Evidence of feminization in wild Elliptio complanata mussels in the receiving waters downstream of a municipal effluent outfall

The endocrine-disrupting activity of municipal effluents has the potential to alter the reproductive system and induce feminization to aquatic organisms. The purpose of this study was to examine the sex ratio, vitellogenin (Vtg)-like proteins, serotonin, arachidonate cyclooxygenase (COX) activity and dopamine status in wild mussels living at sites upstream and downstream of two municipal effluent outfalls in the Mille-Îles River (Quebec, Canada). Gonad integrity was also studied by monitoring the gonado-somatic index (GSI), the activity of the rate-limiting enzyme aspartate transcarbamoylase (ATC) for purine synthesis, and changes in lipid peroxidation (LPO). The results showed that the proportion of females was dramatically increased from 30% at the upstream sites to 80% at the downstream sites. The levels of Vtg-like proteins were significantly elevated in the male mussels only. Male mussels downstream of the municipal effluent plumes expressed female-specific protein bands (Vtg-like), as determined by high-resolution gel electrophoresis and silver staining. The serotonin/dopamine ratio was significantly decreased in the downstream mussels, indicating that the gonad was in a state of early vitellogenesis. However, this change was not accompanied by changes in ATC, suggesting no significant egg production was underway; this was confirmed by the observation that the downstream mussels displayed significantly low GSIs. GSIs were rather dependent on the serotonin/dopamine ratio (r = 0.44; p < 0.001), while Vtg-like proteins were dependent on dopamine levels (r = 0.50; p < 0.001). The increase in COX activity at the downstream sites and its close relationship with increased serotonin levels suggest a concomitant serotonergic signalling in addition to VTG production. The production of Vtg-like proteins combined with the serotonergic effects of the municipal effluents was associated with oxidative damage (LPO) in the gonad. This study provides the first evidence of feminization in wild mussel populations and the disruption in gonad physiology by exposure to municipal effluents.     

Inflammatory properties of municipal effluents to Elliptio complanata mussels--lack of effects from anti-inflammatory drugs

The purpose of this study was to identify the pharmacological effects of anti-inflammatory drugs in freshwater mussels (Elliptio complanata) exposed to a primary-treated municipal effluent. Mussel specimens were injected with either increasing concentrations of ibuprofen or with a municipal effluent extract, and then left to stand for 24 h at 15 degrees C. They were also exposed to dilutions of a primary-treated effluent for 30 days at 15 degrees C under semi-static conditions. Gill and gonad cylcooxygenase (COX) were then determined after the incubation period. The influence of various drugs found in municipal effluents on serotonin and dopamine synaptosome transport was determined in visceral ganglia. The results show that injections of ibuprofen reduced COX activity nearly 4-fold in gills and 1.4-fold in gonads. However, COX activity was induced in both tissues after 24 h in mussels injected with a municipal effluent extract and after 30 days in those exposed to dilutions of the effluent. Moreover, synaptosomal dopamine transport activity was increased by ibuprofen, aspirin, caffeine and estradiol-17beta (E2), and decreased by loperamide and carbamazepine, suggesting increased and decreased turnover rates of this catecholamine, respectively. Serotonin transport activity was much less affected, decreasing with high doses of loperamide and increasing with ibuprofen, but with less intensity than with dopamine. The results suggest that although ibuprofen can effectively reduce COX activity in gill and gonadal tissues, exposure to both the municipal effluent and its organic extract increased COX activities, indicating the absence of NSAID (non-steroidal anti-inflammatory drugs)-related effects. Besides their known estrogenic and serotonergic properties, municipal effluents appear to elicit a state similar to inflammation in freshwater mussels.     

Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year

Freshwater mussels, Elliptio complanata, were caged in special benthic pens and were immersed at one upstream (Ups) site and two downstream sites (8 and 11 km) of a primary-treated municipal effluent plume for 1 year. The levels of metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) activity were assayed in digestive gland, gill and gonad tissues to evaluate biological effects and damage. The levels of monoamines (serotonin and dopamine) in nerve ganglia, ATP-dependent transport activity and monoamine oxidase (MAO) activity were also investigated in the homogenates, synaptosomes and mitochondria, respectively. Results showed that significant amounts of sediment accumulated in cages and 82% of mussels survived the yearlong exposure period at the downstream sites. MT-like proteins were induced in all tissues with the following response intensity: gill (3-fold), digestive gland (1.4-fold) and gonad tissues (1.3-fold). Lipid peroxidation decreased (2.5-fold) in digestive gland but increased in gill (1.6-fold) and in gonad tissues (1.5-fold). GST activity was readily increased in digestive gland (2.5-fold), suggesting the presence of organic contaminants in the plume. Levels of protein-free DNA strands did not vary significantly in digestive gland and gill tissues but were significantly reduced in gonad tissues (2.5-fold) relative to the upstream site. In visceral nerve ganglia, both serotonin and ATP-dependent serotonin transport decreased 1.7-fold with a 4-fold increase of 5-hydroxyindole acetate (5-HIAA, a serotonin metabolite) level relative to the upstream site. However, MAO activity was somewhat reduced at downstream sites (0.7- to 0.9-fold of the activity at the upstream site). Dopamine levels were found to be decreased (1.5-fold), but dopamine ATP-dependent transport activity was increased 1.8-fold, suggesting reduced dopaminergic activity. These results indicate that estrogenic chemicals are likely at play, and the increased dopamine and decreased serotonin ATP-dependent transport suggest that the municipal plume was serotonergic for mussels located at the downstream sites. Mussels exposed for 1 year display a complex but characteristic pattern of responses that could lead to harmful health effects including neuroendocrine disruption of reproduction.     

Effects of municipal effluents on serotonin and dopamine levels in the freshwater mussel Elliptio complanata

Sex differentiation and gametogenesis represent critical steps in the reproductive process and are subject to hormonal control by serotonin, dopamine and steroids such as estradiol-17beta and testosterone. The purpose of this study sought to examine the endocrine-disrupting activity that a primary-treated municipal effluent might have on the metabolism of biogenic amine levels. First, serotonin receptors transfected in Chinese hamster ovary (CHO) cells were used to screen for the presence of serotonin receptor agonist or antagonist. Second, one group of Elliptio complanata mussels were exposed to single compounds likely to be found in municipal wastewaters and another group was exposed in situ to the municipal effluent plume for 90 days in experimental cages. Results showed that solid phase C-8 extracts of surface water downstream a municipal effluent could activate the transport of serotonin by receptors at a distance of at least 5 km from its outfall thereby indicating the presence of serotonin mimics in the effluent dispersion plume. Levels of serotonin and monoamine oxidase (MAO) activity in nerve ganglia of mussels exposed for 90 days to the municipal effluent were, respectively, reduced and increased at a distance 10-km downstream. Injections of estradiol-17beta and nonylphenol in mussels decreased the levels of serotonin and dopamine, but increased MAO activity in the gonad and nerve ganglia. Exposure to estrogenic chemicals present in municipal effluents may therefore alter the normal metabolism of serotonin and dopamine, both of which are involved in sexual differentiation in bivalves and fish. Chemicals acting through E2 receptor-mediated pathways and serotonin receptors are likely to cause the observed effects.     

Alterations in DNA metabolism in Elliptio complanata mussels after exposure to municipal effluents

This study sought to examine the genotoxic potential in Elliptio complanata freshwater mussels exposed to a physically and chemically treated municipal effluent before and after ozone treatment. Mussels were continuously exposed to increasing concentrations of the effluents for 14 days. Genotoxicity was determined by tracking changes in key enzymes for purine and pyrimidine synthesis (dehydrofolate reductase and aspartate transcarbamoylase), catabolism of purines (xanthine oxido-reductase) and DNA strand-break levels as determined by the alkaline precipitation assay. Other biomarkers related to xenobiotic biotransformation (cytochrome P4503A and glutathione S-transferase activities), metal metabolism (labile zinc and redox state of metathioneins) and oxidative stress (superoxide dismutase activity) were also determined in the mussels. The data revealed that dehydrofolate reductase activity was reduced by the initial effluent and increased by the ozonated effluent. Aspartate transcarbamoylase activity was significantly induced only with the ozonated effluent. The levels of DNA strand breaks responded in a biphasic manner in mussels exposed to the physically and chemically treated effluent where an initial decrease was observed at a low effluent concentration (3% v/v) followed by an increase in DNA strand breaks at a higher effluent concentration (20%). This response pattern was lost in the ozonated effluent, where only a decrease in DNA breaks was found. Xanthine oxidoreductase activity was not significantly affected but did correlate significantly with dehydrofolate reductase activity. Multivariate factorial and canonical analyses revealed that oxidative stress and metal/xenobiotic metabolism markers were strongly correlated with DNA strand breaks in mussels, suggesting that the presence of metals (zinc) and planar hydroxylated hydrocarbons present in these effluents were strong contributors to the observed response. We conclude that municipal effluents contain a complex mixture of pollutants that could modulate DNA synthesis and repair mechanisms in mussels.     

