Selected endocrine disrupting compounds (Vinclozolin,Flutamide, Ketoconazole and Dicofol): Effects on survival,occurrence of males,growth, molting and reproduction of <Emphasis Type="Italic">Daphnia magna</Emphasis>

BACKGROUND, AIM AND SCOPE: Pollution-induced endocrine disruption in vertebrates and invertebrates is a worldwide environmental problem, but relatively little is known about effects of endocrine disrupting compounds (EDCs) in planktonic crustaceans (including Daphnia magna). Aims of the present study were to investigate acute 48 h toxicity and sub-chronic (4-6 days) and chronic (21 days) effects of selected EDCs in D. magna. We have investigated both traditional endpoints as well as other parameters such as sex determination, maturation, molting or embryogenesis in order to evaluate the sensitivity and possible use of these endpoints in ecological risk assessment. MATERIALS AND METHODS: We have studied effects of four model EDCs (vinclozolin, flutamide, ketoconazole and dicofol) on D. magna using (i) an acute 48 h immobilization assay, (ii) a sub-chronic, 4-6 day assay evaluating development and the sex ratio of neonates, and (iii) a chronic, 21 day assay studying number of neonates, sex of neonates, molting frequency, day of maturation and the growth of maternal organisms. RESULTS: Acute EC50 values in the 48 h immobilization test were as follows (mg/L): dicofol 0.2, ketoconazole 1.5, flutamide 2.7, vinclozolin >3. Short-term, 4-6 day assays with sublethal concentrations showed that the sex ratio in Daphnia was modulated by vinclozolin (decreased number of neonate males at 1 mg/L) and dicofol (increase in males at 0.1 mg/L). Flutamide (up to 1 mg/L) had no effect on the sex of neonates, but inhibited embryonic development at certain stages during chronic assay, resulting in abortions. Ketoconazole had no significant effects on the studied processes up to 1 mg/L. DISCUSSION: Sex ratio modulations by some chemicals (vinclozolin and dicofol) corresponded to the known action of these compounds in vertebrates (i.e. anti-androgenicity and anti-oestrogenicity, respectively). Our study revealed that some chemicals known to affect steroid-regulated processes in vertebrates can also affect sublethal endpoints (e.g. embryonic sex determination and/or reproduction) in invertebrates such as D. magna. CONCLUSIONS: A series of model vertebrate endocrine disrupters affected various sub-chronic and chronic parameters in D. magna including several endpoints that have not been previously studied in detail (such as sex determination in neonates, embryogenesis, molting and maturation). Evaluations of traditional reproduction parameters (obtained from the 21 day chronic assay). as well as the results from a rapid, 4-6 day, sub-chronic assay provide complementary information on non-lethal effects of suspected organic endocrine disrupters. RECOMMENDATIONS AND PERSPECTIVES: It seems that there are analogies between vertebrates and invertebrates in toxicity mechanisms and in vivo effects of endocrine disruptors. However, general physiological status of organisms may also indirectly affect endpoints that are traditionally considered 'hormone regulated' (especially at higher effective concentrations as observed in this study) and these factors should be carefully considered. Further research of D. magna physiology and comparative studies with various EDCs will help to understand mechanisms of action as well as ecological risks of EDCs in the environment.     

Evaluation of the fish short term reproduction assay for detecting endocrine disrupters

In a fish testing strategy, positive results of the fish short term reproduction assay (FSTR), often trigger a definitive test like the fish sexual development test (FSDT) or the fish full life cycle test (FFLC), entailing ethical and economic problems. This study analysed 137 studies encompassing 35 chemicals with different modes of actions (MOAs). Variability is quantified for MOA endpoints vitellogenin (VTG) and secondary sex characteristics (SSCs) as well as for apical endpoints. Two MOA endpoints could indicate estrogenic, anti-estrogenic, androgenic, anti-androgenic and steroidogenesis activities. Great variability, however, has been observed for chemicals with anti-androgenic and steroidogenesis activities, suggesting that TG229/230 may not be sensitive enough to detect these types of chemicals and may produce false negatives. Changes in apical endpoints like fecundity are not limited to endocrine disrupting chemicals (EDCs). Non-EDCs could induce the similar effects on these apical endpoints. If elucidating MOA is needed, targeted in vitro MOA tests are suggested. Positive in vitro MOA results trigger a definitive test, which could be used for confirmation of the MOA in vivo and for deriving a no observed effect concentration (NOEC). Based on positive MOA results of TG229, a definitive test such as the FSDT or the FFLC is still needed, because the current TG229 has limitation on the derivation of a NOEC. An extended TG229 with more power to detect reproduction effects, as recently proposed in the OECD test guideline program, would improve the possibility to derive a NOEC and increase its usefulness in risk assessment.     

