Oxygen Flux As an Indicator of Physiological Stress in Fathead Minnow

Aug 6, 2008 - Horticulture & Landscape Architecture, Purdue University, 225. South University Street, West Lafayette, Indiana 47907. Received November...
2 downloads 0 Views 4MB Size
Download PDF
Environ. Sci. Technol. 2008, 42, 7010–7017

Oxygen Flux As an Indicator of Physiological Stress in Fathead Minnow (Pimephales promelas) Embryos: A Real-Time Biomonitoring System of Water Quality B R I A N C . S A N C H E Z , †,‡ HUGO OCHOA-ACUÑA,§ D . M A R S H A L L P O R T E R F I E L D , * ,‡,| A N D ´ L V E D A * ,† M A R ´I A S . S E P U Department of Forestry and Natural Resources, 715 West State Street, Purdue University, West Lafayette, Indiana 47907, The Bindley Bioscience Center Physiological Sensing Facility, Purdue University, West Lafayette, Indiana 47907, Department of Comparative Pathobiology, Purdue University, 725 Harrison Street, West Lafayette, Indiana 47907, and Departments of Agricultural & Biological Engineering, and Horticulture & Landscape Architecture, Purdue University, 225 South University Street, West Lafayette, Indiana 47907

Received November 16, 2007. Revised manuscript received June 5, 2008. Accepted June 19, 2008.

The detection of harmful chemicals and biological agents in real time is a critical need for protecting freshwater ecosystems. We studied the real-time effects of five environmental contaminants with differing modes of action (atrazine, cadmium chloride, pentachlorophenol, malathion, and potassium cyanide) on respiratory oxygen consumption in 2-day postfertilization fathead minnow (Pimephales promelas) eggs. Our objective was to assess the sensitivity of fathead minnow eggs using the self-referencing micro-optrode technique to detect instantaneous changes in oxygen consumption after brief exposures to low concentrations of contaminants. Oxygen consumption data indicated that the technique is indeed sensitive enough to reliably detect physiological alterations induced by four of the five contaminants. After 2 h of exposure, we identified significant increases in oxygen consumption upon exposure to pentachlorophenol (100 and 1000 µg/L), cadmium chloride (0.0002 and 0.002 µg/L), and atrazine (150 µg/L). In contrast, we observed a significant decrease in oxygen flux after exposures to potassium cyanide (44 and 66 µg/L) and atrazine (1500 µg/L). No effects were detected after exposures to malathion (200 and 340 µg/L). Our work is the first step in development of a new technique for physiologically coupled biomonitoring as a sensitive and reliable tool for the detection of environmental toxicants.

* Address correspondence to either author. E-mail: porterf@ purdue.edu (D.M.P.), [email protected] (M.S.S.). Phone: 765494-1190(D.M.P.), 765-496-3428(M.S.S.). † Department of Forestry and Natural Resources. ‡ The Bindley Bioscience Center Physiological Sensing Facility. § Department of Comparative Pathobiology. | Departments of Agricultural & Biological Engineering, and Horticulture & Landscape Architecture. 7010

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 18, 2008

Introduction The use of biological early warning systems to continuously monitor water quality has been investigated since the early 1970s (1). Systems have exploited stress responses to contaminants in freshwater and saltwater bacteria (2, 3), cladocerans (4), amphipods (5), freshwater and marine bivalves (6, 7), and several species of fish (8–10). The quantification of stress in fish models is often measured in terms of changes in activity (9), opercular movement (1, 11), and electric organ discharge (8). One of the more sophisticated systems was developed by Shedd et al. (12) whereby they recorded irregularity in body movement, cough rate, and ventilatory rate and depth of bluegill (Lepomis macrochirus) exposed to test water (10, 12). Similar to other biomonitors, pairs of bulk electrodes in each test chamber were used to measure changes in impedance patterns, which were recorded and associated with aberrant behavior due to degraded water quality. This system has been used to monitor water quality in the field for a long period (2 y) (13). Respiratory activity of a fish is often the first physiological response to be affected by the presence of contaminants in the aquatic environment (14). Although many biological early warning systems monitor abnormal opercular movement (i.e., ventilation depth, rate, and coughing) as an indicator of respiratory stress, a more direct measurement of stress in this sense necessitates the quantification of oxygen consumed by the fish. Although oxygen consumption is not often used as a bioindicator of pollution-associated stress in biological early warning systems, it has recently been applied to a system utilizing the cladoceran, Daphnia magna, with relatively good success (4). The paucity of biomonitoring systems based on oxygen consumption to date may have been due to the unavailability of technology that would allow measuring these fluxes in real time. Fiber optic biosensing technology has yielded the ability to measure minute fluxes of oxygen in real time at the cellular level (15). By sequentially measuring the concentration of oxygen at different distances from the cell or organism, this technology allows measurement of oxygen flux (i.e., consumption) in real time. Although measurement of oxygen flux in extracellular microgradients using polarographic microelectrodes is not new (16–18), the use of optically transduced sensors (optrodes) for this purpose is rather novel. Optrodes are superior because their measurements do not consume oxygen, are not disturbed by external electromagnetic interference, and do not require the use of a reference electrode (15, 19). Because of these advantages, optrodes provide an optimal platform for developing sensitive and reliable biological early warning systems based on measuring oxygen consumption. The objective of this study was to evaluate the performance of a biosensing approach based on measuring the respiratory responses of fathead minnow (Pimephales promelas) embryos as quantified by their real-time oxygen consumption (pmol/ cm2/s). Specifically, we wanted to determine if our proposed technique was sensitive enough to detect instantaneous changes in oxygen consumption after exposure to low concentrations of contaminants.

