Early Life Stage Toxicity Tests Research Articles

1,2,3-benzotriazole adversely affects early-life stage of Oryzias latipes

1,2,3-benzotriazole (BT) is used in large amounts around the world and is one of the substances derived from household chemicals that are of concern for risk when discharged to aquatic environments. Therefore, several studies have been conducted on the aquatic toxicity effects of BT, but the chronic impact assessment studies to evaluate the developmental effects on the early-life stage of fish are insufficient. In this study, the acute toxicity test and subchronic toxicity test (fish, early-life stage toxicity test, ELS test) using embryos of Japanese medaka (Oryzias latipes) were performed to evaluate the acute toxicity, developmental toxicity, growth (indicated by total length and weight at the end of the test), and histopathological effect of BT. In the short-term toxicity test on embryo and sac-fry stage, toxicity value was calculated to be 41 mg/L (NOEC). Based on this value, the exposure concentration of the ELS test was determined as 0.04, 0.4, 4 and 40 mg/L, and total exposure duration was 42 days. At the highest concentration group (40 mg/L), failure of swim bladder inflation and decrease of survival and size (total length and weight) were observed. Moreover, in the histopathological analysis, abnormal findings were detected in swim bladders from the 40 mg/L group such as inflammation and tumor changes. On the other hands, condition index (weight-length relationships, CI) was statistically significantly lower in all exposed groups compared to the control group. NOEC for the survival of BT was calculated to be 4 mg/L. LOEC for CI was 0.04 mg/L, which means BT inhibited weight gain relative to its length on larvae of medaka.

Use of pufferfish, Takifugu niphobles, for early-life stage toxicity test as a marine fish model

Use of pufferfish, <i>Takifugu niphobles</i>, for early-life stage toxicity test as a marine fish model

Development of an exposure protocol for toxicity test (FEET) for a marine species: the European sea bass (Dicentrarchus labrax).

Regulatory assessment of the effects of chemicals requires the availability of validated tests representing different environments and organisms. In this context, developing new tests is particularly needed for marine species from temperate environments. It is also important to evaluate effects that are generally poorly characterized and seldom included in regulatory tests. In this study, we designed an exposure protocol using European sea bass (Dicentrarchus labrax) larvae. We examined classical toxicological values (LCx) as well as behavioral responses. By comparing different hatching and breeding strategies, we defined the optimal conditions of exposure as non-agitated conditions in 24- or 48-well microplates. Our exposure protocol was then tested with 3,4-dichloroaniline (3,4-DCA), a recommended reference molecule. Based on our results, the 96 h LC50 for 3,4-DCA corresponded to 2.04 mg/L while the 168 h LC50 to 0.79 mg/L. Behavioral analyses showed no effect of 3,4-DCA at low concentration (0.25 mg/L). In conclusion, the present work established the basis for a new test which includes behavioral analysis and shows that the use of sea bass is suitable to early-life stage toxicity tests.

Effects on Apical Outcomes of Regulatory Relevance of Early-Life Stage Exposure of Double-Crested Cormorant Embryos to 4 Environmental Chemicals.

