistence and greater potential for the bioaccumulation of fluoxetine and norfluoxetine in fish tissues than would be predicted from prior half life estimates derived using mammalian species.     

Waterborne and sediment toxicity of fluoxetine to select organisms

Ecological risk assessments of pharmaceuticals are currently difficult because little-to-no aquatic hazard and exposure information exists in the peer-reviewed literature for most therapeutics. Recently several studies have identified fluoxetine, a widely prescribed antidepressant, in municipal effluents. To evaluate the potential aquatic toxicity of fluoxetine, single species laboratory toxicity tests were performed to assess hazard to aquatic biota. Average LC(50) values for Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas were 0.756 (234 microg/l), 2.65 (820 microg/l), and 2.28 microM (705 microg/l), respectively. Pseudokirchneriella subcapitata growth and C. dubia fecundity were decreased by 0.044 (14 microg/l) and 0.72 microM (223 microg/l) fluoxetine treatments, respectively. Oryias latipes survival was not affected by fluoxteine exposure up to a concentration of 28.9 microM (8.9 mg/l). An LC(50) of 15.2 mg/kg was estimated for Chironomus tentans. Hyalella azteca survival was not affected up to 43 mg/kg fluoxetine sediment exposure. Growth lowest observed effect concentrations for C. tentans and H. azteca were 1.3 and 5.6 mg/kg, respectively. Our findings indicate that lowest measured fluoxetine effect levels are an order of magnitude higher than highest reported municipal effluent concentrations.     

Toxicity of o,p'-DDE to medaka d-rR strain after a one-time embryonic exposure by in ovo nanoinjection: an early through juvenile life cycle assessment

The toxicity of o,p'-DDE (1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethylene) was evaluated in embryos of medaka (Oryzias latipes) following a one time exposure via nanoinjection. Medaka eggs (early gastrula) were injected with 0.5 nl of triolein (vehicle control) or 0.5 nl of 4 graded doses (0.0005-0.5 ng/egg) of o,p'-DDE in triolein. Embryos were allowed to develop, and fry were reared. Embryonic survival was monitored daily during the first 10 d until hatching and thereafter, on a weekly basis until day 59, at which time the fish were monitored for sexual maturity until day 107. In general, o,p'-DDE caused a dose- and time-dependent mortality. No changes in mortality were observed between the last two time points (day 38 and 59, respectively), and hence a 59 day-LD50 of 346 ng o,p'-DDE/egg was derived from the linear dose-response relationship. Prior to late stage death, only isolated cases of cardiovascular lesions and spinal deformities were observed, but were not dose-dependent. The lowest observable adverse effect level (LOAEL), based on upper 95% CI for regression line=0.0018 mg/kg, and the LOAEL based on exposure doses=0.5 mg/kg. Likewise, the no observable adverse effect level (NOAEL) based on linear extrapolation to 100% survival=0.0000388 mg/kg, while the NOAEL based on exposure doses=0.05 mg/kg. The nanoinjection medaka model has potential in the study of hormonally active compounds in the environment.     

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.     

Copper regulation and homeostasis of Daphnia magna and Pseudokirchneriella subcapitata: influence of acclimation

This study aimed to evaluate (1) the capacity of the green alga Pseudokirchneriella subcapitata and the waterflea Daphnia magna to regulate copper when exposed to environmentally realistic copper concentrations and (2) the influence of multi-generation acclimation to these copper concentrations on copper bioaccumulation and homeostasis. Based on bioconcentration factors, active copper regulation was observed in algae up to 5 microg Cu L(-1) and in daphnids up to 35 mug Cu L(-1). Constant body copper concentrations (13+/-4 microg Cu g DW(-1)) were observed in algae exposed to 1 through 5 microg Cu L(-1) and in daphnids exposed to 1 through 12 microg Cu L(-1). At higher exposure concentrations, there was an increase in internal body copper concentration, while no increase was observed in bioconcentration factors, suggesting the presence of a storage mechanism. At copper concentrations of 100 microg Cu L(-1) (P. subcapitata) and 150 microg Cu L(-1) (D. magna), the significant increases observed in body copper concentrations and in bioconcentration factors may be related to a failure of this regulation mechanism. For both organisms, internal body copper concentrations lower than 13 microg Cu g DW(-1) may result in copper deficiency. For P. subcapitata acclimated to 0.5 and 100 microg Cu L(-1), body copper concentrations ranged (mean+/-standard deviation) between 5+/-2 microg Cu g DW(-1) and 1300+/-197 microg Cu g DW(-1), respectively. For D. magna, this value ranged between 9+/-2 microg Cu g DW(-1) and 175+/-17 microg Cu g DW(-1) for daphnids acclimated to 0.5 and 150 microg Cu L(-1). Multi-generation acclimation to copper concentrations >or =12 microg Cu L(-1) resulted in a decrease (up to 40%) in body copper concentrations for both organisms compared to the body copper concentration of the first generation. It can be concluded that there is an indication that P. subcapitata and D. magna can regulate their whole body copper concentration to maintain copper homeostasis within their optimal copper range and acclimation enhances these mechanisms.