Waterborne Concentrations Research Articles

Limitations of waterborne exposure of fish early life stages to BDE-47

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is acknowledged as the most abundant congener of all polybrominated diphenyl ethers (PBDEs). Despite its limited residence in the water column, most ecotoxicological research using fish early life stages (ELS) has focused on its waterborne bioavailability. These studies have been supported either by chemical analysis in solutions or in tissues after ≤168h exposures to relatively high waterborne concentrations with dimethyl sulfoxide (DMSO) as solvent carrier (≤0.5%). Using noninvasive physiological and anatomical features in medaka ELS, we investigated the viability of waterborne BDE-47 exposures (100–10,000μg/L; 1% DMSO) and evaluated the developmental effects in relation to the actual BDE-47 present in water. Embryos were exposed for 10 days under semi-static (24-h renewal) conditions and waterborne BDE-47 concentrations (i.e., dissolved) were quantitated daily and their accumulation in eleutheroembryonic tissues was analyzed 4 days after exposures finished. BDE-47 in solution rapidly decreased after each renewal by >50% in 24h. This was confirmed by discernible precipitation occurring at ≥5000μg/L on the bottom of the container and attached to the chorionic filaments of eggshell. The fast dissipation from water may explain why, besides the subtle, yet significant effects on post-hatching growth (short length at ≥5000μg/L), no other significant deleterious developmental effects were observed despite the fact that BDE-47 accumulated in tissues in response to BDE-47 treatment. Waterborne BDE-47 exposure was unachievable under traditional semi-static exposure conditions, but was achievable in repeated pulse exposures lasting a few hours whenever the medium was renewed. Hence, this research encourages the use of alternate – more realistic – exposure routes (e.g., particulate matter or sediments) when evaluating early developmental toxicity of BDE-47 or any other PBDE sharing similar properties.

Critical body residues, Michaelis–Menten analysis of bioaccumulation, lethality and behaviour as endpoints of waterborne Ni toxicity in two teleosts

Traditionally, water quality guidelines/criteria are based on lethality tests where results are expressed as a function of waterborne concentrations (e.g. LC50). However, there is growing interest in the use of uptake and binding relationships, such as biotic ligand models (BLM), and in bioaccumulation parameters, such as critical body residue values (e.g. CBR50), to predict metal toxicity in aquatic organisms. Nevertheless, all these approaches only protect species against physiological death (e.g. mortality, failed recruitment), and do not consider ecological death which can occur at much lower concentrations when the animal cannot perform normal behaviours essential for survival. Therefore, we investigated acute (96h) Ni toxicity in two freshwater fish species, the round goby (Neogobius melanostomus) and rainbow trout (Oncorhynchus mykiss) and compared LC, BLM, and CBR parameters for various organs, as well as behavioural responses (spontaneous activity). In general, round goby were more sensitive. Ni bioaccumulation displayed Michaelis-Menten kinetics in most tissues, and round goby gills had lower Kd (higher binding affinity) but similar Bmax (binding site density) values relative to rainbow trout gills. Round goby also accumulated more Ni than did trout in most tissues at a given exposure concentration. Organ-specific 96h acute CBR values tended to be higher in round goby but 96h acute CBR50 and CBR10 values in the gills were very similar in the two species. In contrast, LC50 and LC10 values were significantly higher in rainbow trout. With respect to BLM parameters, gill log KNiBL values for bioaccumulation were higher by 0.4-0.8 log units than the log KNiBL values for toxicity in both species, and both values were higher in goby (more sensitive). Round goby were also more sensitive with respect to the behavioural response, exhibiting a significant decline of 63-75% in movements per minute at Ni concentrations at and above only 8% of the LC50 value; trout exhibited no clear behavioural response. Across species, diverse behavioral responses may be more closely related to tissue Ni burdens than to waterborne Ni concentrations. To our knowledge, this is the first study to link Ni bioaccumulation with behavioural endpoints. In future it would be beneficial to expand these analyses to a wider range of species to determine whether Ni bioaccumulation, specifically in the gills, gut and whole fish, may be a good predictor of behavioural changes from metal exposure; which in the wild can lead to ecological death.