Environmental risk assessment is often challenged by a lack of toxicity data for ecological species. The overall goal of the present study was to employ an avian early-life stage toxicity test to determine the effects of 4 chemicals (benzo[a]pyrene [BaP], chlorpyrifos, fluoxetine hydrochloride [FLX], and ethinyl estradiol [EE2]) on an ecologically relevant avian species, the double-crested cormorant (Phalacrocorax auritus), and to compare our results with those we previously reported for a laboratory model species, Japanese quail. Chemicals were dissolved in dimethyl sulfoxide and administered via air cell injection to fertilized, unincubated double-crested cormorant eggs at 3 nominal concentrations, the highest selected to approximate the 20% lethal dose. Of the 4 chemicals, only chlorpyrifos and FLX were detected in liver tissue of embryos at midincubation (day 14) and termination (day 26; 1-2 d prior to hatch); EE2 and BaP were not detectable, suggesting embryonic clearance/metabolism. No apical effects were observed in double-crested cormorant embryos up to the highest concentrations of chlorpyrifos (no-observed-effect level [NOEL] = 25 µg/g) or FLX (NOEL = 18 µg/g). Exposure to EE2 reduced embryonic viability and increased deformities at a concentration of 2.3 µg/g (NOEL = 0.18 µg/g), and BaP decreased embryonic viability (median lethal dose = 0.015 µg/g; NOEL = 0.0027 µg/g). Compared with Japanese quail, double-crested cormorant were more sensitive with regard to embryolethality and deformities for EE2 and embryolethality for BaP, whereas they were less sensitive to embryonic deformities associated with chlorpyrifos exposure. These data reinforce the idea that standardized toxicity tests using a laboratory model species may not always be protective of wild birds, and thus they stress the importance of developing such alternative testing strategies (e.g., the EcoToxChip Project) for ecologically relevant species to augment risk assessment efforts. Environ Toxicol Chem 2021;40:390-401. © 2020 SETAC.

Predicting ZnO nanoparticle and ZnSO4 toxicity from zebrafish embryo gene expression

The potential to replace the early-life-stage toxicity test (ELS) with the gene expression toxicity test in zebrafish (Danio rerio) embryos (gene-DarT) was evaluated. We compared the lowest-observable- effect concentrations (LOECs) for each. For ELS, zebrafish were exposed to ZnO nanoparticles (NPs) and ZnSO4 at 0-0.25 and 0-16 mg/L, respectively, for 28 d. For 48-h gene-DarT tests, expression of potential marker genes (opioid growth factor receptor-like 2 [ogfrl2], cytochrome b5 domain-containing 1 [cyb5d1], and intelectin 2 [intl2] for ZnO NPs and trace amine-associated receptor 10c, CaM kinase-like vesicle-associated, and interferon regulatory factor 2a [irf2a] for ZnSO4) was investigated. The ELS LOECs for ZnO NP and ZnSO4 were 0.016 and 1 mg/L, respectively. Gene-DartT cyb5d1 and irf2a LOECs were 4-16 times lower than ELS LOECs. Gene-DarT ogfrl2 and intl2 and ELS LOECs were similar. Our results suggest the gene-DarT is an effective alternative for predicting ZnO NP and ZnSO4 LOECs.

Simulated developmental and reproductive impacts on amphibian populations and implications for assessing long-term effects

Fish endpoints measured in early life stage toxicity tests are often used as representative of larval amphibian sensitivity in Ecological Risk Assessment (ERA). This application potentially overlooks the impact of developmental delays on amphibian metamorphosis, and thereby reduced survival, in amphibian populations constrained by habitat availability. Likewise, the effects of reduced productivity or altered sexual development as a result of chemical exposure are not presented in terms of lower population fecundity in these surrogate tests. Translating endpoints measured in toxicity tests to those that are more representative of amphibian ecology and population dynamics provides a means of identifying how developmental effects result in long-term impacts. Here we compare effects of developmental delay on metamorphosis success in six anuran species and simulate population-level impacts of subsequent reductions in larval survival as well as potential reductions in fecundity as a result of developmental impacts. We use deterministic matrix models to compare realistic combinations of amphibian demographic rates and relative impacts of reduced growth on larval survival and subsequently on population growth. Developmental delays are less detrimental in species with longer and less synchronous larval periods. All six species were most sensitive to changes in first-year survival, and damping ratios were generally a good indicator of resilience to perturbations in both larval survival and fecundity. Further identification of species and population-level vulnerabilities can improve the evaluation of sublethal effects in relevant context for ERA.

