Static Exposure System Research Articles

Exposure paradigm of fluoxetine impacted the Faxonius virilis agonistic behavior differently

Pharmaceutically active compounds are major contaminants of aquatic environments. The effects on aquatic organisms have been assessed mostly through eco-toxicological tests performed using static exposure systems or flow through systems with constant concentrations. Yet, constant concentration exposures ignore the spatio-temporal dynamics of chemicals in flowing environments. In dynamic systems, a chemical's effect on an organism will vary due to fluctuations in the frequency, magnitude, and duration of the chemical concentration within the plume, which develops due to turbulence interacting with the geomorphology of habitat. The aim of this study was to analyze how different exposure dynamics to the antidepressant fluoxetine might alter the agonistic behavior of aquatic organisms. Male crayfishes, Faxonius virilis, were subjected to 23 h exposures at different concentrations of fluoxetine (control, 0.05, 0.5, 1, 10 and 100 μg/l) in both static and dynamic mesocosm systems. After exposure, size-matched crayfishes, from the same exposure system and fluoxetine concentration, underwent a fifteen minute fight trial. The aggressive intensities and duration of agonistic interactions were quantified. The time spent performing a tailflip was significantly longer for fluoxetine concentrations of 1, 10, 100 μg/l in the static exposure than in the dynamic exposure. On other hand, the time spent at higher intensities and the time to escalate to the highest intensity of interactions in control treatments were significantly lower in the dynamic exposure than in the static exposure. Whereas, in elevated fluoxetine concentrations, these times were significantly higher in the dynamic than in static treatments. Therefore, we could conclude that the fight dynamics and duration of agonistic behavior in crayfish were affected by static and dynamic exposure paradigms differently. Despite these behavioral changes, serotonin levels in fluoxetine-exposed crayfish did not differ significantly between exposure paradigms. Future research should incorporate different exposure methods that more accurately represent chemical exposure in natural habitats.

A novel system for embryo-larval toxicity testing of pelagic fish: Applications for impact assessment of Deepwater Horizon crude oil

Key differences in the developmental process of pelagic fish embryos, in comparison to embryos of standard test fish species, present challenges to obtaining sufficient control survival needed to successfully perform traditional toxicity testing bioassays. Many of these challenges relate to the change in buoyancy, from positive to negative, of pelagic fish embryos that occurs just prior to hatch. A novel exposure system, the pelagic embryo-larval exposure chamber (PELEC), has been developed to conduct successful bioassays on the early life stages (ELSs; embryos/larvae) of pelagic fish. Using this unique recirculating upwelling system, it was possible to significantly improve control survival in pelagic fish ELS bioassays compared to commonly used static exposure methods. Results demonstrate that control performance of mahi-mahi (Coryphaena hippurus) embryos in the PELEC system, measured as percent survival after 96-hrs, significantly outperformed agitated static exposure and static exposure systems. Similar significant improvements in 72-hr control survival were obtained with yellowfin tuna (Thunnus albacares). The PELEC system was subsequently used to test the effects of photo-induced toxicity of crude oil to mahi-mahi ELSs over the course of 96-hrs. Results indicate a greater than 9-fold increase in toxicity of Deepwater Horizon (DWH) crude oil during co-exposure to ambient sunlight compared to filtered ambient sunlight, revealing the importance of including natural sunlight in 96-hr DWH crude oil bioassays as well as the PELEC system's potential application in ecotoxicological assessments.

The relationship of cytotoxic and genotoxic damage with blood aluminum levels and oxidative stress induced by this metal in common carp (Cyprinus carpio) erythrocytes

Aluminum is one of the most abundant elements in nature and is used in diverse industrial processes. As a result, it contaminates aquatic ecosystems, inducing damage on associated biota. In fish, it has been observed to induce hypoxia, hypercapnia, metabolic acidosis and respiratory arrest. Although there is little information on Al-induced cytotoxicity and DNA damage, this type of studies are essential in order to identify the mechanisms of action of this metal. The cytotoxic and genotoxic effects induced by Al on common carp (Cyprinus carpio) erythrocytes were determined in specimens exposed to 0.05, 120 and 239mgAlL−1 in static exposure systems. Blood samples were taken at 12, 24, 48, 72 and 96h, erythrocytes were separated, and the following were evaluated: frequency of micronuclei and frequency of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, blood Al levels, lipid peroxidation, protein carbonyl content, and activity of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase. The results show that tested aluminum concentrations produces oxidative stress (increase in lipid peroxidation degree and oxidized proteins content, as well as decrease in antioxidant enzymes activity) and induced higher frequencies of micronuclei and TUNEL-positive cells, so this metal can be considered as a cytotoxic and genotoxic agent for erythrocytes of common carp.