The Impact of Water Exchange Rate and Treatment Processes on Water-Borne Hormones in Recirculation Aquaculture Systems Containing Sexually Maturing Atlantic Salmon Salmo salar

A controlled seven-month study was conducted in six replicated water recirculation aquaculture systems (WRAS) to assess post-smolt Atlantic salmon (Salmo salar) performance in relation to WRAS water exchange rate. Unexpectedly high numbers of precocious sexually mature fish were observed in all WRAS toward the end of the study period; therefore, a separate investigation was conducted to quantify the levels of water-borne hormones (cortisol (C), testosterone (T), 11-ketotestosterone (11-KT), progesterone (P), and estradiol (E2)) to determine the impact of WRAS exchange rate, as well as transit through the unit processes, on soluble hormone concentrations.Triplicate water samples were collected at three separate sites in each of the six WRAS: pre-unit processes, post-unit processes, and at the makeup water influent. Water samples were concentrated and separate quantifications were carried out for each target hormone using enzyme immunoassay kits. Results indicated that among the hormones examined, only T was associated with higher concentrations in low exchange WRAS compared to high exchange WRAS. Water passage through the unit processes was associated with a significant reduction in concentration of 11-KT, in both high and low exchange WRAS. Water-borne concentrations of T, 11-KT, and E2 were significantly higher than influent makeup water; the majority of C and P concentrations were not significantly different between WRAS and makeup water samples. No significant differences were noted in the prevalence of apparently sexually mature fish or gonadosomatic indices in either sex between treatments, except a significantly higher prevalence of apparently mature female fish in low exchange WRAS. Overall, these findings suggest that, under the conditions of this study, C, P, E2, and 11-KT do not accumulate in lower exchange WRAS, and that, aside from 11-KT, the WRAS unit processes do not impact hormone concentration. Furthermore, the observed precocious sexual maturation was mostly unrelated to WRAS exchange rate.

Isotopically modified silver nanoparticles to assess nanosilver bioavailability and toxicity at environmentally relevant exposures

Environmental context Predicting the environmental implications of nanotechnology is complex in part because of the difficulty in studying nanoparticle uptake in organisms at environmentally realistic exposures. Typically, high exposure concentrations are needed to detect accumulation and effects. We use labelled Ag nanoparticles to determine whether Ag bioaccumulation responses are linear over concentrations likely to occur in the environment, and whether concentration-dependent changes in agglomeration and dissolution affect bioavailability. Abstract A major challenge in understanding the environmental implications of nanotechnology lies in studying nanoparticle uptake in organisms at environmentally realistic exposure concentrations. Typically, high exposure concentrations are needed to trigger measurable effects and to detect accumulation above background. But application of tracer techniques can overcome these limitations. Here we synthesised, for the first time, citrate-coated Ag nanoparticles using Ag that was 99.7% 109Ag. In addition to conducting reactivity and dissolution studies, we assessed the bioavailability and toxicity of these isotopically modified Ag nanoparticles (109Ag NPs) to a freshwater snail under conditions typical of nature. We showed that accumulation of 109Ag from 109Ag NPs is detectable in the tissues of Lymnaea stagnalis after 24-h exposure to aqueous concentrations as low as 6ngL–1 as well as after 3h of dietary exposure to concentrations as low as 0.07μgg–1. Silver uptake from unlabelled Ag NPs would not have been detected under similar exposure conditions. Uptake rates of 109Ag from 109Ag NPs mixed with food or dispersed in water were largely linear over a wide range of concentrations. Particle dissolution was most important at low waterborne concentrations. We estimated that 70% of the bioaccumulated 109Ag concentration in L. stagnalis at exposures <0.1µgL–1 originated from the newly solubilised Ag. Above this concentration, we predicted that 80% of the bioaccumulated 109Ag concentration originated from the 109Ag NPs. It was not clear if agglomeration had a major influence on uptake rates.

Study of genetic damage in the Japanese oyster induced by an environmentally-relevant exposure to diuron: Evidence of vertical transmission of DNA damage