Neurochemical effects of benzodiazepine and morphine on freshwater mussels

The purpose of this study was to examine the neurochemical effects of morphine, diazepam, a common benzodiazepine, and an effluent concentrate on the endemic freshwater mussel Elliptio complanata. Mussels were exposed to the drugs and to the solid-phase concentrate of a municipal effluent and left to stand at 15 °C for 48 h. Neurochemical effects were determined by monitoring changes in dopamine, serotonin, glutamate and γ-aminobutyric acid (GABA) levels in the visceral mass (containing the nerve ganglia) of mussels. The activities of acetylcholinesterase (AChE), dopamine and serotonin-dependent adenylyl cyclase (ADC) were also determined in the mussels. Oxidative stress was determined by tracking changes in lipid peroxidation (LPO) in the mitochondrial and post-mitochondrial fractions. The results revealed that the drugs and the effluent extract were biologically active in mussels. Morphine reduced serotonin and increased dopamine in mussel tissues while reducing AChE activity and increasing GABA levels. This suggests the induction of a relaxation state in mussels. Diazepam also reduced serotonin levels but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, indicating the potential effect on opiate signaling. Diazepam increased glutamate levels slightly, but AChE remained stable. The increase in both dopamine ADC activity and glutamate concentrations was also associated with greater oxidative stress on the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity to morphine. In conclusion, the data warrant more studies on the analysis of opiates and benzodiazepines in municipal effluents to better address the potential environmental hazard of these neuroactive drug classes to aquatic organisms.     

Survival,Growth and Condition of Freshwater Mussels: Effects of Municipal Wastewater Effluent

Freshwater mussels (Family Unionidae) are among the most imperiled group of organisms in the world, with nearly 65% of North American species considered endangered. Anthropogenic disturbances, including altered flow regimes, habitat alteration, and pollution, are the major driver of this group''s decline. We investigated the effects of tertiary treated municipal wastewater effluent on survivorship, growth, and condition of freshwater mussels in experimental cages in a small Central Texas stream. We tested the effluent effects by measuring basic physical parameters of native three ridge mussels (Amblema plicata) and of non-native Asian clams (Corbicula fluminea), before and after 72-day exposure at four sites above and below a municipal wastewater treatment plant outfall. Survivorship and growth of the non-native Asian clams and growth and condition indices of the native three ridge mussels were significantly higher at the reference site above the outfall than in downstream sites. We attribute this reduction in fitness below the outfall to elevated nutrient and heavy metal concentrations, and the potential presence of other untested-for compounds commonly found in municipal effluent. These results, along with an absence of native mussels below the discharge, indicate a significant negative impact of wastewater effluent on both native and non-native mussels in the stream.     

Vitellogenin levels in mussel hemolymph—a suitable biomarker for the exposure to estrogens?

Increased vitellogenin (vtg) levels in the blood of male fish are frequently used as an indicator of estrogenic exposure. Similar responses are expected for mussels, where the concentration of vtg-like proteins has been reported to depend on estrogens. To verify the role of hemolymph during vitellogenesis of mussels, the saltwater mussel Mytilus edulis and the freshwater mussel Anodonta cygnea were exposed to 17beta-estradiol (E2) and wastewater treatment plant effluents, known for their estrogenic potential. Gel electrophoresis did not reveal any significant induction (or repression) of plasma proteins compared to control plasma. Our results do not support the hypothesis that mussel hemolymph is a carrier of estrogen-dependent major egg-yolk precursors (vtg-like proteins). However, additional information on a 35+/-2-kDa hemolymph protein, previously reported to bind heavy metals, was obtained by high-resolution two-dimensional electrophoresis. It was resolved in a cluster of single proteins with properties that match the characteristics of a previously reported histidine-rich glycoprotein.     