Dermal and ocular exposure systems for the development of models of sulfur mustard-induced injury

Sulfur mustard (SM) is a chemical threat agent for which the effects have no current treatment. Due to the ease of synthesis and dispersal of this material, the need to develop therapeutics is evident. The present article details the techniques used to develop SM laboratory exposure systems for the development of animal models of ocular and dermal injury. These models are critical to enable evaluation of SM injury and therapeutics against that injury. Iterative trials were conducted to optimize dermal and ocular injury models in guinea pigs and rabbits respectively. The goal was a homogeneous and diffuse ocular and dermal injury that compares to the human injury. Dermal exposures were conducted by either a flow-past or static vapor cup system. Ocular exposures were conducted by a static exposure system. Ocular and dermal exposures were conducted with vaporized SM. Vapor concentrations increased with time in the dermal and ocular exposure systems but were stable with varying amounts of applied SM. A dermal deposition estimation study was also conducted. Deposited volumes increased with exposure time.

Bioconcentration of the intense sweetener sucralose in a multitrophic battery of aquatic organisms

Reports of the intense (artificial) sweetener sucralose (1,6-dichloro-1,6-dideoxy-β-D-fructo-furanosyl 4-chloro-4-deoxy-α-D-galactopyranoside) in various environmental compartments have led to speculations about biological effects in nontarget species living in areas receiving discharges from anthropogenic activities. We have, as the first step in the risk assessment of this compound, conducted bioaccumulation studies in the freshwater alga Pseudokirchneriella subcapitata, the crustacean Daphnia magna, and zebrafish (Danio rerio). The freshwater algae and the daphnid tests were performed using a 48-h static exposure system, whereas the zebrafish test was performed using a 48-h semi static exposure system followed by 48 h flow-through of clean water for the depuration phase. All three studies were conducted with two exposure concentrations (10 and 100 mg/L), and the concentrations of sucralose in water and biota were verified by liquid chromatography/mass spectrometry. The studies showed that uptake of sucralose was assumed to achieve a steady state within the first 48 h, and the bioconcentration factor at the assumed steady state (BCF(SS) ) was calculated to be less than 1 for algae and between 1.6 to 2.2 for the daphnids. The fish BCF(SS), assumed to occur between 24 to 48 hours, were calculated to be less than 1 for both concentrations tested. A first-order one-compartment (uptake phase) and a first-order two-compartment (elimination phase) model characterized the uptake and depuration kinetics in zebrafish (k(1)=0.027-0.038/h and k(2)=0.206-0.222/h, t(95)=13.5 to 14.6 h, t(50)=3.1 to 3.3 h, and BCF(kinetic)=0.4 to 0.9). The current study shows that sucralose does not bioaccumulate in aquatic organisms from different tiers of the food web, and that the BCF's obtained were considerably lower than the criteria set to identify persistent, bioaccumulative, and toxic substances (i.e., BCF ≥ 2,000).

Aluminum-induced oxidative stress in lymphocytes of common carp (Cyprinus carpio)

Several studies of fish have shown that aluminum may induce hypoxia, hypercapnia, metabolic acidosis, and respiratory failure. In lymphocytes, morphologic abnormalities and reduced immune activity have been observed. Nevertheless, there is little data on oxidative stress and such data are essential in order to identify its mechanism of action. The common carp Cyprinus carpio, an omnivorous fish commonly used in commercial aquaculture, has been proposed as a test organism in toxicologic assays due to its economic importance and wide geographic distribution. The aim of this work was to evaluate Al-induced oxidative stress in lymphocytes of the common carp C. carpio. Specimens were exposed to three different concentrations of Al (0.05, 120, and 239mg/l) in a static exposure system for 96h. At the end of the exposure period, blood was collected and lymphocytes were separated. Lipid peroxidation, oxidized protein content and the activity of superoxide dismutase, catalase, and glutathione peroxidase were measured. Results show that the tested Al concentrations modified the activity of antioxidant enzymes and elicited higher levels of lipid peroxidation and oxidized proteins. The degree of damage induced was concentration and tissue dependent.

