Drifting towards the surface: a shift in newborn pipefish's vertical distribution when exposed to the synthetic steroid ethinylestradiol

Endocrine disrupting chemical (EDC) effects during early life have the potential to modulate population structure, either directly through increased mortality or by causing inappropriate aggregation events, thus affecting the number of young that will reach adulthood. An alteration in the dispersal and recruitment patterns can also impair the connectivity among geographically distant populations. However, the detection of EDC-induced effects occurring after egg hatch, when newborns increase their chances of contacting with environmentally dispersed contaminants, is not a simple process as effects might be masked by the large natural mortality rates that usually occur during fish early life.Since there is a lack of information regarding the impact of EDCs on fish early life dispersal patterns, particularly on vertical migrations patterns, the effects of environmentally relevant concentrations of EE₂ on the vertical distribution of newborn fish was assessed through an ex situ exposure experiment. Syngnathus abaster newborns were exposed to EE₂ (nominal concentrations of 8, 12 and 36 ng L⁻¹) and the dynamics of their vertical distribution was monitored for up to 40 d. No significant differences in overall mortality were observed between treatments or in the dynamics of the registered death curves. Nevertheless, an alteration in the distribution patterns was observed. The commonly benthic newborn tended to shift their vertical distribution towards the surface, in a dose-dependent manner. Curiously, a follow up of the exposed pipefish confirmed that EE₂ effects were also noticeable upon sexual maturity, namely by the alteration of several primary and secondary sexual characters. The observation that vertical distributional patterns, at least in pipefish, are clearly altered at environmentally relevant EE₂ concentrations indicates that EDC’s impact in fish larvae behaviour should be considered when addressing the effects of contaminants, given the obvious implications on population connectivity, stability and persistence.     

Developmental toxicity of cypermethrin in embryo-larval stages of zebrafish

Cypermethrin, a type II pyrethroid insecticide, is widely used throughout the world in agriculture, forestry, horticulture and homes. Though the neurotoxicity of cypermethrin has been thoroughly studied in adult rodents, little is so far available regarding the developmental toxicity of cypermethrin to fish in early life stages. To explore the potential developmental toxicity of cypermethrin, 4-h post-fertilization (hpf) zebrafish embryos were exposed to various concentrations of cypermethrin (0, 25, 50, 100, 200 and 400 μg L⁻¹) until 96 h. Among a suite of morphological abnormalities, the unique phenotype curvature was observed at concentrations as low as 25 μg L⁻¹. Studies revealed that 400 μg L⁻¹ cypermethrin significantly increased malondialdehyde production. In addition, activity of antioxidative enzymes including superoxide dismutase and catalase were significantly induced in zebrafish larvae in a concentration-dependent manner. To further investigate the toxic effects of cypermethrin on fish, acridine orange (AO) staining was performed at 400 μg L⁻¹ cypermethrin and the result showed notable signs of apoptosis mainly in the nervous system. Cypermethrin also down-regulated ogg1 and increased p53 gene expression as well as the caspase-3 activity. Our results demonstrate that cypermethrin was able to induce oxidative stress and produce apoptosis through the involvement of caspases in zebrafish embryos. In this study, we investigated the developmental toxicity of cypermethrin using zebrafish embryos, which could be helpful in fully understanding the potential mechanisms of cypermethrin exposure during embryogenesis and also suggested that zebrafish could serve as an ideal model for studying developmental toxicity of environmental contaminants.     

Assessing estrogenic activity in surface water and sediment of the Liao River system in northeast China using combined chemical and biological tools

This paper investigated some selected estrogenic compounds (4-t-octylphenol: 4-t-OP; 4-nonylphenols: 4-NP; bisphenol-A: BPA; diethylstilbestrol: DES; estrone: E1; 17β-estradiol: E2; 17α-Ethinylestradiol: EE2; triclosan: TCS) and estrogenicity in the Liao River system using the combined chemical and in vitro yeast screen bioassay and assessed their ecological risks to aquatic organisms. The estrogenic compounds 4-t-OP, 4-NP, BPA, E1, E2 and TCS were detected in most of the samples, with their concentrations up to 52.1 2065.7, 755.6, 55.8, 7.4 and 81.3 ng/L in water, and up to 8.6, 558.4, 33.8, 7.9, <LOQ and 33.9 ng/g in sediment, respectively. However, DES and EE2 were not detected in the Liao River. The estrogen equivalents (EEQ) of the water and sediment samples were also measured by the bioassay. High estrogenic risks to aquatic organisms were found in the river sections of metropolitan areas and the lower reach of the river system.     