Toxicity of naproxen sodium and its mixture with tramadol hydrochloride on fish early life stages

Pharmaceuticals occur in water bodies as a consequence of their incomplete removal during waste water treatment processes. The occurence of pharmaceuticals in surface waters as well as their possible impact on aquatic vertebrates have received considerable attention in recent years. However, there is still a lack of informations on the chronic effects of widely used drugs as well as their possible mixture toxicity on non-target aquatic vertebrates as well as their possible mixture toxicity. The aim of this study was to assess the effects of naproxen sodium on early life stages of fish and evaluate its mixture toxicity with tramadol hydrochloride, which was assessed in our earlier study as a single substance.Two embryo-larval toxicity tests with common carp (Cyprinus carpio) were performed according to the OECD guideline 210 (Fish, Early-life Stage Toxicity Test) in order to assess the subchronic toxicity of naproxen sodium and tramadol hydrochlorid-naproxen sodium mixture at the concentrations of 10; 50; 100 and 200 μg/L. These experiments were conducted for 32 days.The subchronic exposure to naproxen sodium and naproxen sodium and tramadol hydrochloride mixture had a strong effect on the early life stages of common carp. Hatching, developmental rate, morphology, histopathology and, in the case of the naproxen sodium and tramadol hydrochloride mixture, mortality were influenced. The bioindicators of oxidative stress were also influenced. The LOEC was determined at 10 μg/L for both naproxen sodium and naproxen sodium and tramadol hydrochloride mixture.

A critical evaluation of the fish early-life stage toxicity test for engineered nanomaterials: experimental modifications and recommendations.

There are concerns that regulatory toxicity tests are not fit for purpose for engineered nanomaterials (ENMs) or need modifications. The aim of the current study was to evaluate the OECD 210 fish, early-life stage toxicity test for use with TiO2 ENMs, Ag ENMs, and MWCNT. Both TiO2 ENMS (≤160mgl(-1)) and MWCNT (≤10mgl(-1)) showed limited acute toxicity, whilst Ag ENMs were acutely toxic to zebrafish, though less so than AgNO3 (6-day LC50 values of 58.6 and 5.0µgl(-1), respectively). Evidence of delayed hatching, decreased body length and increased muscle width in the tail was seen in fish exposed to Ag ENMs. Oedema (swollen yolk sacs) was also seen in fish from both Ag treatments with, for example, mean yolk sac volumes of 17, 35 and 39µm(3) for the control, 100µgl(-1) Ag ENMs and 5µgl(-1) AgNO3 treatments, respectively. Among the problems with the standard test guidelines was the inability to maintain the test solutions within ±20% of nominal concentrations. Pronounced settling of the ENMs in some beakers also made it clear the fish were not being exposed to nominal concentrations. To overcome this, the exposure apparatus was modified with the addition of an exposure chamber that ensured mixing without damaging the delicate embryos/larvae. This allowed more homogeneous ENM exposures, signified by improved measured concentrations in the beakers (up to 85.7 and 88.1% of the nominal concentrations from 10mgl(-1) TiO2 and 50µgl(-1) Ag ENM exposures, respectively) and reduced variance between measurements compared to the original method. The recommendations include: that the test is conducted using exposure chambers, the use of quantitative measurements for assessing hatching and morphometrics, and where there is increased sensitivity of larvae over embryos to conduct a shorter, larvae-only toxicity test with the ENMs.

The effect of tramadol hydrochloride on early life stages of fish.

The aim of this study was to perform the fish embryo acute toxicity test (FET) on zebrafish (Danio rerio) and the early-life stage toxicity test on common carp (Cyprinus carpio) with tramadol hydrochloride. The FET was performed using the method inspired by the OECD guideline 236. Newly fertilized zebrafish eggs were exposed to tramadol hydrochloride at concentrations of 10; 50; 100 and 200μg/l for a period of 144h. An embryo-larval toxicity test on C. carpio was performed according to OECD guideline 210 also with tramadol hydrochloride at concentrations 10; 50; 100 and 200μg/l for a period of 32 days. Hatching was significantly influenced in both acute and subchronic toxicity assays. Subchronic exposure also influenced early ontogeny, both morphometric and condition characteristics and caused changes in antioxidant enzyme activity. The LOEC value was found to be 10μg/l tramadol hydrochloride.