Abuse pattern of gestational toluene exposure alters behavior in rats in a “waiting-for-reward” task

Toluene abuse during pregnancy is a world-wide public health concern although the neurobehavioral teratogenic effects of toluene at the high concentrations and binge-like exposure patterns typical of abuse remain understudied. We assessed the effects of binge prenatal toluene exposure on behavior reflective of impulsivity in rat offspring using a “waiting-for-reward” operant task. Timed-pregnant Sprague–Dawley rats were exposed for 15 min, twice daily, from gestational day (GD) 8 through GD20 to either air, 8000 ppm, 12,000 ppm or 16,000 ppm toluene in a static exposure system. At postnatal day 60, male and female offspring were trained to stable lever pressing in a standard fixed-ratio 50 (FR50) paradigm. A wait requirement was then introduced such that after each FR completion, a “free” pellet was delivered at increasing time intervals (2 s, 4 s, 6 s, etc.) until the rat pressed another lever which reinstated the FR50 component (“FR Reset”). A pattern of increased FR Resets and fewer total pellets received overall and during the wait component is interpretable as “impulsivity.” The “wait” component assessment was repeated after the rats had been injected with varying doses of amphetamine. Consistent with our hypotheses, repeated binge prenatal toluene exposure appeared to increase impulsivity based upon decreases in the total number of free pellets received and mean waiting time. However, a toluene dose-dependent decrease in the number of FR Resets and in response rates under all conditions indicated there was a general impairment in performance in rats exposed prenatally to higher doses of toluene. Also, prenatal exposure to 12,000 ppm and 16,000 ppm toluene resulted in a hyposensitivity to the stimulatory effects of the amphetamine challenge in male rats. For female rats, amphetamine further interfered with performance on the task. These results suggest that acute binge prenatal toluene exposure alters performance in this task but the results are not consistent with increased impulsivity or impaired behavioral inhibition. These outcomes in young adult rats also suggest that there may be long-term behavioral deficits due to prenatal toluene exposure.

Influence of Humic Acid on Bioavailability and Toxicity of Benzo[k]fluoranthene to Japanese Medaka

Japanese medakas (Oryzias latipes) and triolein-embedded cellulose acetate membranes (TECAMs) were exposed simultaneously to benzo[k]fluoranthene (BkF) in the static exposure system containing different concentrations of humic acid (HA). The concentration-response relationships of induced hepatic 7-ethoxysorufin-o-deethylase (EROD) activity were established in regard to the nominal water concentration of BkF and the free concentration estimated using TECAM, as well as the body residues, respectively. In general, bioaccumulation of BkF and EROD activity in medaka were reduced with an increase of HA concentration in the exposure medium. The concentration-response relationships varied with HA concentration when expressed in nominal concentration. However, these relationships overlapped completely and partially when expressed in body BkF residue and in free BkF concentration estimated by TECAM, respectively. HA treatments were slightly beyond the 0.95 confidence band of HA-free control thereby indicating the participation of BkF-HA complex to the bioavailability and toxicity. On the basis of the bioavailability model, it was estimated that approximately 17-22% and 13-18% of BkF-HA complex contributed to the bioaccumulation and/or to the induced toxic effect, correspondingly.

Accurate quantification of freely dissolved organochlorine pesticides in water in the presence of dissolved organic matter using triolein-embedded cellulose acetate membrane