Pesticides represent a major proportion of the chemical pollutants detected in French coastal waters and hence a significant environmental risk with regards to marine organisms. Commercially-raised bivalves are particularly exposed to pollutants, among them pesticides, as shellfish farming zones are subject to considerable pressure from agricultural activities on the mainland.The aims of this study were to determine (1) the genotoxic effects of diuron exposure on oyster genitors and (2) the possible transmission of damaged DNA to offspring and its repercussions on oyster fitness. To investigate these points, oysters were exposed to concentrations of diuron close to those detected in the Marennes–Oleron Basin (two 7-day exposure pulses at 0.4 and 0.6μgL−1) during the gametogenesis period. Genomic abnormalities were characterized using two complementary approaches. The Comet assay was applied for the measurement of early and reversible primary DNA damage, whereas flow cytometry was used to assess the clastogenic and aneugenic effect of diuron exposure. Polar Organic Chemical Integrative Samplers (POCIS) were used in exposed and assay tanks to confirm the waterborne concentration of diuron reached during the experiment.The results obtained by the Comet assay clearly showed a higher level of DNA strand breaks in both the hemocytes and spermatozoa of diuron-exposed genitors. The transmission of damaged genetic material to gamete cells could be responsible for the genetic damage measured in offspring. Indeed, flow cytometry analyses showed the presence of DNA breakage and a significant decrease in DNA content in spat from diuron-exposed genitors. The transmission of DNA damage to the offspring could be involved in the negative effects observed on offspring development (decrease in hatching rate, higher level of larval abnormalities, delay in metamorphosis) and growth.In this study, the vertical transmission of DNA damage was so highlighted by subjecting oyster genitors to short exposures to diuron at medium environmental concentrations. The analysis of POCIS showed that oysters were exposed to integrated concentrations as low as 0.2 and 0.3μgL−1, emphasizing the relevance of the results obtained and the risk associated to chemical contamination for oyster recruitment and fitness.

A Single Metal, Metal Mixture, and Whole-Effluent Approach to Investigate Causes of Metal Mine Effluent Effects on Fathead Minnows (Pimephales promelas)

Although many studies have examined effects of metal mine effluents (MMEs) on receiving environments, few have compared the roles of individual and mixed metals relative to whole effluents. The objective of the present set of studies was to examine whether Cu, Ni, or Se, alone or in a mixture, causes comparable effects to those observed in fathead minnows (Pimephales promelas) exposed to an environmentally relevant MME (45 % process water effluent [PWE]). Metal bioaccumulation, fathead minnow (FHM) morphometrics, and egg production were compared between treatments over a 21-day exposure. FHMs were exposed to similar waterborne concentrations and species of metals in single and mixed metal treatments relative to 45 % PWE. FHMs were also exposed to similar concentrations of metals in single and mixed metal treatments relative to 45 % PWE through the diet (Chironomus dilutus — a representative prey species). However, only FHMs exposed to 45 % PWE had reduced egg production (60–80 % less than controls). Our findings indicate that Cu, Ni, and Se exposures and bioaccumulation did not contribute to decreased reproductive output in FHMs under the conditions that were examined. We also found no evidence to believe that these metals were responsible for decreased egg production in PWE. It is therefore reasonable to suggest that these metals have limited potential to cause reproductive effects in MMEs with similar composition and water chemistry conditions. Overall, this study highlights the importance of examining single and mixed metal exposures prior to suggesting that adverse effects in fish exposed to MMEs occur due to bioaccumulation of metal(s).

Waterborne versus Dietary Zinc Accumulation and Toxicity in Daphnia magna: a Synchrotron Radiation Based X-ray Fluorescence Imaging Approach

Recent studies have suggested that exposure of the freshwater invertebrate Daphnia magna to dietary Zn may selectively affect reproduction without an associated increase of whole body bioaccumulation of Zn. The aim of the current research was therefore to investigate the hypothesis that dietary Zn toxicity is the result of selective accumulation in tissues that are directly involved in reproduction. Since under field conditions simultaneous exposure to both waterborne and dietary Zn is likely to occur, it was also tested if accumulation and toxicity under combined waterborne and dietary Zn exposure is the result of interactive effects. To this purpose, D. magna was exposed during a 16-day reproduction assay to Zn following a 5 × 2 factorial design, comprising five waterborne concentrations (12, 65, 137, 207, and 281 μg Zn/L) and two dietary Zn levels (49.6 and 495.9 μg Zn/g dry wt.). Tissue-specific Zn distribution was quantified by synchrotron radiation based confocal X-ray fluorescence (XRF). It was observed that the occurrence of reproductive inhibition due to increasing waterborne Zn exposure (from 65 μg/L to 281 μg/L) was accompanied by a relative increase of the Zn burdens which was similar in all tissues considered (i.e., the carapax, eggs, thoracic appendages with gills and the cluster comprising gut epithelium, storage cells and ovaries). In contrast, the impairment of reproduction during dietary Zn exposure was accompanied by a clearly discernible Zn accumulation in the eggs only (at 65 μg/L of waterborne Zn). During simultaneous exposure, bioaccumulation and toxicity were the result of interaction, which implies that the tissue-specific bioaccumulation and toxicity following dietary Zn exposure are dependent on the Zn concentration in the water. Our findings emphasize that (i) effects of dietary Zn exposure should preferably not be investigated in isolation from waterborne Zn exposure, and that (ii) XRF enabled us to provide possible links between tissue-specific bioaccumulation and reproductive effects of Zn.