Acute and sub-chronic toxicity of four cytostatic drugs in zebrafish.

The acute and sub-chronic effects of four cytostatic drugs-5-fluorouracil (5-FU), cisplatin (CisPt), etoposide (ET) and imatinib mesylate (IM)-on zebrafish (Danio rerio) were investigated. Acute tests were carried out in a static system in accordance with the OECD guideline 203 for adult fish and the draft guideline for fish embryos (FET test) in order to find the LC50 values of the four cytostatic drugs. Early-life stage toxicity test on zebrafish was conducted according the OECD guideline 210 using the cytostatic drugs 5-FU and IM in a semistatic system with the objective of investigating the sub-chronic effects of the cytostatic drugs on fish. In adult fish, the cytostatic drugs 5-FU and ET did not pass the limit test, thus, are considered non-toxic. In case of cisplatin, LC50 was calculated at 64.5 mg L(-1), whereas in case of IM, LC50 was at 70.8 mg L(-1). In the FET test, LC50 of 5-FU at 72-h post fertilization (hpf) was 2441.6 mg L(-1). In case of CisPt, LC50 was 349.9 mg L(-1) at 48 hpf and it progressively decreased to 81.3 mg L(-1) at 120 hpf. In addition, CisPt caused a significant delay in the hatch of larvae. In case of ET, LC50 values were not calculable as they were higher than 300 mg L(-1) at which concentration the substance crystallized in the solution. LC50 values of IM were 48 hpf; 158.3 mg L(-1) , 72 hpf; 141.6 mg L(-1), 96 hpf; 118.0 mg L(-1), and 120 hpf; 65.9 mg L(-1). In the Early-life Stage Test with 5-FU, embryonic deformities were not detected during the tests. Regarding mortalities, the 10 mg L(-1) concentration can be considered as LOEC, as statistically significant difference in mortalities was detected in this group alone. Concerning dry body weight and standard length, 1 mg L(-1) is the LOEC. In case of IM, the highest tested concentration (10 mg L(-1)) can be considered LOEC for mortalities, however, the treatment did not have an effect on the other investigated parameters (dry and wet weight, standard length). All four cytostatic drugs were characterized by low toxicity in zebrafish in acute and sub-chronic tests.

Effect of salicylic acid on early life stages of common carp (Cyprinus carpio)

Environmental concentrations of pharmaceutical residues are often low; nevertheless, they are designed to have biological effects at low doses. The aim of this study was to assess the effects of salicylic acid on the growth and development of common carp (Cyprinus carpio) early life stages with respect to antioxidant defence enzymes. An embryo-larval toxicity test lasting 34 days was performed according to OECD guidelines 210 (Fish, Early-life Stage Toxicity Test). The tested concentrations were 0.004, 0.04, 0.4, 4 and 20mg/l of salicylic acid.Hatching, early ontogeny, and both morphometric and condition characteristics were significantly influenced by subchronic exposure to salicylic acid. Also, changes in antioxidant enzyme activity and an increase in lipid peroxidation were observed. The LOEC value was found to be 0.004mg/l salicylic acid.The results of our study confirm the suggestion that subchronic exposure to salicylic acid at environmental concentrations can have significant effects on aquatic vertebrates.

NeemAzal T/S - toxicity to early-life stages of common carp (Cyprinus carpio L.)