Production of male neonates in Daphnia magna (Cladocera, Crustacea) exposed to juvenile hormones and their analogs

We exposed the water flea Daphnia magna (Cladocera, Crustacea) to either juvenile hormone I (JH I), juvenile hormone II (JH II), or the juvenile hormone-mimicking insecticides kinoprene, hydroprene, epofenonane, or fenoxycarb. By 21-day reproduction tests, we investigated the effects on the number of neonates born per female and the offspring sex ratio. All six chemicals induced D. magna to produce male neonates; the male sex ratio of the offspring increased as the chemical concentration increased. EC50 values for production of male neonates were estimated as 400 (JH I), 410 (JH II), 190 (kinoprene), 2.9 (hydroprene), 64 (epofenonane), and 0.92 (fenoxycarb) microg/l. The number of neonates produced was reduced with all chemicals at the concentrations investigated. At the EC50 for male production, five of the six chemicals reduced the reproductive rate to less than 50%; the exception was epofenonane, which caused only a slight reduction in reproductive rate. These results were similar to those obtained for five juvenoids studied previously, one of which was studied here again. There are now 10 chemical substances--all juvenile hormones or their analogs-that are known to induce D. magna to produce male neonates. This suggests that juvenile hormone is involved in initiating male production followed by sexual reproduction in D. magna, and probably in most cladocerans that exhibit cyclic parthenogenesis.     

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.     

Juvenile hormone agonists affect the occurrence of male Daphnia

The water flea Daphnia magna reproduces primarily by cyclic parthenogenesis. Environmental stimuli that signal a change to adverse conditions induce the organisms to switch from parthenogenesis to gamogenetic reproduction. During the gamogenetic period, they produce male daphnids and dormant resting eggs, which can survive prolonged periods of environmental adversity. However, little is known about the mechanisms associated with the switch from parthenogenesis to gamogenetic reproduction. We investigated the effects of several juvenoids on sex determination in Daphnia. Females less than 24 h old were exposed to various concentrations of the test substance and were observed for 21 days. It was found that they can trigger the appearance of male daphnids: the percentage of males in the population increases to a level greater than what occurs under ordinary environmental conditions. We found that methylfarnesoate, juvenile hormone III, methoprene, and the phenoxyphenoxy derivatives pyriproxyfen and fenoxycarb (both insecticides) reduced the production of offspring and produced sex ratios dominated by male daphnids. Pyriproxyfen and fenoxycarb showed striking effects at low concentrations. Exposure to either of these chemicals at a concentration of 330 ngl(-1) caused adult females to produce almost all male neonates. Methylfarnesoate, juvenile hormone III, and methoprene showed an effect in inducing male production at higher concentrations (3.7 x 10(3), 3.3 x 10(5), and 1.3 x 10(5) ngl(-1), respectively). Our findings suggest that juvenile hormone agonists, including some insecticides, affect the chemical signaling responsible for inducing the production of male offspring.     

Genetic differences in the production of male neonates in Daphnia magna exposed to juvenile hormone analogs

We studied the susceptibility of three genetically different strains of the cyclical parthenogen Daphnia magna (Cladocera, Crustacea) in producing male neonates following exposure to juvenile hormone analogs. In experiment 1, NIES, Clone A, and Belgium A strains were exposed to the insect growth regulators (IGRs) fenoxycarb or epofenonane in a 21-day reproduction experiment. Fenoxycarb exposure decreased the total number of neonates and increased production of male neonates in a concentration-dependent manner in the NIES strain. The decrease in the total number of neonates was so great in Clone A following fenoxycarb exposure that male neonates were not observed, even at the highest concentration, where the total number of neonates was only 2% of the control. In the Belgium A strain, male neonates were observed at a rate of about 20% following exposure to the highest fenoxycarb concentration, but the total number observed was small. Epofenonane did not decrease reproduction in the NIES and Belgium A strains as dramatically as did fenoxycarb, but the neonatal sex ratio changed in a concentration-dependent manner. Although the ratio of males was as low as about 10%, induction of male neonates was also observed in Clone A following epofenonane exposure. In experiment 2, gravid females were exposed to high concentrations (5 or 10 microg/l) of fenoxycarb or pyriproxyfen for 12h. These treatments induced the production of male neonates in all strains, with a small decrease in the total number of neonates. Although induction of male neonates by juvenile hormones and their analogs was universal among genetically different strains, care is needed in interpreting the results of the 21-day reproduction tests, because decreased numbers of neonates at higher concentrations could obscure the presence of male neonates.     