Materials and Methods Fathead Minnows. We chose to use fathead minnows because they are a commonly used model organism in ecotoxicological studies and are relatively easy to breed in captivity. Fathead minnow embryos are ideal for this application, because although they are complex organisms 10.1021/es702879t CCC: $40.75

 2008 American Chemical Society

Published on Web 08/06/2008

FIGURE 1. Diagram of the self-referencing oxygen optrode system. From the test medium, an optrode is coupled to a digital signal processor (DSP)-based frequency domain lifetime fluorometer (Presens, Regansburg, Germany). The fluorometer excites the fluorophore and measures its luminescence decay time in the frequency domain as a phase angle (Φ) shift. The output from the fluorometer is transferred to a DC-coupled lock-in differential amplifier (Applicable Electronics, Inc. Forestdale, MA) that subtracts the baseline signal (offset potential) from the optrode baseline signal (offset subtraction) prior to amplification. The mV output values are digitally logged within the Automated Scanning Electrode Technique software (ASET, Science Wares, East Falmouth, MA) installed on a personal computer. Analog-to-digital (A/D) and digital-to-analog (D/A) converters are incorporated into the system to allow effective communication between the personal computer and the differential amplifier. The zoom microscope and optrode are operated with 2- and 3-dimensional (2D and 3D) stepper motors controlled through the software via motor controllers and interfaced to the personal computer through two parallel ports. A frame grabber linked to a video camera mounted on the zoom microscope enables the operator to visualize the position of the optrode within the ASET on-screen display. during testing (2 d postfertilization), they are immobile, thereby allowing accurate flux measurements to be taken. There is also considerable evidence showing that early life stages of fish are the most sensitive to many, perhaps most, toxic agents (20–23). As such, the system should prove responsive to low levels of aquatic contamination. Pairs of breeding fathead minnows were held in static 9.4 L aquaria that underwent 50% water changes three times per week. Water was maintained at 25 °C and a 16:8 h (light/ dark) photoperiod was imposed. Breeding pairs were fed frozen brine shrimp (Artemia salina) twice daily until satiation. Each aquarium was supplied with a semicircle of polyvinylchloride (PVC) pipe matrix (7.6 cm diameter, 12 cm length) on which the fish were able to spawn and fertilize eggs. Aquaria were checked for eggs daily and when present, the matrix was removed and placed in a separate 9.4 L aquarium until used for experimental trials. Oxygen Optrode. The setup of our system is illustrated in Figure 1. The basic components of the self-referencing optrode setup include a microscope with a head-stage sensor amplifier driven by a translational motion control system. The microscope allows visualization of the position of the optrode relative to the cell or organism being studied (Figure 2). Although initial positioning of the probe is done by visual observation, final positioning was done by controlling the probe position with a computer-driven motion control system. The microscope, camera, and motion control system were mounted on an antivibration table within a Faraday cage. The motion control system allowed the optrode tip to be moved through the gradient at a known frequency and between known points (commonly 10-50 µm apart). The tip of the probe is located on the end of a fiber optic cable that is approximately 5 µm in diameter. It is coated with an immobilized, quenchable fluorophore, platinum tetrakis (pentafluorophenyl) porphyrin (PtTFPP). The probe

is housed within a tapered pipet held by the motion control system. The probe is coupled to a digital signal processor (DSP)-based lifetime fluorometer (Presense, Regansburg, Germany), which excites the fluorophore, measures its luminescence decay time and phase angle (Φ), and transfers the analog signal to a DC-coupled, lock-in amplifier (10-100×). The amplifier discretely subtracts the baseline signal (offset potential) from the optrode baseline signal (offset subtraction) prior to amplification. The mV output values are digitally logged within the automated scanning electrode technique software (Science Wares, East Falmouth, MA) installed on a personal computer. The zoom microscope and optrode are operated with 2- and 3-dimensional (2D and 3D) stepper motors controlled through the software. Calculation of Oxygen Flux. Oxygen flux was calculated using the Fick Equation: J ) -D(∆C ⁄ ∆r)