Tracking losses of brevetoxins on exposure to phytoplankton competitors: Ecological impacts

The frequent occurrence of devastating blooms of the harmful dinoflagellate Karenia brevis in the Gulf of Mexico has motivated research into bloom dynamics and potential mitigation strategies. The use of competing phytoplankton to lower waterborne concentrations of the most abundant and toxic brevetoxins produced during these blooms has been proposed. However the ecological impacts of using such biocontrol agents have not been addressed. This study investigated the impact on marine invertebrates of lowered brevetoxin concentrations due to the presence of competing phytoplankton. Even at low brevetoxin concentrations, the presence of the common diatom Skeletonema grethae ameliorated harmful toxic effects of brevetoxins upon the brine shrimp, Artemia salina, and reduced the incidence of negative physiological and morphological responses of the sea anemone Aiptasia pallida. In addition, brevetoxin biotransformation products formed by competing phytoplankton appear to be non-toxic or do not trigger the same physiological responses as brevetoxins in the model organisms used. These findings may impact the interpretation of ecotoxicological data gathered during bloom events, since the presence of phytoplankton competitors in Karenia blooms is likely to reduce the harmful effects seen on many marine invertebrates.

Predictive modeling of selenium accumulation in brine shrimp in saline environments

Great Salt Lake, Utah, is a large, terminal, hypersaline lake consisting of a northern more saline arm and a southern arm that is less saline. The southern arm supports a seasonally abundant fauna of low diversity consisting of brine shrimp (Artemia franciscana), 7 species of brine flies, and multiple species of algae. Although fish cannot survive in the main body of the lake, the lake is highly productive, and brine shrimp and brine fly populations support large numbers of migratory waterfowl and shorebirds, as well as resident waterfowl, shorebirds, and gulls. Selenium and other trace elements, metals, and nutrients are contaminants of concern for the lake because of their concentrations in municipal and industrial outfalls and runoff from local agriculture and the large urban area of Salt Lake City. As a consequence, the State of Utah recently recommended water quality standards for Se for the southern arm of Great Salt Lake based on exposure and risk to birds. The tissue-based recommendations (as measured in bird eggs) were based on the understanding that Se toxicity is predominately expressed through dietary exposure, and that the breeding shorebirds, waterfowl, and gulls of the lake are the receptors of most concern. The bird egg-based recommended standards for Se require a model to link bird egg Se concentrations to their dietary concentrations and water column values. This study analyzes available brine shrimp tissue Se data from a variety of sources, along with waterborne and water particulate (potential brine shrimp diet) Se concentrations, in an attempt to develop a model to predict brine shrimp Se concentrations from the Se concentrations in surrounding water. The model can serve as a tool for linking the tissue-based water quality standards of a key dietary item to waterborne concentrations. The results were compared to other laboratory and field-based models to predict brine shrimp tissue Se concentrations from ambient water and their diet. No significant relationships were found between brine shrimp and their dietary Se, as measured by seston concentrations. The final linear and piecewise regression models showed significant positive relationships between waterborne and brine shrimp tissue Se concentrations but with a very weak predictive ability for waterborne concentrations<10 µg/L.

Conducting site‐specific assessments of selenium bioaccumulation in aquatic systems

