Comparative toxicity of urban wastewater and rainfall overflow in caged freshwater mussel Elliptio complanata.

Municipal effluents are well-recognized as disrupting sexual differentiation and reproduction in mussels. However, the contribution to this problem made by rainfall combined with sewer overflow (increased by rain due to climate change) is not well understood. The purpose of this study was to compare the neuroendocrine effects of municipal discharge and rainfall overflow on caged endemic mussel Elliptio complanata. To this end, mussels were experimentally caged and placed for 3 months at a municipal effluent dispersion plume site and at overflow sites. Data revealed that downstream surface water contained some pharmaceuticals (caffeine and carbamazepine) and accumulated significant levels of heterotrophic bacteria, but these effects were not observed at the overflow sites. The principal effects observed at the downstream site were increased soft tissue mass (and gonad index), inflammation, and Vtg proteins in male mussels as determined by a novel immunostaining methodology. The rainfall overflow sites had no effects on these markers, but were specifically associated with reduced Vtg proteins in females, dopamine (Dop), gonad lipids, and DNA strand breaks, with increased metallothioneins. In conclusion, the observed feminizing effects of municipal effluent were not additionally observed in mussels caged at rainfall overflow sites, although the latter exhibited a different pattern of toxicity.

An introduction to the sources, fate, occurrence and effects of endocrine disrupting chemicals released into the environment.

Many classes of compounds are known or suspected to disrupt the endocrine system of vertebrate and invertebrate organisms. This review of the sources and fate of selected endocrine disrupting chemicals (EDCs) in the environment includes classes of compounds that are "legacy" contaminants, as well as contaminants of emerging concern. EDCs included for discussion are organochlorine compounds, halogenated aromatic hydrocarbons, brominated flame retardants, per- and polyfluoroalkyl substances, alkylphenols, phthalates, bisphenol A and analogues, pharmaceuticals, drugs of abuse and steroid hormones, personal care products, and organotins. An exhaustive survey of the fate of these contaminants in all environmental media (e.g., air, water, soil, biota, foods and beverages) is beyond the scope of this review, so the priority is to highlight the fate of EDCs in environmental media for which there is a clear link between exposure and endocrine effects in humans or in biota from other taxa. Where appropriate, linkages are also made between the fate of EDCs and regulatory limits such as environmental quality guidelines for water and sediments and total daily intake values for humans.

Methods for the analysis of endocrine disrupting chemicals in selected environmental matrixes.

Endocrine disrupting chemicals (EDCs) are heterogenous in structure, chemical and physical properties, and their capacity to partition into various environmental matrixes. In many cases, these chemicals can disrupt the endocrine systems of vertebrate and invertebrate organisms when present at very low concentrations. Therefore, sensitive and varied analytical methods are required to detect these compounds in the environment. This review summarizes the analytical methods and instruments that are most used to monitor for EDCs in selected environmental matrixes. Only those matrixes for which there is a clear link between exposures and endocrine effects are included in this review. Also discussed are emerging methods for sample preparation and advanced analytical instruments that provide greater selectivity and sensitivity.

The differential effect of sub-micron level HA aggregates on influenza potency assays.

Influenza vaccines remain the most effective public health measure for the prevention of influenza-related illnesses. The primary immunogen in inactivated influenza vaccines is hemagglutinin (HA), the receptor binding protein of influenza. The concentration of HA during vaccine production and testing is standardized according to the level of antigen as measured by Single Radial Immunodiffusion Assay (SRID). This allows vaccine potency to be controlled such that individuals receive a dose known to provoke a clinically protective immune response. As compared to alternatives, SRID has the advantage of quantifying immunologically relevant forms of HA, but it depends on timely generation of novel reagents for each new vaccine strain. In recent years, a number of alternative assays have been suggested based on either epitope recognition, receptor binding or protection from proteolysis but it is unclear how they relate to vaccine potency in clinical trials. In this report we describe the development of a lectin-based, ELISA-type assay for HA potency and find it provides similar potency estimates to SRID except in the case of a vaccine with aggregated HA and other viral proteins. In that case, SRID predicted the immunologically active HA present and ELISA techniques did not. This difference was due to tested antibodies failing to pull down or bind to the HA present unless particle aggregates were first dissociated. Furthermore, detergent treatment alone was insufficient to complete this dissociation. While others have previously demonstrated that immunocapture-based techniques can misestimate the potency of influenza vaccines depending on the individual antibodies used we demonstrate that in this case the failure was due to an inability of all antibodies to capture HA contained in the aggregated influenza vaccine.