(1)

2/s),

D is the diffusion coefficient of where J is flux (mol/cm oxygen in water (0.0000242 cm2/s at 25 °C; ref 24), ∆C is the difference in oxygen concentration at two points in a concentration gradient adjacent to the embryo chorion (mol/ mL), and ∆r is the distance between the two points within the gradient (cm). The two measurement points were (1) directly adjacent to the chorion, and (2) 20 µm perpendicular to the chorion (i.e., ∆r ) 0.02 mm). Prior to each trial, the optrode underwent a two-point calibration at 100% oxygen saturation in deionized water and 0% oxygen concentration in nitrogen-bubbled deionized water. Experimental Trials. Fathead minnow eggs (2 d postfertilization) were removed from the matrix and adhered to the bottom of a BD Falcon tissue culture dish (35 × 10 mm, Franklin Lakes, NJ). Each dish had been previously treated with 350 µL of 0.1 mg/mL poly L-lysine solution, a commonly used adhesive in cell and tissue culturing, and allowed to dry VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

7011

FIGURE 2. Image of fathead minnow embryo and oxygen optrode during a monitoring event. for a minimum of 6 h (25). Prior to use, the dishes were rinsed 5 times with a phosphate-buffered saline solution (150 mM NaCl, 2.2 mM NaH2PO4, 8.1 mM Na2HPO4) followed by a single rinse with deionized water. The dish was filled with 4.9 mL of deionized water (pH ) 6.5, free of CaCO3). Eggs were numbered 1 through 5 and pre-exposure oxygen consumption measurements were taken from eggs 1 through 4 in sequence for 10 min each to yield their pre-exposure oxygen consumption measurements. Egg 5 was typically monitored continuously for 1 h prior to exposure and the final 10 min of data were used for egg 5 pre-exposure statistical analysis. Therefore, oxygen consumption of each egg was always measured when exposed to uncontaminated water (i.e., served as its own control) before being exposed to a given chemical. After 1 h, 100 µL of a spike solution was added to the 4.9 mL of deionized water to yield the target chemical concentration in 5 mL of solution. Data on egg 5 were typically recorded for another 2 h, and the final 10 min of data were used for egg 5 postexposure statistical analysis. Eggs 1 though 4 were subsequently monitored in reverse order for 10 min each to yield their postexposure flux measurements. Experimental Chemicals and Concentrations. We chose four common environmental contaminants (atrazine; cadmiumchloride,CdCl2;pentachlorophenol,PCP;andmalathion) that vary in mode of toxic action, in order to assess the utility of our technique (see Table 1). We also utilized potassium cyanide (KCN), a known inhibitor of oxygen consumption, as a negative control (26). Carbonyl cyanide m-chlorophenylhydazone (CCCP, 10 mM, 1023 mg/L) and rotenone (5 µM, 3944 mg/L) were also used in successive exposure events to demonstrate the system’s ability to detect instantaneous 7012

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 18, 2008

oxygen consumption changes upon embryo exposure to chemicals with mitochondrial effects known to both increase and decrease oxygen consumption, respectively (26). Our test concentrations were based on those used by other realtime water quality monitoring systems reported in the literature or on established water quality guidelines for atrazine, CdCl2, PCP, malathion, and KCN (Table 1). Additional concentrations of KCN (44 µg/L and 66 µg/L) were included post hoc (see Results). Statistical Analysis. We used SAS statistical analysis software (SAS Institute Inc., Cary, NC) for all analyses. We calculated mean oxygen consumption and standard errors for each embryo for the pre- and postexposure measurements. We conducted a repeated measures analysis of variance (ANOVA) to test for significant differences (R ) 0.05) among oxygen consumption rates measured during 10 min of pre-exposure and 10 min of postexposure for each embryo on a chemical by dose basis. This was done because each embryo served as its own control. Oxygen consumption values were recorded every 10 s; therefore approximately 60 points per condition were compared.

Results As can be seen in Figure 3, our system was able to detect CCCP and rotenone in water in near-real time. Introduction of 5 µM CCCP in the test media resulted in an almost immediate increasing trend in oxygen consumption until adding 10 mM of rotenone resulted in a decrease in oxygen consumption. A similar time trend was observed with the 66 µg/L KCN test solution (Figure 4A). In this situation, a decrease in oxygen consumption began soon after exposure.