Selenium (Se) is a chemical of concern at many locations across North America and elsewhere, and site-specific conditions are important when evaluating its bioaccumulation and effects in aquatic ecosystems. Most regulatory criteria and guidelines are based on waterborne Se concentrations. In contrast, the draft water quality chronic criterion of the US Environmental Protection Agency (USEPA) is based on Se concentrations in whole-body fish, and current information suggests the agency will issue a new draft criterion based in part on fish egg and/or ovary Se concentrations. However, implementation guidance is not available from the agency for either of these tissue-based criteria. Therefore, we describe a phased approach for field and laboratory assessments of Se bioaccumulation in fish and aquatic-dependent birds that can be applied in different environmental settings with the goal of developing and interpreting a tissue-based Se value. We recommend here the use of decision trees, conceptual models, and data quality objectives toward defining what should be done during the assessment, plus sampling and monitoring procedures for the assessment. First, available tissue or waterborne Se concentrations should be compared to tissue residue guidelines or adopted water quality criteria and guidelines. When needed, reproductive toxicity testing and assessment of fish populations should also be conducted in the area of interest. In addition, extensive data on the effects of Se on fish and bird species have been developed, and describing the associations between fish or bird egg tissue, aqueous Se, and potential effects is important for sites where Se may be a concern. Selenium bioaccumulation and toxicity also are of concern for amphibians and oviparous reptiles, but interpretive information is very limited for those species. Recent science indicates that effects are more strongly related to tissue concentrations of Se (especially in the eggs or ovaries of oviparous vertebrates) than to waterborne concentrations. Overall, we conclude that the approach for site-specific assessment must be flexible enough to allow what is appropriate for the situation. Furthermore, risk management and remediation decisions should be based on combined biology and chemistry data, using multiple lines of evidence in the assessment.

Tracking losses of brevetoxins on exposure to phytoplankton competitors: Mechanistic insights

The increasing frequency of devastating blooms of the harmful dinoflagellate Karenia brevis has motivated investigations into understanding bloom dynamics and the potential for mitigation. Previous findings indicate that waterborne concentrations of the most abundant brevetoxin (brevetoxin B or PbTx-2) associated with these blooms decrease in the presence of other phytoplankton species. The current study explores the mechanism of brevetoxin removal from seawater upon exposure to phytoplankton competitors. Live phytoplankton removed waterborne brevetoxins more rapidly than lysates, but phytoplankton did not need to be in a state of active metabolism. Biomolecules, probably proteins, exuded from phytoplankton appeared to be responsible for the loss of brevetoxins, either by irreversible complexation or by degradation. Selective removal of PbTx-2 and -1, but not PbTx-3, -9 or BTX-B5, by cultured phytoplankton revealed that brevetoxin removal is dependent upon the presence of an α,β-unsaturated aldehyde functionality. The mechanism of biotransformation appears to be common among phytoplankton, since members of various taxonomic groups including diatoms, dinoflagellates, and a cryptophyte each caused 75–90% decrease in PbTx-2 concentration, as did a generic protein (bovine serum albumin) added to seawater at high concentration. These findings support the concept of potentially using competitor phytoplankton species or compounds derived from phytoplankton as biocontrol agents for waterborne toxins associated with red tide.

In situ biomonitoring of juvenile Chinook salmon ( Onchorhynchus tshawytscha) using biomarkers of chemical exposures and effects in a partially remediated urbanized waterway of the Puget Sound, WA

In situ biomonitoring has been used to assess the effects of pollution on aquatic species in heavily polluted waterways. In the current study, we used in situ biomonitoring in conjunction with molecular biomarker analysis to determine the effects of pollutant exposure in salmon caged in the Duwamish waterway, a Pacific Northwest Superfund site that has been subject to remediation. The Duwamish waterway is an important migratory route for Pacific salmon and has received historic inputs of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Juvenile pre-smolt Chinook salmon ( Oncorhynchus tshawytscha) caged for 8 days in the three contaminated sites in close proximity within the Duwamish were analyzed for steady state hepatic mRNA expression of 7 exposure biomarker genes encompassing several gene families and known to be responsive to pollutants, including cytochrome P4501A (CYP1A) and CYP2K1, glutathione S-transferase π class (GST-π), microsomal GST (mGST), glutamylcysteine ligase catalytic subunit (GCLC), UDP-glucuronyltransferase family 1 (UDPGT), and type 2 deiodinase (type 2 DI, or D2). Quantitation of gene expression was accomplished by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in assays developed specifically for Chinook salmon genes. Gill PAH-DNA adducts were assessed as a chemical effects biomarker using 32P-postlabeling. The biomarkers in the field-caged fish were analyzed with respect to caged animals maintained at the hatchery receiving flow-through water. Chemical analysis of sediment samples from three field sampling sites revealed relatively high concentrations of total PAHs in one site (site B2, 6711 ng/g dry weight) and somewhat lower concentrations of PAHs in two adjacent sites (sites B3 and B4, 1482 and 1987 ng/g, respectively). In contrast, waterborne PAHs at all of the sampling sites were relatively low (<1 ng/L). Sediment PCBs at the sites ranged from a low of 421 ng/g at site B3 to 1160 ng/g at site B4, and there were no detectable waterborne PCBs at any of the sites (detection limit=10 ng/L). There were no significant differences ( p<0.05) in biomarker gene expression in the Duwamish-caged fish relative to controls, although there was a pattern of gene expression suppression at site B3, the most heavily PAH-enriched site. The lack of a marked perturbation of mRNA biomarkers was consistent with relatively low levels of gill PAH-DNA adduct levels that did not differ among caged reference and field fish, and which were also consistent with relatively low waterborne concentrations of chemicals. The results of our study suggest a low bioavailability of sediment pollutants in caged juvenile Chinook potentially reflecting low waterborne exposures occurring at contaminated sites within the Duwamish waterway that have undergone partial remediation.