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.     

Reproduction disturbances of Brachionus calyciflorus (rotifer) for the screening of environmental endocrine disrupters

Rotifers and especially the Brachionus calyciflorus species have already shown advantages (e.g. good sensitivity, simplicity, ecological relevance, short generation time) for the chronic toxicity assessment of water pollutants. In this study, the B. calyciflorus three-day test was evaluated on ethinylestradiol, nonylphenol and testosterone for the determination of endocrine disrupting (reproductive) effects. Reproduction (intrinsic rate of population increase) and sex ratio (males/females and mictic females/amictic females) were explored and discussed as suitable endpoints. B. calyciflorus has shown a fair sensitivity to detect reproductive disrupting effects after exposure to ethinylestradiol, nonylphenol and testosterone. Population growth (intrinsic rate of population increase, r) and the ratio ovigerous females/non-ovigerous females are both suitable endpoints, whereas the occurrence of males was not frequent enough to be used as endpoint.     

Chronic toxicity tests with Daphnia magna for examination of river water quality

Chronic toxicity tests with Daphnia magna were applied for examination of river water quality. Water was sampled from the Maioka River in Yokohama City on May 14, 20, and 27, 1999, and used for the test after solid-phase extraction. The chronic test was carried out according to the OECD method. The duration was 21 days and the total number of live offspring produced per parent animal was counted. The results of the tests showed, survival rates of 100% using river water sampled on May 14 and 20 and the total numbers of live offspring produced per parent animal did not differ from the control. However, the survival rate of the sample collected on May 27 was 0% and the pesticides, fenitrothion, and thiobencarb were detected in the water. In addition to the river water samples, reconstituted water (Elendt M7) with additions of fenitrothion and thiobencarb was prepared to investigate mortality. When the reconstituted water with thiobencarb was applied to the test, the total number of live offspring produced per parent animal did not differ from the control. In contrast, when reconstituted water with fenitrothion was applied to the test, most parents were alive, but the total number of live offspring produced per parent animal was apparently different. The results of the above tests indicate that D. magna was affected not only by fenitrothion in the river water collected on May 27, but also by other factors that were not clarified in this study.     

Aquatic toxicity of cartap and cypermethrin to different life stages of Daphnia magna and Oryzias latipes

Cartap and cypermethrin, which are among the most widely used pesticides in many countries, are considered safe because of their low mammalian toxicity and their low persistence in the environment. However, recent findings of endocrine-disrupting effects and developmental neurotoxicity have raised concerns about the potential ecological impacts of these pesticides. We evaluated the aquatic toxicity of cartap [S,S'-(2-dimethylaminotrimethylene) bis(thiocarbamate), unspecified hydrochloride] and cypermethrin [(RS)-alpha-cyano-3-phenoxybenzyl-(1RS,3RS,1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate], both individually and combined, on different life stages of the freshwater cladoceran Daphnia magna and a freshwater teleost, Japanese medaka (Oryzias latipes). The 96-hr Daphnia median effective concentrations (EC50s) for cartap and cypermethrin were 91.0 microg/L and 0.00061 microg/L, respectively. Rapid recovery of Daphnia was observed after short-term pulsed exposure to cartap and cypermethrin; there were no adverse effects on reproduction or survival 20 d after a 24 hr exposure to cartap up to 1240 microg/L and cypermethrin up to 1.9 microg/L. Chronic continuous exposure (for 21 d) of 7-d-old Daphnia to cypermethrin significantly reduced the intrinsic population growth rate in a concentration-dependent manner. However, because the intrinsic population growth rates were all above zero, populations did not decrease even at the highest experimental concentration of 200 ng/L. Exposure of Daphnia neonates (< 24 hr old) to cypermethrin for 21 d caused significant, sub-lethal reproduction-related problems, such as increased time to first brood, reduced brood size, and reduced total brood number, at 0.0002, 0.002, and 0.2 ng/L cypermethrin, but the intrinsic population growth rate was not significantly affected. Oryzias latipes was relatively more resistant to both pesticides. In particular, embryos appeared to be more resistant than juveniles or adults, which may be partly due to the protective role of the chorion. The incidence of larval fish deformity was significantly higher after a 96 hr exposure to as low as 250 microg/L of cartap or 40 microg/L of cypermethrin. The mixture of both compounds showed no synergistic toxicity. The extremely high acute-to-chronic ratio suggests that the standard acute lethal toxicity assessment might not reflect the true environmental hazards of these frequently used pesticides. Ecological hazard assessments of long-term low dose or pulsed exposures to cartap and cypermethrin may reveal more realistic consequences of these compounds in surface water.     