The distribution dynamics and desorption behaviour of mobile pharmaceuticals and caffeine to combined sewer sediments.

Pharmaceuticals are discharged to the environment from wastewater resource recovery facilities, sewer overflows, and illicit sewer connections. To understand the fate of pharmaceuticals, there is a need to better understand their sorption dynamics to suspended sediments (SS) and settled sediments (StS) in sewer systems. In this study, such sorption dynamics to both SS and StS were assessed using a batch equilibrium method under both static and dynamic conditions. Experiments were performed with natively occurring and artificially modified concentrations of sewer pharmaceuticals (acetaminophen, theophylline, carbamazepine, and a metabolite of carbamazepine) and caffeine. Differences in apparent distribution coefficients, Kd,app, between SS and StS were related to differences in their organic carbon (OC) content, and the practice of artificially modifying the concentration. Kd,app values of modified contaminant concentrations and high OC sediments were substantially higher. Pseudo-second order desorption rates for these mobile compounds were also quantified. Successive flushing events to simulate the addition of stormwater to sewer networks revealed that aqueous concentrations would not necessarily decrease, because the added water will rapidly return to equilibrium concentrations with the sediments. Sorption and desorption kinetics must be considered in addition to dilution, to avoid underestimating the influence of dilution on concentrations of pharmaceuticals discharged to the environment.

Analysis of steroid hormones and their conjugated forms in water and urine by on-line solid-phase extraction coupled to liquid chromatography tandem mass spectrometry.

BACKGROUND: In recent years, endocrine disrupting compounds (EDCs) have been found in rivers that receive significant inputs of wastewater. Among EDCs, natural and synthetic steroid hormones are recognized for their potential to mimic or interfere with normal hormonal functions (development, growth and reproduction), even at ultratrace levels (ng L(-1)). Although conjugated hormones are less active than free hormones, they can be cleaved and release the unconjugated estrogens through microbial processes before or during the treatment of wastewater. Due to the need to identify and quantify these compounds, a new fully automated method was developed for the simultaneous determination of the two forms of several steroid hormones (free and conjugated) in different water matrixes and in urine. RESULTS: The method is based on online solid phase extraction coupled with liquid chromatography and tandem mass spectrometry (SPE-LC-MS/MS). Several parameters were assessed in order to optimize the efficiency of the method, such as the type and flow rate of the mobile phase, the various SPE columns, chromatography as well as different sources and ionization modes for MS. The method demonstrated good linearity (R(2) > 0.993) and precision with a coefficient of variance of less than 10 %. The quantification limits vary from a minimum of 3-15 ng L(-1) for an injection volume of 1 and 5 mL, respectively, with the recovery values of the compounds varying from 72 to 117 %. CONCLUSION: The suggested method has been validated and successfully applied for the simultaneous analysis of several steroid hormones in different water matrixes and in urine.

Sorption and desorption of diverse contaminants of varying polarity in wastewater sludge with and without alum.

Sewage sludge sorption and desorption measurements were conducted for nine diverse contaminants of varying polarity: caffeine, sulfamethoxazole, carbamazepine, atrazine, estradiol, ethinylestradiol, diclofenac, and, for the first time desethylatrazine and norethindrone. Two types of sorption behaviour were observed. Compounds with a log octanol-water partition coefficient, log Kow, below 3 showed little or no sorption over 48 hours of shaking, while compounds with log Kow over 3 showed 30 to 90% sorption within the first few minutes. After 6 hours of shaking, mass loss through suspected biotransformation became evident for some compounds. At the pH range considered (5.7-6.7), diclofenac (pKa 4.0, log Kow 4.5) was the only compound in which pH dependent sorption could be quantified. The log sewage sludge-water distribution coefficients, log Kd, ranged from 0.2 to 2.9, and, as expected, increased with increasing log Kow of the compound and organic carbon (OC) content of the sewage sludge. A sewage sludge precipitated with alum had a substantially lower Kd values, as well as lower OC content, compared to alum-free sludge. Desorption was studied by sequentially replacing supernatant water. With each water replacement, log Kd values tended to either remain similar (following a linear isotherm) or in some cases increase (following a Freundlich-type isotherm). The length of time required to restore equilibrium increased with each rinsing step. A literature review of reported Kd values compared well with the alum-free sludge data, but not the alum-sludge data. Sewage sludge Kd across the literature appear more consistent with increasing Kow.