Spatial, Phase, And Temporal Distributions of Perfluorooctane Sulfonate (PFOS) and Perfluorooctanoate (PFOA) in Tokyo Bay, Japan

The spatial distribution, partitioning, and time trends of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) were investigated in the water column and bottom sediment of Tokyo Bay, Japan, during 2004-2006. A total of 480 water and 60 sediment samples obtained by regular 20-station samplings ascertained the three-dimensional distributions of these compounds and changes in the seawater structure in the whole bay. The median of volume-based average water-borne concentrations of PFOS and PFOA was 3.7 and 12 ng/L, respectively. The median concentrations in sediment were 0.61 (PFOS) and 0.20 (PFOA) ng/g-dry. Vertical mixing of the water column probably affected the vertical distribution of these compounds. The negative correlations between PFOS and PFOA concentrations and water salinity and the horizontal distributions of their concentrations suggested that freshwater inputs into the bay were the source of these compounds. A mixing model estimated the average PFOS concentration in the freshwater inputs to be 29 ng/L. The common logarithm of the partition coefficients between the dissolved and suspended-particle-sorbed phases varied among samples, with the average of 4.2 (PFOS) and 3.5 (PFOA). Our analyses indicated no apparent time trends in the concentrations of these compounds during 2004-2006 in either the freshwater input or the bay.

Application of Solid-Phase Microextraction for In Vivo Laboratory and Field Sampling of Pharmaceuticals in Fish

Previous field studies utilizing solid-phase microextraction (SPME) predominantly focused on volatile and semivolatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to the liquid matrix (blood). The present study has expanded the SPME technique to semisolid tissues under laboratory and field conditions through the investigation of both theoretical and applied experimental approaches. Pre-equilibrium extraction and desorption were performed in vivo in two separate animals. Excellent linearity was found between the amounts extracted by SPME from the muscle of living fish and the waterborne concentrations of pharmaceuticals. A simple SPME method is also described to simultaneously determine free and total analyte concentrations in living tissue. The utility of in vivo SPME sampling was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory and in vivo field sampling conditions.

Molecular and cellular detection of expression of vitellogenin and zona radiata protein in liver and skin of juvenile salmon (Salmo salar) exposed to nonylphenol

In developing bioassays for estrogenic effects, vitellogenin (Vtg) induction and zona radiata protein (Zr-protein) induction in males and juveniles of oviparous vertebrates have been used as sensitive biomarkers for estrogenicity. Nonylphenol (NP) produces similar and parallel expression patterns of Vtg and Zr-protein levels in plasma and surface mucus of salmon, the response being concentration- and time-dependent. We have explored the potential mechanisms of Vtg and Zr-protein expression in surface mucus by comparative molecular and cellular approaches. Liver, skin, blood, and surface mucus samples were collected from fish exposed to a single waterborne concentration of NP (10 and 60 microg/l), 3, 7, and 10 days post-exposure, for gene expression analysis (liver and skin; quantitative real-time polymerase chain reaction) and protein analysis (blood and surface mucus; enzyme-linked immunosorbent assay). Protein expression was localized by immunohistochemistry. NP produced concentration- and time-dependent increases of hepatic estrogen receptors (ERalpha and ERbeta), Vtg, and Zr-protein mRNA and plasma protein levels. These responses paralleled cellular detection of Vtg and Zr-protein in the liver with unique expression patterns in the cytoplasm of hepatocytes, hepatic sinusoids, and endothelial cells. ERalpha, Vtg, and Zr-protein mRNA were detectable in the skin. ERbeta was the only skin response that was NP-concentration-dependent, especially at day 10 post-exposure. Immunohistochemistry for Vtg and Zr-protein in skin showed unique expression patterns in mucus vacuoles, epidermal cells, and scales in an NP-concentration- and time-specific manner. Thus, analysis of skin mRNA levels for xenoestrogen biomarker responses is a less-promising approach than protein analysis. The immunohistochemical localization of Vtg and Zr-protein levels in the skin further validates surface mucus as a sensitive biomarker source for estrogenic compounds. These responses represent an improvement for the detection of endocrine-disrupting compounds and related pollutants in the environment.