In the European Union, the use of the insecticide NeemAzal T/S (standardised variant 1% of the active ingredient azadirachtin) is authorised in organic agriculture. The objective of this study was to determine the toxic effects of NeemAzal T/S at concentrations of 3, 10, 30, and 60 mg/lon the morphometric and condition characteristics, mortality, hatching, and histopathology of early-life stages of common carp (Cyprinus carpio L.) as a non-target aquatic organism, as well as related effects of NeemAzal T/S on selected indices of oxidative stress in the same organism. The embryo-larval toxicity test was performed according to OECD Guidelines 210 (Fish, Early-life Stage Toxicity Test). NeemAzal T/S exposure induced slow hatching on the first day and increased cumulative mortality in groups exposed to the insecticide. No effect on morphometric or condition characteristics was observed after 31 days of exposure. Histopathological changes of the gills were found at the highest concentration of 60 mg/lof NeemAzal T/S. Exposure at 30 mg/lwas associated with significantly (P < 0.01) increased glutathione peroxidase and glutathione S-transferase (P < 0.05) activities compared to the control group. The content of oxidised lipids was significantly higher (P < 0.05) in the 3, 10, and 30 mg/l experimental groups than in the controls. NeemAzal T/S exerted a significant negative influence on histopathological parameters in the embryo and larvae of common carp, as well as on the indices of oxidative stress in the same organism.

Early Life Stage (ELS) Toxicity of Sucralose to Fathead Minnows, Pimephales promelas

Sucralose, an intense artificial sweetener, has been detected in wastewater and surface waters at concentrations ranging from ng/L to low µg/L. Although over a hundred studies have been conducted to evaluate the safety of sucralose for human consumption, few studies have focused on the chronic ecotoxicological effects of this compound in fish. As a remedy to this data gap, an early-life stage toxicity test was conducted to assess the effects of sucralose on hatching, survival, and growth of fathead minnows (Pimephales promelas). Hatching, survival, and growth were unaffected by 98 mg/L of sucralose. The Lowest-Observed-Effect Concentration (LOEC) and the No-Observed-Effect Concentration (NOEC) for fathead minnows determined by this study are >98 and 98 mg/L, respectively. The results from this study suggest that the concentrations of sucralose detected in the environment are well below those required to cause adverse effects to developing aquatic organisms.

Effects of mixture of pharmaceuticals on early life stages of tench (Tinca tinca).

Ubiquitous occurrence of pharmaceuticals in aquatic environment results in concern about potential adverse the effects on nontarget organisms. In water, drugs are present in complex mixtures, in which complicated interactions affect toxicity of single components. The purpose of this study was to examine effect of 35-day-long exposure to mixture of ibuprofen, diclofenac, and carbamazepine on the mortality, growth, early ontogeny, and histopathological changes in tench (Tinca tinca). Early life stage toxicity test was carried out using a modified protocol according to OECD guideline 210. Exposure to mixture of pharmaceuticals at concentration of 60 μg·L−1 for each substance was associated with significant increase in mortality, as well as significant increase in growth and elevated incidence of malformations. Any of the tested concentrations resulted in histopathological changes of liver, kidney, skin, or gill. After fourteen days of exposure there was short-term delay of development related to increased concentrations of pharmaceuticals in the mixture (2, 20, and 60 μg·L−1). Environmentally relevant concentrations (0.02; and 0.2 μg·L−1) used in this experiment did not result in toxic impairment of tench.

Selecting Optimal Eggs and Embryonic Developmental Stages of Fathead Minnow (Pimephales promelas) for Early Life-Stage Toxicity Tests

Aquaculture research has indicated that fish embryo hatching success and larval survival can sometimes be predicted by embryo characteristics, such as blastomere cleavage patterns. An analogous strategy of individual assessment of spawned eggs could also be used to improve the quality of toxicity tests using early life-stages of fish where control-group survival determines experimental validity. Here we explored whether a simple method of assessing fathead minnow eggs and embryos for abnormalities could predict hatch success, and larval size at hatch, as indicators of embryo larval quality. Embryos were classified according to both their developmental stage and the presence of any abnormalities: uneven blastomere cleavage, atypical embryo size or shape, and the presence of inclusions in the yolk. Clutch size and fertilization rate did not predict embryo larval quality. Fewer abnormalities in embryos with ≤32 cells correlated with longer larvae at hatch. Normal embryos were more likely to hatch successfully than abnormal embryos of the same clutch, but because abnormality rates were generally low, much of the variation in hatch success could not be attributed to visible embryo malformations. Blastomere symmetry may be a useful selection criterion in embryos <3 h postfertilization. Where toxicant exposures early in embryonic development are not required or possible, hatch success could be increased by using older embryos that have survived gastrulation. Purposeful selection of embryos with at least two blastomeres, blastomere symmetry, and few inclusions can improve control survival and improve the quality of any generated (sub)lethality data. In our laboratory, application of the egg-selection criteria significantly improved control group hatch success increasing it from a mean of 84.4 to 94.2%.