A novel method is described using triolein-embedded cellulose acetate membrane (TECAM) for accurate determination of the freely dissolved fraction of organochlorine pesticides (OCPs) in waters rich in dissolved organic matter (DOM). The performance of the method was tested with an air-bridge system for extracting OCPs from aqueous solutions with and without humic acid. In addition, the partition coefficients between humic acid and water (K (doc)s) for 20 OCPs were determined by TECAM with negligible depletion extraction. Results show that TECAM predominantly extracts the freely dissolved compounds and its extraction efficiency decreases significantly with an increase in concentration of humic acid in water. The proposed methodology is suitable for facile laboratory K (doc) measurement for moderate to high hydrophobic compounds (log K (ow) > 4). The linear relationship between log K (ow) and log K (doc) obtained in this study agrees well with the results reported earlier. The kinetic uptake rate constants (k (u)s) and TECAM-water partition coefficients (K (TECAM)s) for the 20 OCPs were obtained using the controlled laboratory continuous-flow and static exposure system, respectively. These calibration parameters were used in the field experiment to estimate the freely dissolved concentrations of OCPs in the water of Taihu Lake in China. Our results show that TECAM can be used successfully to determine the freely dissolved OCPs in aquatic environments containing DOM, and the method is particularly suited for long-term water sampling. Figure Schematic diagram of water sampling with a triolein-embedded cellulose acetate membrane (TECAM).

Rapid bioconcentration of steroids in the plasma of three‐spined sticklebackGasterosteus aculeatusexposed to waterborne testosterone and 17β‐oestradiol

The relationship over time between the concentrations of two steroids, singly and in combination, in a static exposure system and in the blood of three‐spined sticklebackGasterosteus aculeatus, held within the exposure system was investigated. Groups of three‐spined stickleback were exposed (nominally) to either 1000 ng l−117β‐oestradiol (E2), testosterone (T) or E2 and T in combination at the same concentrations for 6 days. Both water and fish were sampled at intervals and steroid concentrations in both compartments were determined. The plasma steroid time profile revealed a rapid bioconcentration within the first 6 h of exposure. The plasma steroid levels attained at this time point (20–90 ng ml−1) were up to 50‐fold (E2) and 200‐fold (T) greater than the actual levels of steroid measured in the exposure water, while levels in the blood of control fish did not exceed 4 ng ml−1. The substantial elevation of plasma steroid levels relative to the concentrations of steroid to which the fish were exposed in the ambient water gives scope for delivery of the steroids to target endocrine tissues at levels far in excess of what might be predicted on the basis of passive branchial uptake alone. These results are discussed in relation to endocrine disruption, and in particular the occurrence of effects in fish exposed to levels of endocrine active substances that are seemingly physiologically irrelevant.

Estrogenic, anti-estrogenic and cytotoxic effects elicited by water from the type localities of the endangered goodeid fish Girardinichthys viviparus

Girardinichthys viviparus is a Mexican endangered endemic fish species living only in Lake Texcoco (LTX), one of two extant type localities for this species. The other type locality is Lake Zumpango (LZ). LTX and LZ are fed by wastewater treated at secondary level that contains several endocrine disrupting chemicals. Our goal was to assess the estrogenic and anti-estrogenic effects elicited in G. viviparus by water from the two type localities and by these same matrices enriched with PCBs in order to understand potential damage due to increased xenobiotic levels. Estrogenic and anti-estrogenic effects were evaluated in vitro by E-screen assay in MCF-7 cells and cytotoxicity by MTT assay. PCBs were quantified in type localities. In vivo vitellogenin (VTG) induction was determined by a hybrid ELISA in adult laboratory-born fish exposed during 1, 2, 4, 8 and 16 days to LTX or LZ water in static exposure systems, and by the same matrices enriched with PCBs. We found PCBs only in LTX, but the water from both type localities elicit estrogenic and anti-estrogenic effects in vitro. Cytotoxicity was not observed in MCF-7 cells exposed to LTX or LZ water. VTG induction was higher with LTX water than with LZ water; also the response of males was greater than in females. In the PCB-enriched matrices, VTG induction in both sexes exposed to LTX water was reduced compared to un-enriched matrices. Thus, the sublethal increases in PCB levels may be hazardous to both sexes since they are linked probably to hepatotoxicity.

Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations

Protein expression and lysosomal stability were observed in Mytilus edulis exposed to 0, 20, 40, 60, and 80 ppb copper sulfate for 24 h in a static exposure system. Haemolymph was removed from each mussel and analyzed for lysosomal damage using the Neutral Red retention assay expressed as the ratio of lysosomal size to cell area, the lysosomal destabilization ratio. Gill tissue from the same individuals was dissected, homogenized and the proteins extracted and then separated using two-dimensional gel electrophoresis. Two-dimensional protein maps from each concentration were analyzed using MELANIE II 2D gel analysis software (Biorad). Lysosomal damage increased with each dosage, with LDRs ranging from 0.18±0.01 at 0 ppb copper sulfate to 0.32±0.11 at 80 ppb copper sulfate. Orthogonal comparisons showed that the control, 20 and 40 ppb classes together differed significantly ( P<0.05) from the 60 and 80 ppb classes together. Specific protein expression signatures were identified at each dosage.

Hepatic CYP1A induction in rainbow trout (Oncorhynchus mykiss) after exposure to benzo[a]pyrene in water

Juvenile rainbow trout were exposed to unlabelled benzo[a]pyrene BaP and 3H benzo a pyrene (3H BaP), in a static exposure system for 2 days. The initial concentration was 30 μg l-1 and 0.625 μCi l-1, corresponding to 6 mg kg-1 body weight and 125 μCi kg-1 body weight. Hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity was measured during the exposure and depuration periods, elucidating the time course pattern of CYP1A induction. Maximum induction (11-fold) of EROD activity was observed on day 2 after addition of BaP to the water. Tissue distribution of 3H-BaP was studied by liquid scintillation counting and whole body autoradiography. The concentration of 3H-BaP-derived radioactivity was highest in the bile at all sampling times. High levels of radiolabelled compound were also present in the gills, liver and the olfactory organ. There was an overall decrease in all tissues during the depuration period. The elimination of 3H-BaP-derived radioactivity from the gills, however, was slow compared with liver and blood (6.2 days vs 2.7 and 2.9 days, respectively).

Biotransformation of monochloroanilines in guppy,Poecilia reticulata

1. Guppies (Poecilia reticulata) were exposed for 96 h to 4-chloroaniline (4CA), 3-chloroaniline (3CA) and 2-chloroaniline (2CA) in a static exposure system. 4-Chloroacetanilide (N4CA), 3-chloroacetanilide (N3CA), and 2-chloroacetanilide (N2CA) were identified in test water as the respective biotransformation products. 2. Dependence of biotransformation on aqueous concentration was investigated with 4CA. The measured biotransformation product in water (N4CA) increased with concentration increase of parent compound, 4CA, and with exposure time. 3. Quantitative differences in net acetylation of the three monochlorinated anilines were examined. The amount of acetanilide found in water increased in the following order: N2CA < N3CA < N4CA. Mass balance calculations indicated the highest loss of aniline occurred for 2CA. This compound, which is highest in toxicity, had the smallest amount of acetylated product formed. 4. This study proves, for the first time, that the acetylation reaction in fish is reversible. Quantitative differences in the net deacetylation of monochloroacetanilides were observed. The amount of deacetylated product found in water increased in the order 4CA < 3CA < 2CA.

Thermal modulation of benzo[ a]pyrene metabolism by the gulf toadfish, Opsanus beta

Gulf toadfish, Opsanus beta, were exposed to initial [ 14 C] benzo[a] pyrene (BaP) concentrations of 5 μg 1 −1 at acclimation temperatures (18 or 28°C) or following an acute temperature change (18 to 28°C or 28 to 18°C) in a simple static exposure system. At 24 h, fish were sacrificed and the bile was analyzed by HPLC for Phase I and Phase II metabolites of BaP. Toadfish can metabolize BaP rapidly, as very low levels of parent compound were detected, and the overall rate of uptake and metabolic processing of [ 14C]BaP was higher in fish exposed at high temperatures. There were no significant differences between temperature treatments in the proportions of organic soluble and aqueous soluble metabolites, which contributed approximately 7.8 and 92%, respectively. A variety of Phase I metabolites were produced by toadfish, and the major Phase I metabolite identified was benzo[ a]pyrene 7,8-dihydrodiol. There were significant quantitative differences between temperature treatments in the classes of Phase I metabolites produced. Fish acclimated to high temperature produced more triols and tetrols (breakdown products of highly carcinogenic BaP diol epoxides) than did fish acclimated at low temperature regardless of exposure temperature. Thus, in addition to increasing rate of uptake, higher temperatures appear to lead to a shift in metabolism towards potentially more carcinogenic intermediates.