Toxicological effects of primary-treated urban wastewaters, before and after ozone treatment, on freshwater mussels (Elliptio complanata)

This study examined the toxic potential of a primary-treated municipal effluent, before and after ozonation, in freshwater mussels. Animals were exposed to various concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent and also after a treatment with ozone at 10 mg/L in continuous flow-through mode for seven weeks. A suite of biomarkers was used to assess the potential toxic effects of various contaminants typically present in municipal wastewaters: heavy metal metabolism (metallothioneins and labile zinc), cytochrome P4501A1 and 3A4, glutathione S-transferase activities (biotransformation of organic compounds), lipid peroxidation and xanthine oxidoreductase (oxygen radical scavenging), DNA damage, mitochondrial electron transport activity at various temperatures and gonad lipid levels (cellular energy allocation) and aspartate transcarbamoylase and dihydrofolate reductase (gonad activity). On the one hand, some biomarkers, including metallothioneins, labile zinc, glutathione S-transferase, cytochrome P4503A4 activity, dehydrofolate reductase and aspartate transcarbamoylase, were readily decreased. In contrast, these biomarkers, cytochrome P4501A1, gill lipid peroxidation, DNA strand breaks in gills and digestive gland, mitochondrial electron transport at high and low temperatures (temperature-dependent activity) and total gonad lipids, were readily increased. In general, ozone treatment reduced adverse effects by either decreasing the intensity of the toxic responses or increasing the threshold concentration. For gill lipid peroxidation, however, intensity was greater at a higher threshold concentration. Ozone treatment eliminated the temperature sensitivity of the mitochondrial electron transport system, indicating a loss of interaction between temperature and urban pollution in terms of energy expenditure in mussels. Ozone treatment could significantly decrease either the toxic potency or intensity of urban pollutants at the expense of increased oxidative stress in gills of freshwater mussels.     

Effects of pharmaceutical products and municipal wastewaters on temperature-dependent mitochondrial electron transport activity in Elliptio complanata mussels

The advent of global warming has given rise to the issue on how temperature impacts the susceptibility of ectothermic organisms to pollution. The purpose of this study was to examine the effects of pharmaceutical products and domestic wastewaters on temperature-dependent mitochondrial electron transport activity in the freshwater mussel Elliptio complanata. Mitochondria from mussels were freshly prepared and exposed to increasing concentrations of various pharmaceutical products known to be found in municipal effluents for 30 min at 4, 12 and 20 degrees C. Electron transport activity as well as lipid peroxidation and DNA strand breaks were determined in the mitochondria. Next, mussels were placed in the aeration lagoons of two municipal wastewater treatment plants for 30 days. Mitochondrial electron transport (MET), temperature-dependent MET (MET(T)) and lipid peroxidation in gonad were then determined. The results show that all products were able to increase MET activity, but at two different ranges of threshold concentration. Certain pharmaceutical products (i.e., ibuprofen, cotinine, fluoxetine, coprostanol and trimethoprim) increased MET(T) at a lower threshold concentration than observed at 20 degrees C. Products of lesser potency in reducing lipid peroxidation were those that produced more DNA strand breaks in mitochondria. Both MET and MET(T) were significantly increased in mussels exposed to aeration lagoon effluents. Lipid peroxidation was also increased in the gonad and was significantly correlated with MET and MET(T) activities. The data indicate that pharmaceutical products and municipal effluents increase respiration rates in isolated mitochondria, such that interaction with temperature could enhance the susceptibility of mitochondrial energy production and oxidative stress in environments contaminated by domestic wastewater.     

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.     

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.     