Wastewater micropollutants as tracers of sewage contamination: analysis of combined sewer overflow and stream sediments.

A sensitive method was developed to measure the sediment concentration of 10 wastewater micropollutants selected as potential sanitary tracers of sewage contamination and include: nonsteroidal anti-inflammatory drugs (acetaminophen - ACE and diclofenac - DIC), an anti-epileptic drug (carbamazepine - CBZ), a ß-blocker (atenolol - ATL), a stimulant (caffeine - CAF), a bronchodilator (theophylline - THEO), steroid hormones (progesterone - PRO and medroxyprogesterone - MedP), an artificial sweetener (aspartame - APM) and personal care products (N,N-diethyl-3-methylbenzamide - DEET). Natural sediments (combined sewer overflow and stream sediments) were extracted by ultrasonic-assisted extraction followed by solid-phase extraction. Analyses were performed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) using atmospheric pressure chemical ionisation in positive mode (APCI+) with a total analysis time of 4.5 min. Method detection limits were in the range of 0.01 to 15 ng g(-1) dry weight (dw) for the compounds of interest, with recoveries ranging from 75% to 156%. Matrix effects were observed for some compounds, never exceeding |±18%|. All results displayed a good degree of reproducibility and repeatability, with relative standard deviations (RSD) of less than 23% for all compounds. The method was applied to an investigation of stream and combined sewer overflow sediment samples that differed in organic carbon contents and particle size distributions. Acetaminophen, caffeine and theophylline (as confounded with paraxanthine) were ubiquitously detected at 0.13-22 ng g(-1) dw in stream bed sediment samples and 98-427 ng g(-1) dw in combined sewer overflow sediment samples. Atenolol (80.5 ng g(-1) dw) and carbamazepine (54 ng g(-1) dw) were quantified only in combined sewer overflow sediment samples. The highest concentrations were recorded for DEET (14 ng g(-1) dw) and progesterone (11.5 ng g(-1) dw) in stream bed and combined sewer overflow sediment samples, respectively. The ratio of concentration to its limit of detection (C : LOD) in sediments for a subset of compounds were compared to their C : LOD in water. In waters with a large capacity for dilution relative to fecal sources, the C : LOD ranges in sediments were greater than in water. Thus monitoring programs for fecal source tracking using wastewater micropollutants should consider sediment sampling, particularly for waters with highly diluted sources of fecal contamination.

Using a novel sol-gel stir bar sorptive extraction method for the analysis of steroid hormones in water by laser diode thermal desorption/atmospheric chemical ionization tandem mass spectrometry.

A new coating material was used for a stir bar sorptive extraction (SBSE) method coupled to a high throughput sample analysis technique. This allowed for a simple procedure for fast determinations of eight steroid hormones (estriol, estradiol, ethynylestradiol, estrone, progesterone, medroxyprogesterone, levonorgestrel, northindrone) in water. Sample pre-treatment was performed using an in-house SBSE method based on a polydimethylsiloxane/phenyltrimethylsiloxane/ß-cyclodextrin sol-gel material. The analytes were desorbed by liquid extraction prior to their analysis by laser diode thermal desorption/atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS). Several parameters, including ionic strength, volume and time of extraction as well as volume and time of desorption, were investigated to maximize extraction efficiency by SBSE in aqueous solutions. The in-house stir bar showed good reproducibility and could be used for at least 50 extractions without affecting analytical performance. The recoveries of the spiked steroid hormones ranged from 55% to 96% in all water matrices studied (HPLC grade water, tap water and raw wastewater). Only one compound showed poor recovery values (<2% for estriol) in all matrices. The method detection limits (MDLs) in real matrices were within the range of 0.1-0.3 µg L(-1) except for estriol at 48 µg L(-1). The extraction performance of the in-house SBSE for the eight selected hormones was also compared with that of a commercially-available stir bar coated with polydimethylsiloxane (PDMS). This novel stir bar coating could prove to be useful method for the detection and quantification of trace levels of steroid hormones.

Distribution of antidepressant residues in wastewater and biosolids following different treatment processes by municipal wastewater treatment plants in Canada.