Evaluating the efficiency of advanced wastewater treatment: Target analysis of organic contaminants and (geno-)toxicity assessment tell a different story

At a pilot scale wastewater treatment plant ozonation and powdered activated carbon filtration were assessed for their efficacy to remove trace organic contaminants from secondary treated effluents. A chemical analysis of 16 organic compounds was accompanied by a comprehensive suite of in vitro and in vivo bioassays with the focus on genotoxicity to account for the potential formation of reactive oxidation products. In vitro experiments were performed with solid phase extracted water samples, in vivo experiments with native wastewater in a flow through test system on site at the treatment plant. The chemical evaluation revealed an efficient oxidation of about half of the selected compounds by more than 90% at an ozone dose of 0.7 g/g DOC. A lower oxidizing efficiency was observed for the iodinated X-ray contrast media (49–55%). Activated carbon treatment (20 mg/L) was less effective for the removal of most pharmaceuticals monitored. The umuC assay on genotoxicity delivered results with about 90% decrease of the effects by ozonation and slightly lower efficiency for PAC treatment. However, the Ames test on mutagenicity with the strain YG7108 revealed a consistent and ozone-dose dependent increase of mutagenicity after wastewater ozonation compared to secondary treatment. Sand filtration as post treatment step reduced the ozone induced mutagenicity only partly. Also the fish early life stage toxicity test revealed an increase in mortality after ozonation and a reduced effect after sand filtration. Only activated carbon treatment reduced the fish mortality compared to conventional treatment on control level. Likewise the in vivo genotoxicity detected with the comet assay using fish erythrocytes confirmed an increased (geno-)toxicity after ozonation, an effect decrease after sand-filtration and no toxic effects after activated carbon treatment.This study demonstrates the need for a cautious selection of methods for the evaluation of advanced (oxidative) treatment technologies and of the effectiveness of post-treatments for elimination of adverse effects caused by oxidative treatments case by case.

Molecular Cloning and Gene Expression of Mummichog (Fundulus heteroclitus) Runx2 During Embryogenesis

In our previous study, we clarified the toxicity of 2,2'-dipyridyldisulfide [(PS)2], one of photodegradation products of a metal pyrithione that is used as an alternative antifouling paint biocides to organotin compounds in Japan. In early life stage toxicity tests, we exposed the mummichog, (Fundulus heteroclitus) to (PS)2, and the hatched larvae subsequently displayed notochord undulations and skeletal deformities ( Mochida et al., 2012 ). Runx2, a transcription factor of the runt family, is a key regulator in skeletal development in mammals. It is possible that (PS)2 inhibits Runx2 gene expression, inducing the skeletal deformities in mummichog. In the present study, we cloned two Runx2 cDNAs (type I and type II) from mummichog embryos. The deduced amino acid sequences of type I and type II contain an open reading frame encoding 450 and 464 amino acid residues, respectively. The derived amino acid sequence of Fundulus Runx2 type I showed the highest identity (93.8%) with Takifugu Runx2 type I, and Fundulus Runx2 type II showed 94.6% homology with medaka Runx2. The expression level of Runx2 mRNA in the early stage series was measured using a real-time quantitative PCR assay. Expression levels tended to increase in both the blastula-gastrula and the retinal pigmentation stage. To examine the effect of toxic compounds on skeletal formation, mummichog embryos in the late blastula to retinal pigmentation stage were exposed to (PS)2. After exposure to (PS)2 for one week, the expression level of Runx2 mRNA was unchanged. These results suggest that there is no inhibition of Runx2 gene expression due to (PS)2 exposure.