The freshwater planarian Polycelis felina as a sensitive species to assess the long-term toxicity of ammonia

Behavioural endpoints are a good link between physiological and ecological effects. However long-term behavioural endpoints are not uniformly studied over all different organism groups. For example behaviour has been scarcely studied in planarians. Unionized ammonia (NH₃) is one of the most widespread pollutants in developed countries, and is known to alter animal behaviour. In this study a long-term (30 d) bioassay was conducted to assess the effect of this pollutant on survival and behavioural activity (e.g. locomotion activity) of the freshwater planarian Polycelis felina. One control and three environmentally-realistic concentrations of unionized ammonia (treatments of 0.02, 0.05, and 0.09 mg N-NH₃ L⁻¹) were used in quintuplicate. The behaviour of planarians was measured after 0, 10, 20 and 30 d of ammonia exposure. Mortality was recorded every 2 d. Unionized ammonia increased mortality in the two highest NH₃ concentrations and the locomotory activity was depressed in all treatments after 20 d of exposure. Behavioural effect was observed at concentrations 20 times lower than the short-term LC50 for this species. Previous studies proposed safe concentrations of unionized ammonia of 0.01-0.10 mg N-NH₃ L⁻¹ to aquatic ecosystems, but our study has shown that these concentrations will affect planarians. Because planarians play a key role in streams (as predator/scavenger), safe concentrations should be below 0.02 mg N-NH₃ L⁻¹ to protect this species in the freshwater community. Our results can contribute to improve the knowledge about ammonia toxicity to freshwater ecosystems, we recommend that safe concentrations of unionized ammonia should be based on very sensitive species.     

Integrated biological responses of zebrafish (Danio rerio) to analyze water quality in regions under anthropogenic influence

This study analyzed water quality in regions around Patos lagoon (Southern Brazil) that are under anthropogenic pressure. Water samples were collected from five different sites, including one used as a source for human consumption (COR) and others known to be influenced by human activities (IP). Danio rerio (Teleostei, Cyprinidae) organisms were exposed for 24 h to these water samples, plus a control group. It was observed that: (1) reactive oxygen species levels were lower in COR and IP than in the control group; (2) glutamate-cysteine ligase (catalytic subunit) expression was higher in COR than in other sites; (3) exposure to all water samples affected long-term memory (LTM) when compared to control group. Thus, some water samples possess the ability to modulate the antioxidant system and to induce a decline in cognitive functions, as measured by LTM. The obtained results indicate that a combination of variables of different organization level (molecular, biochemical and behavioral) can be employed to analyze water quality in impacted regions.     

Amphibians and agricultural chemicals: Review of the risks in a complex environment

Agricultural landscapes, although often highly altered in nature, provide habitat for many species of amphibian. However, the persistence and health of amphibian populations are likely to be compromised by the escalating use of pesticides and other agricultural chemicals. This review examines some of the issues relating to exposure of amphibian populations to these chemicals and places emphasis on mechanisms of toxicity. Several mechanisms are highlighted, including those that may disrupt thyroid activity, retinoid pathways, and sexual differentiation. Special emphasis is also placed on the various interactions that may occur between different agro-chemicals and between chemicals and other environmental factors. We also examine the indirect effects on amphibian populations that occur when their surrounding pond communities are altered by chemicals.     

Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Danio rerio) embryos

It is reported that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils are cardiotoxic. However, the action mechanism of PAHs on vertebrate cardiovascular development and disease is unclear. In the present study, the cardiac morphology and functioning of zebrafish embryos exposed to benzo[a]pyrene [B(a)P], as a high-ring PAHs, for 72 h were observed and determined. The results showed that B(a)P exposure resulted in cardiac developmental defects in zebrafish embryos. Significant changes in expression level of multiple genes potentially critical for regulating the B(a)P-induced cardiovascular developmental defects were also found. A gene network regulating cardiac development perturbed by B(a)P exposure was identified and established by computational analysis and employment of some databases. The information from the network could provide a clue for further mechanistic studies explaining molecular events regulating B(a)P-mediated cardiovascular defects and consequences.     