The fate of 14 antidepressants along with their respective N-desmethyl metabolites and the anticonvulsive drug carbamazepine (CBZ) was studied in 5 different sewage treatment plants (STPs) across Canada. Using two validated LC-MS/MS analytical methods, the concentrations of the different compounds were determined in raw influent, final effluent and treated biosolids samples. Out of the 15 compounds investigated, 13 were positively detected in most 24-h composite raw influent samples. Analysis showed that venlafaxine (VEN), its metabolite O-desmethylvenlafaxine (DVEN), citalopram (CIT), and CBZ were detected at the highest concentrations in raw influent (up to 4.3 µg L(-1) for DVEN). Cumulated results showed strong evidence that primary treatment and trickling filter/solids contact has limited capacity to remove antidepressants from sewage, while activated sludge, biological aerated filter, and biological nutrient removal processes yielded moderate results (mean removal rates: 30%). The more recalcitrant compounds to be eliminated from secondary STPs were VEN, DVEN and CBZ with mean removal rates close to 12%. Parent compounds were removed to a greater degree than their metabolites. The highest mean concentrations in treated biosolids samples were found for CIT (1033 ng g(-1)), amitriptyline (768 ng g(-1)), and VEN (833 ng g(-1)). Experimental sorption coefficients (K(d)) were also determined. The lowest K(d) values were obtained with VEN, DVEN, and CBZ (67-490 L kg(-1)). Sorption of these compounds on solids was assumed negligible (log K(d) ≤ 2). However, important sorption on solids was observed for sertraline, desmethylsertraline, paroxetine and fluoxetine (log K(d) > 4).

Ozone oxidation of pharmaceuticals, endocrine disruptors and pesticides during drinking water treatment.

This study investigates the oxidation of pharmaceuticals, endocrine disrupting compounds and pesticides during ozonation applied in drinking water treatment. In the first step, second-order rate constants for the reactions of selected compounds with molecular ozone (k(O3)) were determined in bench-scale experiments at pH 8.10: caffeine (650+/-22M(-1)s(-1)), progesterone (601+/-9M(-1)s(-1)), medroxyprogesterone (558+/-9M(-1)s(-1)), norethindrone (2215+/-76M(-1)s(-1)) and levonorgestrel (1427+/-62M(-1)s(-1)). Compared to phenolic estrogens (estrone, 17beta-estradiol, estriol and 17alpha-ethinylestradiol), the selected progestogen endocrine disruptors reacted far slower with ozone. In the second part of the study, bench-scale experiments were conducted with surface waters spiked with 16 target compounds to assess their oxidative removal using ozone and determine if bench-scale results would accurately predict full-scale removal data. Overall, the data provided evidence that ozone is effective for removing trace organic contaminants from water with ozone doses typically applied in drinking water treatment. Ozonation removed over 80% of caffeine, pharmaceuticals and endocrine disruptors within the CT value of about 2 mg min L(-1). As expected, pesticides were found to be the most recalcitrant compounds to oxidize. Caffeine can be used as an indicator compound to gauge the efficacy of ozone treatment.

Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS.

This paper describes a fully automated online method for solid-phase extraction coupled with liquid chromatography and tandem mass spectrometry using atmospheric pressure ionization (LC-LC-APPI-MS/MS) to simultaneously detect selected dissolved natural and synthetic hormones at concentrations as low as 5 ng/L from aqueous matrices. The method shows excellent performance for the direct analysis of water and wastewater with respect to calibration curve linearity, analytic specificity, sensitivity, and carryover, as well as overall method accuracy and precision. With a runtime of 15 min, a minimum of 200 samples were analyzed using a single online solid-phase extraction (SPE) column without noticeable differences in system performance. Because of the ruggedness and simplicity of this system, generic methods can be easily developed and applied for the high-throughput analysis of a wide variety of compounds without the need to resort to laborious offline SPE sample preparation.