The effects of zinc oxide nanoparticles on the metallome in freshwater mussels

The use of zinc oxide nanoparticles (nanoZnO) as sunscreens has raised concerns about their safety and release in the aquatic environment through swimming activities and within municipally treated wastewaters. This study's purpose was to examine the effects of nanoZnO on the elemental composition (metallome) in exposed freshwater mussels, Elliptio complanata. Mussels were exposed for 21 days to an environmentally realistic (low) concentration (2 μg/L) of nanoZnO and zinc chloride. The mussels were also exposed to a physically and chemically treated municipal effluent (ME), both alone and in the presence of both forms of Zn. The metallome profile was characterized by the following 15 elements in gills, digestive gland and gonad tissues: Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, V and Zn. The levels of metallothioneins (MT) and lipid peroxidation (LPO) in the digestive gland were also measured as biomarkers of toxic effects. The data revealed that exposure to nanoZnO increased the total levels of Zn, MT and LPO in the digestive gland. Discriminate function analysis revealed that the digestive gland responded the most to exposure to either nanoZnO or Zn^2 +. For nanoZnO, the observed changes in Al, As and Mo in the digestive gland offered the best discrimination from dissolved Zn^2 +. Co-exposure of nanoZnO with the ME changed the metallome profile closer to dissolved Zn^2 +, suggesting a common interaction site within the ME. This was observed in changes in Ni, Cu, Se and Zn in the digestive gland of exposed mussels. Canonical analysis of essential and non-essential elements revealed that exposure to nanoZnO increased the relationships between LPO and the sum of essential elements in the digestive gland. Conversely, exposure to dissolved Zn^2 + and the ME decreased the relationship between the sum of non-essential elements and LPO and MT. In conclusion, the use of a “metallomic” approach was used to discriminate changes following exposure to nanoZnO and dissolved Zn in freshwater mussels and provided insights into the interaction of forms of Zn in ME towards mussels.     

Vitellogenin and egg production in the moth,Heliothis virescens

The characteristics of vitellogenin (Vg) and the relationship between Vg production and egg production in the tobacco budworm, Heliothis virescens, were studied. The relationship between Vg production and juvenile hormone (JH) and the impact of mating on Vg and egg production were also investigated. Vg appears in the hemolymph of H. virescens about 6 h after moth eclosion. Vg may be separated into two apoproteins (ApoVg-I and ApoVg-II) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights were calculated to be 156,065 ± 800 for ApoVg-I and 39,887 ± 323 for ApoVg-II. SDS-PAGE analysis revealed that the female hemolymph Vg polypeptides appear to be identical to those from eggs but are absent in male hemolymph. Vg concentration was significantly higher in mated females than in virgin females of the same age at 48 h after emergence. Rates of egg production increased as Vg production increased; rates of egg production in mated females were significantly higher than those of virgin females at 48, 72, 96, and 120 h postemergence. Vg production is dependent on JH, because hemolymph from decapitated females lacked Vg while that of decapitated females treated with synthetic JH had Vg at levels comparable to similarly aged, normal H. virescens females. Hemolymph JH titers in mated females were significantly higher compared with those in virgin females at all sampling periods. The high JH level in mated females may explain the high Vg and egg production in mated H. virescens. Arch. Insect Biochem. Physiol. 34:287–300, 1997. © 1997 Wiley-Liss, Inc.     

Vitellogenesis in both sexes of gonochoristic mud shrimp, Upogebia major (Crustacea): analyses of vitellogenin gene expression and vitellogenin processing

The mud shrimp, Upogebia major is a gonochoristic species with distinct sexual dimorphism; however, the male has the “ovarian part of testis” in the gonad and mature-looking eggs appear in a similar reproductive cycle to the female. Vitellogenesis of U. major was investigated focusing on the characterization of vitellogenin (Vg) gene expression and Vg processing. Vg cDNA cloned by PCR-based methods was 7799 bp-long, encoding 2568 amino acids in a single open reading frame. The deduced amino acid sequence shared common characteristics conserved in other shrimp Vgs. The Vg gene was expressed in the hepatopancreas of females and males, the ovary, and the ovarian part of testis. Vitellins (Vns) were detected in the gonads of both females and males as three prominent polypeptides with apparent molecular masses of 82 kDa, 100 kDa, and 115 kDa. N-terminal amino acid sequences determined for the three polypeptides were present in the deduced amino acid sequence, demonstrating that they derived from a single long Vg polypeptide. Immunoblot analysis using polyclonal antibodies raised against two Vns (82 kDa and 100 kDa) confirmed Vg processing in the hepatopancreas, in the hemolymph and possibly in the oocytes, similarly in both sexes.     