FIGURE 3. Oxygen consumption time course for a single fathead m-chlorophenylhydrazone (CCCP; 5 µM) and rotenone (10 mM) in succession.

minnow

embryo

exposed

to

carbonyl

cyanide

TABLE 1. Mode of Toxic Action, Concentration, and Basis for Choosing the Concentration of Each Test Chemical Used in the Present Study chemical

mode of toxic action

concentrations (µg/L)

basis

atrazine

oxidative stress

150, 1500*

*maximum 1 h concentration not to be exceeded more than once every 3 years for the protection of aquatic life (73)

cadmium chloride

calcium metabolism interference

0.0002, 0.002*

*criterion maximum concentration for the protection of aquatic life (74)

pentachlorophenol

uncoupler of oxidative phosphorylation

100, 1000*

*1-day human health advisory level (75)

malathion

acetylcholinesterase inhibitor

200*, 340**

*1-day human health advisory level (75), **bluegill LC50 concentration used in van der Shalie et al. (10)

potassium cyanide

electron transport disruption

5.2*, 22**

*criteria continuous concentration and **criteria maximum concentration for the protection of aquatic life (76)

FIGURE 4. Pre- and postexposure oxygen consumption for five fathead minnow embryos exposed to 66 µg/L potassium cyanide (A). Bars represent average 10 min pre- (white bars) and 10 min postexposure (cross-hatched) values (pmol/cm2/s ( 1 standard error) for each embryo. Postexposure measurements were taken 30 min after commencing exposure. Oxygen consumption time course for a single fathead minnow embryo (B). Potassium cyanide (66 µg/L) was added (arrow) 60 min after the trial began and measurements were taken for an additional 30 min. All eggs demonstrated significantly different oxygen consumption after 30 min (Figure 4A). Results from the tests comparing oxygen consumption before and after exposures are presented in Figure 5. No

embryo mortality was observed within the timeframes evaluated. Except for malathion and the two lowest doses of KCN, the system was capable of detecting the test chemicals within 2 h of exposure. Significant increases in oxygen VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

7013

FIGURE 5. Average percent change between 10 min pre- and 10 min postexposure oxygen flux values (pmol/cm2/s) for five embryos exposed to atrazine, cadmium chloride (CdCl2), pentachlorophenol (PCP), malathion, and potassium cyanide (KCN) exposed to chemical concentrations (µg/L) indicated above each bar. Postexposure measurements were taken approximately 2 h after spiking for all chemicals and concentrations with the exception of 66 µg/L KCN (30 min). Asterisks indicate a significant overall difference (repeated measures ANOVA, r ) 0.05) between pre- and postexposure values. consumption after 2 h of exposure were detected for the low dose of atrazine (150 µg/L), CdCl2 (0.0002 and 0.002 µg/L), and PCP (100 and 1000 µg/L). We detected a significant decrease in oxygen consumption after 2 h of exposure to the high dose atrazine (1500 µg/L). There was no significant change in oxygen consumption after 2 h of exposure to malathion (200 and 340 µg/L) or the lowest doses of KCN (5.2 and 22 µg/L). We also meaured oxygen consumption after exposure to 44 and 66 µg/L KCN. Postexposure measurements were recorded after 2 h for the 44 µg/L exposure and 30 min for the 66 µg/L exposure. The exposure time for the 66 µg/L trial was reduced because the real-time data suggested that a significant decrease had occurred prior to the typical 2 h exposure period. Decreases in oxygen flux for the 44 and 66 ug/L doses of KCN were significant. We detected time by treatment effects for atrazine and KCN indicating that the oxygen consumption response was different between the doses of these chemicals.

Discussion We demonstrated that our proposed system may be significantly more sensitive than other systems now in use. Time to alarm reported for the system developed by van der Schiale et al. (10) for some chemicals is rather prolonged. For example, their system required 12.25 h to detect 250 µg/L PCP (10). In constrast, we were able to detect a lower concentration (100 µg/L) of PCP in 2 h. We were also able to detect a comparable concentration of cyanide (66 vs 60 µg/L) as quickly as 15 min after exposure compared to their first alarm time of 30 min. Furthermore, in comparison to several other biomonitor and general stress response studies, our system has proven more responsive to physiological alterations due to the presence of contaminants. Atrazine is a widely used herbicide that blocks photosynthetic electron transport, thereby inhibiting photosynthesis (28). It has been shown to elicit mitochondrial dysfunction and malformation in fish in previous studies (29, 30). It is also known to increase the formation of reactive oxygen species (ROS) in rat heart mitochondria (31) and the production of the superoxide dismutase enzyme in bluegill (32). While the decrease in oxygen flux at the high dose 7014