Determination of basic antidepressants and their N-desmethyl metabolites in raw sewage and wastewater using solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A novel analytical method has been developed for the determination of six basic antidepressants (venlafaxine, sertraline, paroxetine, citalopram, amitriptyline, and fluoxetine) and four of their metabolites (O-desmethylvenlafaxine, desmethylsertraline, nortriptyline, and norfluoxetine) in raw sewage and roughly primary-treated wastewater. For analytical development purposes, two ion exchange solid-phase extraction cartridges were compared. Extracts were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with positive-mode electrospray (+ESI) and selected reaction monitoring transitions. The choice of a basic mobile phase significantly improved the instrumental sensitivity (by up to 14-fold for norfluoxetine) relative to common +ESI acidic mobile phases. In addition to the remarkable gain in sensitivity, negligible matrix effects were also observed in the raw sewage samples. Analyte recoveries ranged from 80 to 103% and effluent detection limits from 0.048 to 0.10 ng/L. Samples collected at the Montreal Wastewater Treatment Plant showed the unequivocal presence of all the target compounds at concentrations of 2-346 ng/L. The target antidepressants were also detected in samples taken from the effluent receiving waters (i.e., the St. Lawrence River) but at lower concentrations (0.41-69 ng/L). The highly sensitive proposed method constitutes one of the best means for monitoring the environmental occurrence of tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs), and some of their metabolites.

Ecotoxicity of CdTe quantum dots to freshwater mussels: impacts on immune system, oxidative stress and genotoxicity.

The purpose of this study was to examine the toxic effects of cadmium-telluride (CdTe) quantum dots on freshwater mussels. Elliption complanata mussels were exposed to increasing concentrations of CdTe (0, 1.6, 4 and 8 mg/L) and cadmium sulfate (CdSO(4), 0.5mg/L) for 24h at 15 degrees C. After the exposure period, they were removed for assessments of immunocompetence, oxidative stress (lipid peroxidation) and genotoxicity (DNA strand breaks). Preliminary experiments revealed that CdTe dissolved in aquarium water tended to aggregate in the particulate phase (85%) while 15% of CdTe was found in the dissolved phase. Immunotoxicity was characterized by a significant decrease in the number of hemocytes capable of ingesting fluorescent beads, and hemocyte viability. The cytotoxic capacity of hemocytes to lyse mammalian K-562 cells was significantly increased, but the number of circulating hemocytes remained unchanged. Lipid peroxidation was significantly increased at a threshold concentration of 5.6 mg/L in gills and significantly reduced in digestive glands at a threshold concentration <1.6 mg/L CdTe. The levels of DNA strand breaks were significantly reduced in gills at <1.6 mg/L CdTe. In digestive glands, a transient but marginal increase in DNA strand breaks occurred at the lowest concentration and dropped significantly at the higher concentrations. A multivariate analysis revealed that the various response patterns differed based on the concentration of CdTe, thus permitting the identification of biomarkers associated with the form (colloidal vs. molecular) of cadmium.

Recycling EDTA solutions used to remediate metal-polluted soils.

The objective of this research was to investigate the recycling of ethylenediamine-tetraacetic acid (EDTA) used for the removal of trace metals from contaminated soils. We successfully used Na2S combined with Ca(OH)2 to precipitate the trace metals allowing us to recycle the EDTA. The results of batch and column leaching experiments show that both Ca-EDTA and Na-EDTA are powerful chelating agents with a similar soil remediation potential. The major advantage of Ca-EDTA is the preservation of soil organic matter. We found that Na2S was capable of separating the metals Cd, Cu and Pb from EDTA; however, the precipitation of Zn required the addition of Ca(OH)2. After reusing the reclaimed EDTA seven times, over a 14-day period, EDTA reagent losses ranged from 19.5% to 23.5%. Successive washing cycles enhanced the removal of trace metals from contaminated soils. The metal sulfide precipitates contain high concentrations of metals and could potentially be recycled.

A new ultrasound protocol for extrusion of coelomocyte cells from the earthworm Eisenia fetida.

There is mounting evidence that earthworms could be used as a sentinel species for soil ecotoxicity evaluation. In this aspect, phagocytosis by coelomocytes was shown to be a sensitive biomarker of exposure to xenobiotics. In this paper, we introduce a simple method for ultrasound extrusion of earthworm coelomocytes that generates a high cell yield, does not interfere with phagocytic competence, and requires a minimum of manipulations. Coelomocytes were extruded from the earthworm Eisenia fetida using this new ultrasound method and compared with ethanol and electrical extrusion. The ultrasonic extrusion showed the highest cell recovery with 3.17 +/ -0.8 x 10(6) cells per earthworm compared with 2.22 +/- 0.8 x 10(6) cells per earthworm for electrical extrusion and 1.57 +/- 0.07 x 10(6) cells per earthworm for ethanol extrusion. No significant differences in the cell viability were observed using propidium iodide and flow cytometry with viability for extrusion with ethanol of 63.8 +/- 12.7%, electrical 76.8 +/- 7.5%, and ultrasound 68.2 +/- 7.8%. To compare the potential effect of extrusion on cell quality, the cells extruded using the three methods were subjected to an 18-h in vitro exposure to methylmercury chloride (MeHgCl; CH3HgCl) with concentrations ranging from 10(-9) to 10(-4)M. The half-maximal effective concentration (EC50) for inhibition of phagocytosis occurred between 10(-7) and 10(-6)M. We found no significant differences among the extrusion methods for the phagocytic potential of the coelomocytes. This method does not harm the worms and can certainly improve collection of coelomocytes from earthworms and therefore contribute to the development of bioassays using invertebrates.