A chronic bioassay with the estuarine amphipod Corophium volutator: test method description and confounding factors

Methods of conducting a chronic sediment toxicity test with the estuarine amphipod Corophium volutator are described. They consist of a 49-day exposure, after which mortality, growth and reproduction are determined. Pilot experiments were used to optimize test design parameters such as temperature, duration, feeding and refreshing regimes, and effects of indigenous organisms. By way of further validation, the present study focused on the effects of four different parameters: oxygen saturation, salinity, ammonium and nitrite. These confounding factors might play an important role especially if the test is used for risk assessment of field-contaminated sediments. It is concluded that the present experimental design is well suited for chronic sediment exposures with C. volutator. The test can be performed at a broad range of salinity values, provided that controls are performed at the same salinity. Results further demonstrate that with the endpoints growth and reproduction this chronic test procedure is a factor 7-18 more sensitive to ammonium and nitrate than the standardized acute bioassay (endpoint mortality).     

Reproduction disturbances of Brachionus calyciflorus (rotifer) for the screening of environmental endocrine disrupters

Rotifers and especially the Brachionus calyciflorus species have already shown advantages (e.g. good sensitivity, simplicity, ecological relevance, short generation time) for the chronic toxicity assessment of water pollutants. In this study, the B. calyciflorus three-day test was evaluated on ethinylestradiol, nonylphenol and testosterone for the determination of endocrine disrupting (reproductive) effects. Reproduction (intrinsic rate of population increase) and sex ratio (males/females and mictic females/amictic females) were explored and discussed as suitable endpoints. B. calyciflorus has shown a fair sensitivity to detect reproductive disrupting effects after exposure to ethinylestradiol, nonylphenol and testosterone. Population growth (intrinsic rate of population increase, r) and the ratio ovigerous females/non-ovigerous females are both suitable endpoints, whereas the occurrence of males was not frequent enough to be used as endpoint.     

Toxicity of two types of silver nanoparticles to aquatic crustaceans Daphnia magna and Thamnocephalus platyurus

Although silver nanoparticles (NPs) are increasingly used in various consumer products and produced in industrial scale, information on harmful effects of nanosilver to environmentally relevant organisms is still scarce. This paper studies the adverse effects of silver NPs to two aquatic crustaceans, Daphnia magna and Thamnocephalus platyurus. For that, silver NPs were synthesized where Ag is covalently attached to poly(vinylpyrrolidone) (PVP). In parallel, the toxicity of collargol (protein-coated nanosilver) and AgNO₃ was analyzed. Both types of silver NPs were highly toxic to both crustaceans: the EC50 values in artificial freshwater were 15–17 ppb for D. magna and 20–27 ppb for T. platyurus. The natural water (five different waters with dissolved organic carbon from 5 to 35 mg C/L were studied) mitigated the toxic effect of studied silver compounds up to 8-fold compared with artificial freshwater. The toxicity of silver NPs in all test media was up to 10-fold lower than that of soluble silver salt, AgNO₃. The pattern of the toxic response of both crustacean species to the silver compounds was almost similar in artificial freshwater and in natural waters. The chronic 21-day toxicity of silver NPs to D. magna in natural water was at the part-per-billion level, and adult mortality was more sensitive toxicity test endpoint than the reproduction (the number of offspring per adult).     