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 18, 2008

concentration (1500 µg/L) may be a direct indication of mitochondrial damage to the extent that oxidative phosphorylation is in decline, the disparity in the increase in oxygen flux at the low dose concentration (150 µg/L) may indicate a general stress response with a subsequent increase in energetic allocation to combating the presence of the chemical. An increase in metabolic rate and oxygen consumption in response to contaminants has been documented for embryos, larvae, and adults of other vertebrate species (33, 34). Although Hussein et al. (35) noted a nonquantitative increase in opercular activity and respiration of adult Nile tilapia (Oreochromis niloticus) and catfish (Chrysichthyes auratus) in response to atrazine exposure (3 and 6 mg/L), another study reported no significant increase in respiration rates of red drum (Sciaenops ocellatus) larvae after 96 h exposures to up to 80 µg/L (36). The inverse response in oxygen flux between the low and high atrazine concentrations in the present study may also indicate the presence of a hermetic response. This phenomenon is not uncommon in ecotoxicological research (37), but would necessitate further experimentation to establish a causative factor. Despite this disparity, our data clearly indicate that our technique for detecting oxygen consumption changes of fathead minnow embryos in response to low concentrations of atrazine is effective as a monitoring system of atrazine concentrations in drinking water. Atrazine’s 1-to10 d Health Advisory Level (HAL), the concentrations in drinking water that should not be exceeded on average for a 1-10 day period, is 100 ug/L. Our system was able to detect a similar atrazine concentration after a 2-h exposure. Other studies have demonstrated lower sensitivity to this chemical. For instance, Liu et al. (30) were not able to detect significant differences in several metabolic parameters in grass carp (Ctenopharygodon idella; i.e., intracellular O2- and H2O2, cellular ATP depletion, and mitochondrial membrane potential disruption) until 24-36 h postexposure to 37.8 mg/L atrazine. Our test concentrations were approximately 25 and 250 times lower. Cadmium is a persistent environmental contaminant that accumulates in the gills, kidneys, and liver of fish (38). It is known to impair respiration and mitochondrial activity of

fish and mammals. For instance, Yilmaz et al. (39) observed that guppies (Poecilia reticulata) exposed to 28.5 mg/L Cd experienced difficulty breathing and attempted to breathe atmospheric air. Increased gill tissue respiration of winter flounder (Pseudopleuronectes americanus) was documented upon exposure to 5 µg/L Cd for 60 d (40) and oxygen consumption rates of grass carp were elevated after 96 h exposure to 500 µg/L Cd (41). Conversely, oxygen consumption rates of striped bass (Morone saxatilis) decreased after 30 d exposure to 0.5, 2.5, and 5 µg/L Cd, but were not different from controls at 90 d or after a 30 d recovery period (42). The gill respiration capacity of wild yellow perch (Perca flavescens) was shown to decrease in relation to an increase in liver Cd concentration (43). Cadmium has also been shown to induce the expression of genes associated with ROS production and mitochondrial metabolism in zebrafish (Danio rerio; 44) and goldfish (45). Achard-Joris et al. (46) observed that the gene encoding for cytochrome oxidase (complex IV of the electron transport chain) in marine bivalves was up-regulated in response to 14.61 µg/L Cd after 10-14 d of exposure. This was interpreted as a compensatory response to restore a decrease in mitochondrial activity (46). Cadmium has also been reported to stimulate ROS production and inhibit the electron transport chain (47) which Oh and Lim (48) tied to a collapse in the mitochondrial membrane potential and the release of cytochrome C into the cytosol. Additionally, these authors noted rapid and transient ROS production by human HepG2 cells within 30 min of exposure to 1.79 mg/L Cd. Our results show that oxygen consumption increased after a 2 h exposure to considerably lower concentrations of CdCl2 compared to previous studies (0.0002 µg/L). The detection capability of our system for cadmium suggest that it could easily detect exceedances of even the rather low ambient water quality criteria developed by the U.S. EPA for this element. Assuming a water hardness of 50 mg/L CaCO3, this criterion equals 0.012 µg/L, which is 60 times higher than the lowest concentration detected by our system. Pentachlorophenol is a biocide commonly used as an antifungal agent for wood preservation (49). It is a strong uncoupler of oxidative phosphorylation (50), increasing the rate of mitochondrial oxygen consumption and inhibiting the production of ATP (27). Juvenile largemouth bass (Micropterus salmoides) had reduced growth rates and food conversion efficiencies at or above 25 µg/L (51), and decreased feeding rates and hyperactivity at 67 µg/L (52). Female rainbow trout (Oncorhynchus mykiss) produced fewer viable oocytes upon exposure to 22 µg/L PCP for 18 d (53). Pentachlorophenol has also been shown to increase oxygen consumption of mature sockeye salmon (O. mykiss) exposured to 20 µg/L for 12-14 h (54) and of river puffer fish (Takifugu obscurus) (50 µg/L for 120 h; 55). Our results are consistent with these findings since a significant oxygen flux difference was detected after 2 h of exposure to 100 µg/L PCP. The U.S. EPA has set a Maximum Contaminant Level (MCL) for PCP in drinking water of 1 µg/L, whereas the 1-d HAL has been set at 1,000 µg/L. More importantly, our detection time was 10.25 h faster than that reported for the bluegill model at 250 µg/L (10). Our results indicate that no difference in oxygen flux was experienced by fathead minnow embryos within 2 h of exposure to either malathion concentration. Supplemental experimentation did not detect a difference after 8 h (p ) 0.96, data not presented). Malathion is an organophosphate insecticide that inhibits cholinesterase activity by binding to its esteratic site. This hinders the enzyme’s ability to scavenge acetylcholine from synaptic clefts and thereby perpetuates uncontrolled nervous signaling (56). In fish, exposure to organophosphate insecticides has been shown to inhibit cholinesterase activity (57) and interfere with behavior (58) and reproduction (59). The effect of malathion exposure on