Immunotoxicological response of the earthworm Lumbricus terrestris following exposure to cement kiln dusts.

Cement kiln dusts are made of a complex mixture of elements. We have evaluated the potential negative impact of those dusts on the immune system of the earthworm Lumbricus terrestris. We specifically studied cell viability and phagocytic activity of coelomocytes extruded during electrical stimulation. We used two modes of exposures: in vitro, and soil incubation using OECD artificial soil media. Extruded coelomocytes were exposed 18 h in vitro to 10, 100, and 500 mg L(-1) of cement kiln dust particles. The phagocytosis and the cell viability were determined using a double-laser-flow acquisition cytometry system. Using the double laser allows us to use a dichlorofluorescein diacetate (DCFDA) marker to discriminate the biological cells from the cement kiln dusts. Dead cells are marked using propidium iodide (PI). All three exposure levels showed highly significant impacts on cell viability and phagocytic activity. The in vivo soil incubation was performed using 10, 100, and 1000 mg kg(-1) of cement kiln dusts incorporated into the OECD media. Here, to discriminate the biological cells from the mineral dusts we only needed to use PI. The day-to-day variability of the in vivo assay was high and although we can observe an overall reduction in cell viability at the highest concentration tested, no statistically significant effects could be observed on either cell viability or phagocytosis.

Angiotensin II is bound to both receptors AT1 and AT2, parallel to the transmembrane domains and in an extended form.

UNLABELLED: We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. CONCLUSIONS: (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.

Phagocytic response of terrestrial and aquatic invertebrates following in vitro exposure to trace elements.

The potential of the trace elements Ag, As, Cd, Hg, Mo, Ni, Pb, Se, and Zn to inhibit the phagocytosis response of extruded coelomocytes of different worm species was tested. We used flow cytometry to evaluate the sensitivity of cell viability and phagocytic potential for Eisenia fetida, Lumbricus terrestris, Aporrectodea turgida, and Tubifex tubifex. Extruded cells were exposed 18 h in vitro to concentrations ranging from 10(-9) to 10(-4) M. Mercury was the most potent immunotoxic element, with 50% inhibition of phagocytosis occurring at concentrations between 10(-7) and 10(-6) M. Cadmium, Cu, Ni, and Zn also showed significant immunosuppressive effects with concentrations inducing 50% inhibition ranging from 10(-5) to 10(-4) M. Species-specific sensitivity varied by about a factor of 10, with no species showing a systematically higher or lower in vitro sensitivity across the range of trace elements tested.

Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn).

We measured non-specific immune function of various bivalves from marine (Cyrtodaria siliqua, Mactromeris polynyma, Mesosdesma arctatum, Mya arenaria, Mya truncata, Mytilus edulis, Serripes groenlandicus, Siliqua costata) and freshwater environments (Dreissena polymorpha and Elliptio complanata). We used flow cytometry to quantify the phagocytosis of fluorescent microspheres by hemocytes exposed in vitro to increasing levels of various metal compounds (AgNO(3), CdCl(2), CH(3)HgCl, HgCl(2) and ZnCl(2)). In some species, low doses of mercury (organic and inorganic) and Zn suggest a hormesis-like stimulation of phagocytic activity. At higher levels of exposure, all metals tested induced a significant dose-related inhibition of hemocyte phagocytosis. The species-specific sensitivity of the assay was determined by comparing the in vitro exposure using the metal concentration inducing a 50% suppression (EC(50)) of the phagocytic activity. Different species expressed different levels of sensitivity. Our results show the variability of the toxic response of different species within a group of similar organisms. It also highlights the need to consider species-species differences in ecotoxicological risk assessment.