Primary production, nutrient dynamics, and accretion of a coastal freshwater forested wetland assimilation system in Louisiana

This study reports on the response of a tidal, freshwater forested wetland ecosystem to long-term input of secondarily treated municipal effluent from the City of Mandeville, LA. Measurements of hydrology, nutrients, and aboveground net primary productivity were made from September 1998 through March 2002. Accretion measurements were made in October 2000 and October 2004. The major hydrologic inputs to the system were the effluent, precipitation, and back water flooding from Lake Pontchartrain. Nutrient levels were generally low except in the immediate vicinity of the outfall. Mean net primary production of the freshwater forest system was significantly higher downstream of the effluent discharge (1202 g m⁻² yr⁻¹) compared to the control site (799 g m⁻² yr⁻¹). Downstream of the outfall, accretion rates were double the rate of relative sea level rise in the area. Removal efficiencies of N and P were as high as 75% and 95%, respectively. The relatively constant flow of secondarily treated municipal effluent buffered the downstream area from salinity intrusion during a region-wide drought. Re-direction of nutrient-enhanced effluents from open water bodies to wetland ecosystems can maintain plant productivity, sequester carbon, and maintain coastal wetland elevations in response to sea-level rise in addition to improving overall surface water quality, reducing energy use, and increasing financial savings.     

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Neurotoxicological effects of a primary and ozonated treated wastewater on freshwater mussels exposed to an experimental flow-through system

The neurotoxic potential of a primary-treated and ozonated municipal effluent was examined using feral freshwater Elliptio complanata mussels. Specimens were exposed to increasing concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent before and after treatment with 10 mg/L of ozone in a mesocosm-type experiment for 30 days. A suite of biomarkers was used to assess the potential neurotoxic stress of the wastewaters on these benthic invertebrates: opiate binding sites, gamma-aminobutyric acid (GABA) metabolism, monoamines levels (serotonin, dopamine), monoamine oxidase, acetylcholinesterase and lipid peroxidation. Gametogenic activity was also determined by the gonado-somatic index and by vitellogenin-like proteins. The results show that the number of opiate binding sites increased slightly, especially after ozonation. GABA metabolism was generally reduced, suggesting higher glutamate stimulation than GABA dampening effects in mussel ganglia. This excitatory state was further confirmed by decreased acetylcholinesterase activity in gonadal tissues. The turnover of dopamine was enhanced with increased serotonin levels, but accompanied by reduced catabolism, as evidenced by decreased monoamine oxidase activity. Moreover, oxidative stress was increased, as determined by lipid peroxidation in the gonad (containing ganglia), which was significantly correlated with acetylcholinesterase activity and dopamine metabolism. The gonado-somatic index was significantly reduced with increased levels of vitellogenin-like proteins, again confirming the estrogenic action of these wastewaters. The data suggest that exposure to a primary-treated municipal effluent before and after ozonation leads to an excitotoxic syndrome implicating perturbations in GABA, dopamine and acetylcholine signaling. The increase in dopamine metabolism may be associated with the occurrence of opiate-like compounds (i.e. morphine) in the effluent. In general, ozonation reduced the severity of the responses, indicating that this disinfection strategy does not increase neurotoxicity to mussels.     

A Conceptual Framework for Using Mussels as Biomonitors in Whole Effluent Toxicity

As a main source of direct and continuous input of pollutants in the aquatic ecosystem, studying the effects of effluents on receiving ecosystems has a high ecological relevance. While ecological risk assessment procedures are usually based on a chemical-based single component approach, their application for complex mixtures and effluents is less straightforward. A chemical-based approach has to rely on the knowledge of what chemicals are present in effluents, their potential toxicity, how all of these individual chemicals interact and what their individual and combined contribution to the mixture is. Whole effluent toxicity (WET) testing, however, is an integrative tool that measures the toxic effect of an effluent as a whole and accounts for uncharacterized sources of toxicity and for interactions. This paper addresses the use of transplanted bivalves, i.e., caged mussels, as a biomonitoring tool in WET testing with special reference to field situations in both freshwater and marine environments. We indicate how endpoints at different levels of biological organization within exposed mussels can give an integrative overview of effects. Finally, we will provide a framework for future research using mussels and discuss a multitude of instream responses for routine, efficient and cost-effective active biomonitoring applications.