the respiratory activity of fish has been addressed in few studies and the results are inconclusive. Two studies found that Mozambique tilapia (Tilapia mossambica) exposed to 122 and 200 µg/L for 48 h experienced an increase in oxygen consumption through hour 12-24, thereafter undergoing a decrease in oxygen consumption through hour 48 (60, 61). Another study found no measurable effect on the respiration of red drum larvae after 96 h exposure to 1 and 10 µg/L malathion (62). McKim et al. (63) noted decreases in heart rate and oxygen utilization with a concomitant increase in ventilation volume, but no detectable changes in oxygen consumption of rainbow trout exposed to 296 µg/L malathion for 24-48 h. The respiration by isolated rat liver mitochondria exposed to malathion (64 µg/L - 27 mg/L) for 20 min was likewise not affected (64). These authors concede, however, that the enzymes required to convert malathion into its activated oxygen analog (i.e., malaoxon, 65) were not present in the assay. Nguyen et al. (66) detected no growth inhibition of larva African catfish (Clarias gariepinus) upon exposure to 5 mg/L malathion when eggs were in the late blastula stage (3 h postfertilization) but significant growth inhibition was observed if larvae were exposed at the 2-4 cell stage (0-1 h postfertilization) and larval stage (g10 h posthatching). The authors suggested that the chorion hindered the influx of malathion after it had undergone water-hardening (assumed to be complete 3 h postfertilization). Our observed lack of a significant response upon malathion exposure may be analogous to this suggested effect of chorion hardening. The bluegill model detected a significant decrease in ventilatory depth after 88.5 h of exposure to 340 µg/L malathion (10). Cyanide’s mode of toxic action toward mitochondria is well documented (26). It acts on cytochrome oxidase (complex IV), competing with oxygen for the heme a3 site. This effectively inhibits the final transfer of electrons to oxygen, destroys the mitochondrial membrane potential, and stalls the production of ATP (50). Brown bullheads (Ictalurus nebulosus) exposed to increasing concentrations of cyanide (200-1800 µg/L) over a 9 h period experienced an increase in oxygen consumption and heart rate through 3 h of exposure, followed by a decrease in both parameters until death at 9 h (67). Brook trout (Salvelinus fontinalis) exposed to 5 µg/L cyanide over 29 d had their swimming performance reduced by 50% (68), while those exposed to 25 µg/L for 5 h underwent an inhibition in oxygen intake (69). Oxygen consumption by liver tissues of white-tailed damselfish (Dascyllus aruanus) was lower for those exposed to 25 and 50 mg/L cyanide for pulses of 10 and 60 s after 2.5 weeks of unstressed recovery. Conversely, liver tissue of fish exposed to cyanide in the same manner, but stressed during recovery, had elevated oxygen consumption rates 2.5 weeks after the exposure (70). Oxygen consumption by unfertilized chinook salmon (O. tshawytscha) ceased upon exposure to 132 mg/L KCN (71). The calculated LC50 (96 h) of cyanide for fathead minnow eggs at 25 °C is 121-202 µg/L (72). Our results indicate that significant decreases in oxygen flux by fathead minnows are detectable within 2 h at concentrations at or above 44 µg/L and that a dose-response relationship exists. This suggests that the classic mode of toxic action by cyanide on cytochrome oxidase may be occurring in fathead minnow embryos. The bluegill model (10) detected a change in movement within 30 min of exposure to 60 µg/L sodium cyanide. We determined an effect for a comparable dose in our study (66 µg/L) at 30 min as well, and a re-examination of the data reveals that a significant decrease in oxygen flux for egg 5 was detected as early as 15 min after exposure (p < 0.01). We demonstrated that our proposed system is effective at detecting the presence of environmentally relevant concentrations of four of five chemicals tested. We believe this is because oxygen consumption measurements provide a robust indicator of whole animal stress and concomitant VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

7015

water quality. Our findings encourage further development of this technology and its ultimate use in water quality monitoring programs and real-time early warning systems.