Enantiospecific sublethal effects of the antidepressant fluoxetine to a model aquatic vertebrate and invertebrate

Many contaminants are chiral compounds with enantiomers that may differ markedly in environmental fate, bioavailability, and toxicity. Enantiospecific environmental fate and ecotoxicological information are lacking for many chiral contaminants. The primary objective of this investigation included an assessment of potential enantiospecific differences in sublethal standardized and behavioral responses of the model organisms Pimephales promelas (teleost) and Daphnia magna (crustacean) to the widely prescribed chiral antidepressant fluoxetine. Endpoints assessed included D. magna immobilization, reproduction, and grazing rate and P. promelas survival, growth, and feeding rate. S-Fluoxetine was found to be more toxic to sublethal standardized and behavioral endpoints in P. promelas, potentially because its primary active metabolite, S-norfluoxetine, is more potent than the same metabolite of R-fluoxetine in mammals. This was not observed for D. magna responses. This differential enantiospecific response between model organisms may have resulted from closer target homology between mammals and fish than between mammals and crustaceans. P. promelas feeding rate, an ecologically relevant and mode-of-action related response, was the most sensitive endpoint tested for R- and S-fluoxetine with 10% effect concentration (EC10) values (+/-SE) of 16.1 (+/-20.2) and 3.7 (+/-4.6) microg l(-1), respectively. Up to a 9.4-fold difference in toxicity between enantiomers was observed; P. promelas growth EC10s (+/-SE) for R- and S-fluoxetine were 132.9 (+/-21.2) and 14.1 (+/-8.1) microg l(-1), respectively. Such differences in sublethal responses to fluoxetine enantiomers suggest that enantiospecific toxicity and mode-of-action related responses that are ecologically relevant (e.g., feeding rate) should be considered in future ecological hazard and risk assessments for chiral contaminants.     

Characterizing the toxicity of pulsed selenium exposure to Daphnia magna

The acute toxicity of selenium (Se) to aquatic biota has been studied extensively for decades. However, most studies have used a constant concentration aqueous exposure of Se to an invertebrate species. Since constant concentration exposure of toxicants to invertebrates is unusual in the environment, episodic exposure or pulsed exposures may represent true risk to aquatic biota more accurately. This research was designed to characterize the toxicity effects of pulsed Se exposure to Daphnia magna. Selenium exposure was varied during a 21-d chronic toxicity test to examine the effects of exposure concentration, duration, and recovery on survival, growth, and reproduction of D. magna. While D. magna did not die during exposures, latent mortality was observed. Latent mortality increased with exposure concentration and duration. Hence, standard toxicity test using continuous exposures would underestimate Se toxicity. Risk assessment method using results of continuous exposure would underestimate risk of Se to biota. For double-pulse exposures, cumulative mortality on day 21 was higher when time interval between pulses was shorter. With the same total exposure time, continuous exposure caused higher toxicity than did pulsed exposures due to recovery and tolerance development in D. magna after earlier pulses. Growth and reproduction of surviving D. magna were not affected by pulsed Se exposure due to recovery of D. magna after removal of the pulses. Based on these results, risk assessment for Se should take latent effects and the effect of recovery in to account.     

Problems in testing and risk assessment of endocrine disrupting chemicals with regard to developmental toxicology.

Endocrine disrupting chemicals (EDCs) may affect mammalian development either indirectly (by impairing implantation, placental development, lactation, etc.) or directly, altering the maturation of target tissues. Current regulatory tests for reproductive/developmental toxicity should be carefully evaluated with regard to risk assessment of EDCs, considering hazard identification (are relevant endpoints being assessed?) and dose-response assessment (are sensitive NOEL/dose-response curves being provided?). Many in vitro and in vivo assays for sex steroid disruption are available; provided that the metabolic capacities of the assays are defined, they could be integrated in a sensitive battery for early detection of steroid-disrupting potentials. The screening battery should address further regulatory in vivo tests (e.g. what specific parameters have to be investigated). As regards dose-response, qualitative differences may be observed between lower and higher exposures, showing primary hormone-related effects and frank embryotoxicity, respectively. Other problems concern (a) the identification of critical developmental windows, according to hormone concentrations and/or receptor levels in the developing target tissues; (b) the potential for interactions between chemicals with common mechanism/target (e.g. xenoestrogens); (c) most important, besides sex steroids more attention should be given to other mechanisms of endocrine disruption, e.g., thyroid effects, which can be highly relevant to prenatal and postnatal development.     