A high-throughput histoarray for quantitative molecular profiling of multiple, uniformly oriented medaka ( Oryzias latipes) embryos

Embryos of aquatic animal model fish have proven to be useful organisms for developmental biology and for early life stage toxicity tests. By virtue of their transparent chorions, imaging of normal and abnormal development can be detected and related to exposure or to alterations due to environmental factors. However, the detection of changes at sub-individual levels of organization is hampered by time required to detect important events within cells and tissues of affected organisms. We describe herein development of a highly cost effective embryo chip enabling stringent inter-individual comparisons and multiplex detection in embryos and eleutheroembryos. As a proof of principle we examine cell proliferation and controlled cell death in normoxic and hypoxic conditions and relate these to tissue turnover in individual organisms. Coupled with a recently developed whole adult animal platform, we can now move beyond the common approach focusing on single target organ to the detection and characterization of systemic phenomena (syndromes) affecting the organism. Taken together, we can now determine adult consequences of early life stage exposure and assess ability of exposed individuals to respond to stresses superimposed along the axis of time.

Toxication or detoxication? In vivo toxicity assessment of ozonation as advanced wastewater treatment with the rainbow trout

Ozonation as advanced wastewater treatment method is an effective technique for micropollutant removal. However, the application of this method carries the inherent danger to produce toxic oxidation byproducts. For an ecotoxicological assessment conventionally treated wastewater, wastewater after ozonation and ozonated wastewater after sand filtration were evaluated in parallel at an operating treatment plant via the fish early life stage toxicity test (FELST) using rainbow trout ( Oncorhynchus mykiss). The FELST revealed a considerable developmental retardation of test organisms exposed to ozonated WW. This was accompanied by a significant decrease in body weight and length compared to reference water, to the conventionally treated WW and to the ozonated water after sand filtration. Hence sand filtration obviously prevents from adverse ecotoxicological effects of ozonation. An additional test with yolk-sac larvae resulted in a significant reduction of vitellogenin levels in fish exposed to ozonated wastewater compared to fish reared in conventionally treated wastewater. This demonstrates the effective removal of estrogenic activity by ozonation. Adverse ozonation effects may have been a result of the conversion of chemicals into more toxic metabolites. However, sand filtration reduced toxication effects indicating that these oxidation byproducts are readily degradable or adsorbable. The results indicate that in any case ozonation should not be applied without subsequent post treatment appropriate for oxidation byproducts removal (e.g. sand filtration).

Java medaka: A proposed new marine test fish for ecotoxicology

Java medaka (Oryzias javanicus), which is distributed in estuarine waters of East to Southeast Asia, is proposed here for consideration as a new marine test fish. Semi-static acute toxicity tests were conducted at 26 degrees C for 1 week, 2 weeks, and 1 month old Java-medaka exposed to 4-chlorophenol and zinc, and 2 weeks olds exposed to potassium dichromate (hexavalent chromium) and sodium hypochlorite (residue chlorine). Ninety-six hours LC50s of 4-chlorophenol, zinc, hexavalent chromium, and residue chlorine were from 3.0 to 4.1, from 12.4 to 14.7, 7.4, and 0.05 mg/L, respectively. There were no large differences in sensitivity for 4-chlorophenol and zinc among the three growth stages. Compared with other fish species, these LC50s were relatively low suggesting that Java medaka has relatively high sensitivity to chemicals. This fish is quite easy to keep in the laboratory, with low mortalities at early stages and it takes about 3 months to mature from hatching, suggesting that this fish can be used not only for short-term toxicity tests but also for early life stage toxicity tests. From these results, Java-medaka is highly recommended as a marine test fish for ecotoxicity tests.