Toxic masking and synergistic modulation of the estrogenic activity of chemical mixtures in a yeast estrogen screen (YES)

Background, aim and scope

Estrogenic and non-estrogenic chemicals typically co-occur in the environment. Interference by non-estrogenic chemicals may confound the assessment of the actual estrogenic activity of complex environmental samples. The aim of the present study was to investigate whether, in which way and how seriously the estrogenic activity of single estrogens and the observed and predicted joint action of estrogenic mixtures is influenced by toxic masking and synergistic modulation caused by non-estrogenic chemical confounders.

Materials and methods

The yeast estrogen screen (YES) was adapted so that toxicity and estrogenicity could be quantified simultaneously in one experimental run. Mercury, two organic solvents (dimethyl sulfoxide (DMSO) and 2,4-dinitroaniline), a surfactant (LAS-12) and the antibiotic cycloheximide were selected as toxic but non-estrogenic test chemicals. The confounding impact of selected concentrations of these toxicants on the estrogenic activity of the hormone 17ß-estradiol was determined by co-incubation experiments. In a second step, the impact of toxic masking and synergistic modulation on the predictability of the joint action of 17ß-estradiol, estrone and estriol mixtures by concentration addition was analysed.

Results

Each of the non-estrogenic chemicals reduced the apparent estrogenicity of both single estrogens and their mixtures if applied at high, toxic concentrations. Besides this common pattern, a highly substance- and concentration-dependent impact of the non-estrogenic toxicants was observable. The activity of 17ß-estradiol was still reduced in the presence of only low or non-toxic concentrations of 2,4-dinitroaniline and cycloheximide, which was not the case for mercury and DMSO. A clear synergistic modulation, i.e. an enhanced estrogenic activity, was induced by the presence of slightly toxic concentrations of LAS-12. The joint estrogenic activity of the mixture of estrogens was affected by toxic masking and synergistic modulation in direct proportion to the single estrogens, which allowed for an adequate adaptation of concentration addition and thus unaffected predictability of the joint estrogenicity in the presence of non-estrogenic confounders.

Discussion

The modified YES proved to be a reliable system for the simultaneous quantification of yeast toxicity and estrogen receptor activation. Experimental results substantiate the available evidence for toxic masking as a relevant phenomenon in estrogenicity assessment of complex environmental samples. Synergistic modulation of estrogenic activity by non-estrogenic confounders might be of lower importance. The concept of concentration addition is discussed as a valuable tool for estrogenicity assessment of complex mixtures, with deviations of the measured joint estrogenicity from predictions indicating the need for refined analyses.

Conclusions

Two major challenges are to be considered simultaneously for a reliable analysis of the estrogenic activity of complex mixtures: the identification of known and suspected estrogenic compounds in the sample as well as the substance- and effect-level-dependent confounding impact of non-estrogenic toxicants.

Recommendations and perspectives

The application of screening assays such as the YES to complex mixtures should be accompanied by measures that safeguard against false negative results which may be caused by non-estrogenic but toxic confounders. Simultaneous assessments of estrogenicity and toxicity are generally advisable.     

U-series disequilibria in a groundwater flow route as an indicator of uranium migration processes

U-series data relating to groundwater, fracture coatings and the adjoining rock matrix in a groundwater flow system at the Palmottu natural analogue site was examined. The aim was to obtain an experimental reference for migration modelling in a transport section defined within the flow system. The U-series reference obtained turned out to be a very useful tool for fine tuning the flow route and for migration mechanism considerations. The U-series data are well in line with other interpretations of the migration system.     

Development of a terrestrial vertebrate model for assessing bioavailability of cadmium in the fence lizard (Sceloporus undulatus) and in ovo effects on hatchling size and thyroid function