Anogenital distance and its application in environmental health research

Anogenital distance (AGD), a useful anthropometric measurement for genital development in both animals and humans, was originally found by reproductive toxicologists in rodent experiments. As an easy-to-measure and sensitive marker, AGD has become a bioassay of fetal androgen action and a well-established reproductive toxicity endpoint in animals. It is generally accepted that AGD is sexually dimorphic in many mammals, with males having longer AGD than females. Exposure to proposed endocrine disruptors may result in reduced AGD; thus, it has been used to measure health effects of compounds with endocrine-altering properties or endocrine-disrupting chemicals (EDCs) in environmental toxicology. Moreover, AGD is an important clinical measure to address endocrine-sensitive endpoints in the first year of life and to assess the adverse impact of in utero exposure to environmental EDCs. Recently, AGD has been identified as one of the endpoints in the US Environmental Protection Agency guidelines for reproductive toxicity studies in humans, but use of AGD in human studies is still rare, and the results remain mixed and inconclusive due to many reasons. In order to achieve a breakthrough, researchers are endeavoring to standardize the measurement of AGD, normalize age-specific population data in different ethnic groups, and conduct more in-depth human researches in this field.     

Determination of pesticides and chronic test with Daphnia magna for rainwater samples

Pesticides contained in rainwater were investigated in June and July 2001 in Yokohama. Although there were few farms and rice paddies near the sampling point, insecticides (fenitrothion and dichlorvos) and fungicides (chlorothalonil and futhalide) were detected by GC-MS and GC-FPD analysis. The maximum concentrations of dichlorvos and fenitrothion were 0.22 microg/L and 0.15 microg/L, respectively. Chronic tests with Daphnia magna were then conducted on two rainwater samples. Test solutions for these chronic tests were prepared by solid-phase extraction. In the case of the rainwater sampled on 25 July, the survival rate of D. magna was 100% after 21 days and no differences were found between the control and sample with regard to the average total number of live offspring produced per parent animal. However, a similar test with rainwater sampled on 14-15 June showed a marked toxic effect, since all the test animals were dead within 2 days. It is concluded that rainwater containing pesticides is an important ecological factor for freshwater plankton.     

Two-generation toxicity study on the copepod model species Tigriopus japonicus

Previous studies on the intertidal copepod Tigriopus japonicus have demonstrated that it is a suitable model species for the assessment of acute toxicities of marine pollutants. In order to standardize T. japonicus for use in environmental risk assessment involving whole life cycle exposure, we tested nine pollutants for their effects on growth and reproduction during a two-generation life cycle exposure test. Nauplii (F 0) were exposed to a range of concentrations of each chemical in a static renewal culture system. Broods of the second generation (F1) were subsequently exposed to the same concentrations for one full life cycle. Of the seven traits (nauplius phase, development time, survival, sex ratio, number of clutch, nauplii per clutch and fecundity), only the length of the nauplius phase and development time showed a greater sensitivity to chemical exposure. Between the two sensitive traits, the period of the nauplius phase was more sensitive than cohort generation time. Biocides significantly increased the maturation period of nauplii as well as copepodids in F 0 generation. In this study, it was demonstrated that T. japonicus could also be used in reproduction and life cycle tests and it provides an opportunity for testing the chronic and subchronic toxic effects of marine pollutants. Further validation and harmonization in a multi-centric study involving other laboratories of the region will strengthen its use as a supplement to existing model species.     

The utility of behavioral studies for aquatic toxicology testing: A meta-analysis

Behavioral responses have been applied for decades as tools for aquatic toxicity testing, but have received far less attention than studies assessing lethality, development or reproduction. With improved visual and non-visual assessment tools and increased knowledge of the importance of behavior for organism health and fitness, interest in behavioral analysis has increased in recent years. However, to our knowledge there has never been a quantitative assessment of the available techniques for organismal toxicity testing, so it is not clear whether behavioral studies represent valuable additions to environmental monitoring. We performed a meta-analysis comparing the relative sensitivities and average durations of behavioral studies to those assessing acute lethality, development and reproduction. Results demonstrate that the average duration of behavioral studies is consistently less than developmental or reproductive studies, and that behavioral endpoints are generally more sensitive than those assessing development or reproduction. We found effect sizes to be lower but power to be higher in behavioral and reproductive studies compared to studies assessing development, which likely relates to low sample sizes commonly used in developmental studies. Overall, we conclude that behavioral studies are comparatively fast and sensitive, and therefore warrant further attention as tools for assessing the toxicological effects of environmental contaminants. We suggest that research aimed at developing and optimizing techniques for behavioral analysis could prove extremely useful to the field of toxicology, but that future work must be directed at determining what specific behaviors are most sensitive to various classes of contaminants, and at understanding the relevance of changes to discrete behaviors for influencing organismal and population-level health and fitness.