Determination of the androgenic potency of whole effluents using mosquitofish and trout bioassays

This study combined bioassay-derived and direct chemical analysis of steroidal compounds in pulp and paper and municipal sewage effluent in order to determine the cause of masculinization of female mosquitofish. The bioassays used in this study consisted of androgen and estrogen receptor binding, and aromatase inhibition using tissues from rainbow trout. This study observed no masculinization of female mosquitofish from a pulp and paper mill effluent that was previously observed to cause this effect. Mosquitofish sampled from the receiving environment of the same mill verified that masculinization was not occurring in the wild. The municipal sewage effluent also had no masculinizing effect. In vitro bioassays indicated significant sources of both androgens and estrogens in the effluents tested with sewage effluent having both the highest estradiol (41 ng/L) and testosterone (182 ng/L) equivalent concentration. These results could not be attributed to any particular compounds measured in the effluents. Two compounds implicated in the masculinization of mosquitofish by pulp and paper effluent, androstenedione and androstadienedione required relatively large (10–100 μg/L) waterborne concentrations to elicit a masculinizing effect and neither of these compounds are likely to occur at levels this high in the natural environment. The potent aromatase inhibitor, 4-hydroxyandrostenedione also did not cause masculinization at 100 μg/L indicating that masculinization did not occur through this mechanism. The mammalian anti-androgen, cyproterone acetate was only partially effective in mosquitofish and reduced the severity of masculinization in the presence of methyl testosterone. While neither effluent masculinized mosquitofish, there was a significant reduction of in vitro ovarian steroid production with the most severe effects observed with the sewage effluent. Overall, this study found the disappearance of a masculinizing effect that had been previously observed; concluded that based on 21 days aqueous exposures androstenedione and androstadienedione are not likely candidates for mosquitofish masculinization, and showed that masculinization and in vitro steroid production are unrelated biological endpoints.

Dietary transfer of fluoranthene from an estuarine oligochaete (Monopylephorus rubroniveus) to grass shrimp (Palaemonetes pugio): Influence of piperonyl butoxide

The objective of this study was to determine the potential for dietary transfer of sediment-associated fluoranthene from tubificid oligochaetes (Monopylephorus rubroniveus) to grass shrimp (Palaemonetes pugio). Grass shrimp, either in the presence or absence of sublethal waterborne concentrations of the metabolic inhibitor, piperonyl butoxide (PBO), were fed fluoranthene-dosed oligochaetes for 5-days. All grass shrimp bioaccumulated fluoranthene; however, bioaccumulation was 3X higher in the presence of PBO. Trophic transfer coefficients (TTCs) were 0.02 and 0.01 in the presence and absence of PBO, respectively. Following the 5-day accumulation period, shrimp in both treatments were allowed to depurate for 3 days. Depuration rates were significantly higher in PBO-exposed shrimp. These results demonstrated that sediment-associated fluoranthene can be transferred through the diet from oligochaetes to grass shrimp, and the presence of PBO enhanced fluoranthene bioaccumulation. However, the comparatively low TTCs suggest that biomagnification of fluoranthene in estuarine food webs is low.

Waterborne gemfibrozil challenges the hepatic antioxidant defense system and down-regulates peroxisome proliferator-activated receptor beta (PPARβ) mRNA levels in male goldfish ( Carassius auratus)