Acknowledgments This work was funded by Purdue University Center for the Environment and a Graduate Assistance in Areas of National Need (GAANN) fellowship awarded to the primary author by the U.S. Department of Education. We thank Nathan Barton, Monica Hensley, Sonia Johns, Geoff Laban, Brett Lowry, Reid Morehouse, and Bob Rode for helping with the maintenance of reproductive fathead minnows. We also thank Rameez Chatni and Aeraj ul Haque for their guidance in operating the micro optrode and associated equipment.

Literature Cited (1) Cairns, J., Jr.; Dickson, K. L.; Sparks, R. E.; Waller, W. T. A preliminary report on rapid biological information for water pollution control. J. Water Pollut. Control Fed. 1970, 42, 685– 703. (2) Munkittrick, K. R.; Power, E. A.; Sergy, G. A. The relative sensitivity of Microtox, Daphnid, rainbow trout, fathead minnow acute lethality tests. Environ. Toxicol. Water Qual. Int. J. 1991, 6, 35– 62. (3) Cho, J.; Park, K.; Ihm, H.; Park, J.; Kim, S.; Kang, I.; Lee, K.; Jahng, D.; Lee, D.; Kim, S. A novel continuous toxicity test system using a luminously modified freshwater bacterium. Biosens. Bioelectron. 2004, 20, 338–344. (4) Martins, J. C.; Saker, M. L.; Teles, L. F. O.; Vasconcelos, V. M. Oxygen consumption by Daphnia magna Straus as a marker of chemical stress in the aquatic environment. Environ. Toxicol. Chem. 2007, 26, 1987–1991. (5) Maltby, L.; Clayton, S. A.; Wood, R. M.; McLoughlin, N. Evaluation of the Gammarus pulex in situ feeding assay as a biomonitor of water quality: robustness, responsiveness, and relevance. Environ. Toxicol. Chem. 2002, 21, 361–368. (6) Sluyts, H.; Van Hoof, F.; Cornet, A.; Paulussen, J. A dynamic new alarm system for use in biological early warning systems. Environ. Toxicol. Chem. 1996, 15, 1371–1323. (7) Kramer, K. J.; Jenner, H. A.; de Zwart, D. The valve movement response of mussels: a tool in biological monitoring. Hydrobiologia 1989, 188/189, 433–443. (8) Thomas, M.; Florion, A.; Chre´tien, D.; Terver, D. Real-time biomonitoring of water contamination by cyanide based on analysis of the continuous electric signal emitted by a tropical fish Apteronotus albifrons. Water Res. 1996, 30, 3083–3091. (9) Gerhardt, A. Whole effluent toxicity with Oncorhynchus mykiss (Walbaum 1792): survival and behavioral responses to a dilution series of a mining effluent in South Africa. Arch. Environ. Contam. Toxicol. 1998, 35, 309–316. (10) van der Shalie, W. H.; Shedd, T. R.; Widder, M. W.; Brenan, L. M. Response characteristics of an aquatic biomonitor used for rapid toxicity detection. J. Appl Toxicol. 2004, 24, 387–394. (11) Cairns, J., Jr.; van der Shalie, W. H. Biological monitoring part I: early warning systems. Water Res. 1980, 14, 1179–1196. (12) Shedd, T. R.; Widder, M. W.; Leach, J. D.; van der Schalie, W. H.; Bishoff, R. C. Apparatus and method for automated biomonitoring of water quality. US Patent 6,393,899 B1, 28, 2000.. (13) Shedd, T. R.; van der Shalie, W. H.; Widder, M. W.; Burton, D. T.; Burrows, E. P. Long-term operation of an automated fish biomonitoring system for continuous effluent acute toxicity surveillance. Bull. Environ. Contam. Toxicol. 2001, 66, 392–399. (14) Heath, A. G. Water Pollution and Fish Physiology, 2nd ed.; Lewis Publishers: New York, 1995. (15) Porterfield, D. M.; Rickus, J. L.; Kopelman, R. Non-invasive approaches to measuring respiratory patterns using a PtTFPP based, phase-lifetime, self-referencing oxygen optrode. Proc. SPIE 2006, 6380, 63800S1–63800S8. (16) Land, S. C.; Porterfield, D. M.; Sanger, R. H.; Smith, P. J. S. The self-referencing oxygen-selective microelectrode: detection of trans-membrane oxygen flux from single cells. J. Exp. Biol. 1999, 202, 211–218. (17) Porterfiled, D. M.; Smith, P. J. S. Characterization of trans-cellular oxygen flux and proton fluxes from Spirogyra grevelleana using self-referencing microelectrodes. Protoplasma 2000, 212, 80–88. (18) Porterfield, D. M. Measuring metabolism and biophysical flux in the tissue, cellular and sub-cellular domains: Recent developments in self-referencing amperometry for physiological sensing. Biosens. Bioelectron. 2007, 22, 1186–1196. 7016