In the terrestrial environment, standardized protocols are available for measuring the exposure and effects of contaminants to invertebrates, but none currently exist for vertebrates. In an effort to address this, we proposed that developing lizard embryos may be used as a terrestrial vertebrate model. Lizard eggs may be particularly susceptible to soil contamination and in ovo exposure may affect hatchling size, mortality, as well as thyroid function. Toxicant-induced perturbations of thyroid function resulting from in ovo chemical exposure may result in toxicity during the critical perinatal period in reptiles. Fertilized Eastern fence lizard (Sceloporus undulatus) eggs were placed in cadmium (Cd)-spiked expanded perlite (0, 1.48, 14.8, 148, 1480, 14 800 μg Cd/g, nominal concentrations), artificially incubated at 28 °C, and examined daily for mortality. Whole lizard hatchlings as well as failed hatches were homogenized in ethanol and the homogenate was divided for Cd body residue analysis and thyroid hormone (triiodothyronine (T₃) and thyroxine (T₄)) analyses. Acute mortality was observed in the two highest doses (1480 and 14 800 μg Cd/g). Cadmium body residues showed a higher internal concentration with increasing exposure concentration indicating uptake of Cd. There was a decrease in T₃:T₄ ratio at the highest surviving dose (148 μg Cd/g), however, there were no differences observed in hatchling size measured as weight and snout-vent length, or in whole body thyroid hormone levels. In summary, this study has shown Cd amended to a solid phase representing soil (perlite) can traverse the thin, parchment-like shell membrane of the fence lizard egg and bioaccumulate in lizard embryos. We believe this study is a good first step in investigating and evaluating this species for use as a model.     

Short-term toxicity of pyraclofos used as a blackfly larvicide on non-target aquatic fauna in a tropical environment

Among the many larvicides tested for the control of s.l. larvae, the vector of human onchocerciasis in West Africa, pyraclofos proved to be 100% effective at 100 μg × L⁻¹ for 10 min in river, with a carry of 20 km at 100 m³ × sec⁻¹. Tests were then performed both in laboratory and field conditions to evaluate its toxicity on the non-target aquatic fauna. In experimental short-term gutter tests, the detachment of the total benthic insects was 35% at 100 μg × L⁻¹ for 10 min against 17% for temephos at the same dose and 59% for chlorphoxim at 50μg × L⁻¹ for 10 min. , and were the most affected organisms. The treatment of a river resulted in a considerable detachment of the same taxonomic groups, plus Orthocladiinae. On the other hand, investigations conducted in tanks showed that the 24-hr LC₅₀ for is 150 μg × L⁻¹ and that for 170 μg × L⁻¹, values which are not very different from the operational dose of the larvicide (100 μg × L⁻¹ for 10 min.). Nevertheless, in a river, no fish mortality was recorded. Based on fish LC₅₀ and drift of benthic insects, pyraclofos at 100 μg × L⁻¹ was judged to be less toxic to aquatic fauna in the short term than permethrin and carbosulfan.     

Monitoring anthropogenic sewage pollution on mangrove creeks in southern Mozambique: a test of Palaemon concinnus Dana, 1852 (Palaemonidae) as a biological indicator

Tropical coastal ecosystems, such as mangroves, have a great ecological and socioeconomic importance for adjacent systems and local populations, but intensive environmental impact monitoring is still lacking, mainly in East Africa. This study evaluated the potential anthropogenic disturbance on Palaemon concinnus population structure and fitness. Palaemon concinnus populations from one peri-urban (domestic sewage impacted) and two pristine mangrove creeks were studied by sampling nearly 100 shrimps per location every 15 days for 12 months. The shrimps at the peri-urban location were larger, experienced longer reproductive periods, presented higher proportion of ovigerous females and better embryo quality when compared with shrimps inhabiting pristine locations. Physiological indices (RNA/DNA ratio) were similar between shrimps at pristine and peri-urban mangroves. However, a higher level of parasitation by a Bopyridae isopod, Pseudione elongata indicated some degree of stress on the host at the peri-urban mangrove, with potential effects on the host population dynamics.     

Euglena gracilis as a model for the study of Cu and Zn toxicity and accumulation in eukaryotic cells

We have observed the effect of copper and zinc on the biology of Euglena gracilis. The cells displayed different sensitivities to these metals, as the apparent LC₅₀ for Cu²⁺ was 0.22 mM, and for Zn²⁺ it was 0.88 mM. While Zn²⁺ was able to increase cell proliferation even at 0.1 mM, the minimal CuCl₂ concentration tested (0.02 mM) was sufficient to impair cell division. Higher concentrations of these metals not only inhibited cell division in a concentration-dependent manner, but also interfered with the metabolism of E. gracilis. A higher accumulation of proteins and lipids per cell was observed at the DI₅₀ concentration for metal-treated cells. These results suggest that the test concentration of both metals leads to a failure in completing cell division. Ultrastructural analysis indicated a chloroplast disorganization in copper-treated cells, as well as the presence of electron dense granules with different shapes and sizes inside vacuoles. Microanalysis of these granules indicated an accumulation of copper, thus suggesting a detoxification role played by the vacuoles. These results indicate that E. gracilis is an efficient biological model for the study of metal poisoning in eukaryotic cells. They also indicate that copper and zinc (copper being more poisonous) had an overall toxic effect on E. gracilis and that part of the effect can be ascribed to defects in the structure of chloroplast membranes.     