The lipid regulator gemfibrozil (GEM) is one of many human pharmaceuticals found in the aquatic environment. We previously demonstrated that GEM bioconcentrates in blood and reduces plasma testosterone levels in goldfish ( Carassius auratus). In this study, we address the potential of an environmentally relevant waterborne concentration of GEM (1.5 μg/l) to induce oxidative stress in goldfish liver and whether this may be linked to GEM acting as a peroxisome proliferator (PP). We also investigate the autoregulation of the peroxisome proliferator-activated receptors (PPARs) as a potential index of exposure. The three PPAR subtypes (α, β, and γ) were amplified from goldfish liver cDNA. Goldfish exposed to a concentration higher (1500 μg/l) than environmentally relevant for 14 and 28 days significantly reduce hepatic PPARβ mRNA levels ( p < 0.001). Levels of CYP1A1 mRNA were unchanged. GEM exposure significantly induced the antioxidant defense enzymes catalase ( p < 0.001), glutathione peroxidase ( p < 0.001) and glutathione- S-transferase ( p = 0.006) but not acyl-CoA oxidase or glutathione reductase. As GEM exposure failed to increase levels of thiobarbituric reactive substances (TBARS), we conclude that a sub-chronic exposure to GEM upregulates the antioxidant defense status of the goldfish as an adaptive response to this human pharmaceutical.

Differential sensitivity of flounder (Platichthys flesus) in response to oestrogenic chemical exposure: An issue for design and interpretation of monitoring and research programmes

This study was conducted as an initial investigation of 'differential response' in one of the main sentinel organisms used for monitoring programmes in United Kingdom estuaries, the flounder Platichthys flesus. It has been hypothesised that monitoring using species with a wide geographical spread and limited migration, such as flounder, might result in the comparison of different genetic stocks and certainly of populations with differing early life stage contaminant exposure histories. Furthermore, it is probable that these pre-exposure and genetic differences could manifest themselves in an ability to respond differently to contaminant exposure, so-called 'differential response'. It is important that the extent and nature of this response is understood, if we want to be able to fully interpret the monitoring data from such programmes. During this study, flounder were collected from four separate sources; wild caught fish from the estuaries of the Rivers Alde, Mersey and Tyne, and farmed flounder from Port Erin Farm, Isle of Man. Under controlled laboratory conditions, groups of fish from each source were exposed to water-borne concentrations of the synthetic oestrogen ethynylestradiol (EE2) at a nominal concentration of 50 ng/l. Plasma was taken from each male fish after 6 and 10 days exposure and analysed for the presence of vitellogenin (VTG) using an ELISA technique. Significant levels of VTG induction were evident in fish from all sources after both 6 and 10 days exposure. Flounder from the Mersey were the only fish with significantly elevated initial background levels of VTG (day 0) and this appeared to be reflected in that these specimens showed the highest induction response after day 6. However, after day 10, fish from all other sites had a slightly higher mean VTG than those from the Mersey which showed significantly (p < 0.05) lower mean plasma VTG. It is suggested that other differential responses may have been masked by the use of a high dose of EE2 which produced maximum induction in nearly all fish. The findings of the study are discussed in terms of implications for further research into the differential response issue and how the initial plasma VTG figures contribute to a time-series from the Mersey, Tyne and Alde estuaries.

Does the red tide dinoflagellate Karenia brevis use allelopathy to outcompete other phytoplankton?

Monospecific blooms of phytoplankton can disrupt pelagic communities and negatively affect human health and economies. Interspecific competition may play an important role in promoting blooms, and so we tested (1) whether the outcome of competition between the red tide dinoflagellate Karenia brevis (ex Gymnodinium breve) and 12 cooccurring phytoplankters could be explained by allelopathic effects of compounds released by K. brevis and (2) whether waterborne, lipophilic molecules, including brevetoxins, are involved. Nine of 12 phytoplankton species were suppressed when grown with live K. brevis at bloom concentrations. K. brevis extracellular filtrates or lipophilic extracts of filtrates inhibited six of these nine species, indicating allelopathy. However, these inhibitory effects were weaker than those experienced by competitors exposed to live K. brevis. Brevetoxins at ecologically reasonable waterborne concentrations accounted for the modest inhibition by K. brevis of only one competitor, Skeletonema costatum. The addition of brevetoxins also caused significant autoinhibition, reducing the maximum concentration of K. brevis. Allelopathy is one mechanism by which K. brevis appears to exhibit competitive advantage over some sympatric phytoplankters, although unidentified compounds other than brevetoxins must be involved, in most cases. K. brevis was also susceptible to competitive exclusion by several species, including Odontella aurita and Prorocentrum minimum, known to thrive during K. brevis blooms. Although field experiments are required to assess whether allelopathy plays a fundamental role in bloom dynamics, our results indicate that allelopathy occurs widely but with species‐specific consequences.