Heavy metals in bark of <Emphasis Type="Italic">Pinus massoniana</Emphasis> (Lamb.) as an indicator of atmospheric deposition near a smeltery at Qujiang,China

Goal, Scope and Background Rapid urbanization and the expansion of industrial activities in the past several decades have led to large increases in emissions of pollutants in the Pearl River Delta of south China. Recent reports have suggested that industrial emission is a major factor contributing to the damages in current natural ecosystem in the Delta area. Tree barks have been used successfully to monitor the levels of atmospheric metal deposition in many areas, but rarely in China. This study aimed at determining whether atmospheric heavy metal deposition from a Pb-Zn smeltery at Qujiang, Guangdong province, could be accurately reflected both in the inner bark and the outer bark of Masson pine (Pinus massoniana L.). The impact of the emission from smeltery on the soils beneath the trees and the relationships of the concentrations between the soils and the barks were also analyzed. Methods Barks around the bole of Pinus massoniana from a pine forest near a Pb-Zn smeltery at Qujiang and a reference forest at Dinghushan natural reserve were sampled with a stainless knife at an average height of 1.5 m above the ground. Mosses and lichens on the surface barks were cleaned prior to sampling. The samples were carefully divided into the inner bark (living part) and the outer bark (dead part) in the laboratory, and dried and ground, respectively. After being dry-ashed, the powder of the barks was dissolved in HNO₃. The solutions were analyzed for iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), chromium (Cr), nickel (Ni) and cobalt (Co) by inductively coupled plasmas emission spectrometry (ICP, PS-1000AT, USA) and Cadmium (Cd) and lead (Pb) by graphite furnace atomic absorption spectrometry (GFAAS, ZEENIT 60, Germany). Surface soils (0–10 cm) beneath the sample trees were also collected and analyzed for the selected metals. Results and Discussion Concentrations of the selected metals in soils at Qujiang were far above their environmental background values in the area, except for Fe and Mn, whilst at Dinghushan, they were far below their background values, except for Cd and Co. Levels of the metals, in particular Pb and Zn, in the soils beneath the sample trees at Qujiang were higher than those at Dinghushan with statistical significance. The result suggested that the pine forest soils at Qujiang had a great input of heavy metals from wet and dry atmospheric deposition, with the Pb-Zn smeltery most probably being the source. Levels of Cu, Fe, Mn, Zn, Ni and Pb at Qujiang, both in the inner and the outer bark, were statistically higher than those at Dinghushan. Higher concentrations of Pb, Fe, Zn and Cu may come from the stem-flow of elements leached from the canopy, soil splash on the 1.5 m height and sorption of metals in the mosses and lichens growing on the bark, which were direct or indirect results from the atmospheric deposition. Levels of heavy metals in the outer barks were associated well with the metal concentrations in the soil, reflecting the close relationships between the metal atmospheric deposition and their accumulation in the outer bark of Masson pine. The significant (p<0.01) correlations of Fe-Cu, Fe-Cr, Fe-Pb, Fe-Ni, Pb-Ni, and Pb-Zn in the outer barks at Qujiang again suggested a common source for the metals. The correlation only occurred between Pb and Ni, Cd and Co in the outer barks at Dinghushan, which suggested that those metals must possibly have other uncommon sources. Conclusions Atmospheric deposition of the selected metals was great at Qujiang, based on the levels in the bark of Pinus massoniana and on the concentrations in the soils beneath the trees compared with that at Dinghushan. Bark of Pinus massoniana, especially the outer bark, was an indicator of metal loading at least at the time of sampling. Recommendations and Perspectives The results from this study and the techniques employed constituted a new contribution to the development of biogeochemical methods for environmental monitoring particularly in areas with high frequency of pollution in China. The method would be of value for follow up studies aimed at the assessment of industrial pollution in other areas similar with the Pearl River Delta.