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 18, 2008

(19) Klimant, I.; Meyer, V.; Ku ¨hl, M. Fiber-optic oxygen microsensors, a new tool in aquatic biology. Limnol. Oceanogr. 1995, 40, 1159– 1165. (20) McKim, J. M. Evaluation of tests with early life stages of fish for predicting long-term toxicity. J. Fish Res. Board Can. 1977, 34, 1148–1154. (21) Luckenbach, T.; Kilian, M.; Triebskorn, R.; Oberemm, A. Fish early life stage tests as a tool to assess embryotoxic potentials in small streams. J. Aquat. Ecosyst. Stress Recov. 2001, 8, 355– 370. (22) Eaton, J. G.; McKim, J. M.; Holcombe, G. W. Metal toxicity to embryos and larvae of seven freshwater fish species. I. Cadmium. Bull. Environ. Contam. Toxicol. 1978, 19, 95–103. (23) Kristensen, P. Sensitivity of embryos and larvae in relation to other stages in the life cycle of fish: a literature review. In Sublethal and Chronic Effects of Pollutants on Freshwater Fish; Mu ¨ ller, R., Lloyd, R., Eds.; FAO: Oxford, UK, 1995; pp155-166.. (24) Fluid properties, Section 6. In CRC Handbook of Chemistry and Physics, 87th ed.; Lide, D. R., Ed.; CRC Press: Boca Raton, FL, 2006. (25) Banker, G.; Goslin, K. Culturing Nerve Cells, 2nd ed.; MIT Press: Cambridge, MA, 1998. (26) Albaum, H. G.; Tepperman, J.; Bodansky, O. The in vivo inactivation by cyanide of brain cytochrome oxidase and its effect on glycolysis and on the high energy phosphorous compounds in brain. J. Biol. Chem. 1946, 164, 45–51. (27) Tzagoloff, A. Mitochondria. In Cellular Oganelles; Siekevitz, P., Ed.; Plenum Press: New York, 1982. (28) Duke, S. O. Overview of herbicide mechanisms of action. Environ. Health Perspect. 1990, 87, 263–271. (29) Biagianti-Risbourg, S.; Bastide, J. Hepatic perturbations induced by a herbicide (atrazine) in juvenile grey mullet Liza ramada (Mugilidae, Teleostei): An ultrastructural study. Aquat. Toxicol. 1995, 31, 217–229. (30) Liu, X.; Shao, J.; Xiang, L.; Chen, X. Cytotoxic effects and apoptosis induction of atrazine in a grass carp (Ctenopharyngodon idellus) cell line. Environ. Toxicol. 2006, 21, 80–89. (31) Stolze, K.; Nohl, H. Effect of xenobiotics on the respiratory activity of rat heart mitochondria and the concomitant formation of superoxide radicals. Environ. Toxicol. Chem. 1994, 13, 499–502. (32) Elia, A. C.; Waller, W. T.; Norton, S. J. Biochemical responses of bluegill sunfish (Lepomis macrochirus, Rafinesque) to atrazine induced oxidative stress. Bull. Environ. Contam. Toxicol. 2002, 68, 809–816. (33) Rowe, C. L.; McKinney, O. M.; Nagle, R. D.; Congdon, J. D. Elevated maintenance costs in an anuran (Rana catesbeiana) exposed to a mixture of trace elements during the embryonic and early larval periods. Physiol. Zool. 1998, 71, 27–35. (34) Hopkins, W. A.; Rowe, C. L.; Congdon, J. D. Elevated trace element concentrations and standard metabolic rate in banded water snakes (Nerodia fasciata) exposed to coal combustion wastes. Environ. Toxicol. Chem. 1999, 18, 1258–1263. (35) Hussein, S. Y.; El-Nasser, M. A.; Ahmed, S. M. Comparative studies on the effects of herbicide atrazine on freshwater fish Oreochromis niloticus and Chrysichthyes auratus at Assiut, Egypt. Environ. Contam. Toxicol. 1996, 57, 503–510. (36) Alvarez, M. C.; Fuiman, L. A. Environmental levels of atrazine and its degradation products impair survival skills and growth of red drum larvae. Aquat. Toxicol. 2005, 74, 229–241. (37) Davis, J. M.; Svendsgaard, D. J. U-shaped curves: their occurrence and implications for risk assessment. J. Toxicol. Environ. Health 1990, 30, 71–83. (38) Kay, J.; Thomas, D. G.; Brown, M. W.; Cryer, A.; Shurben, D.; Solbe, J. F. G.; Garvey, J. S. Cadmium accumulation and protein binding patterns in tissues of the rainbow trout Salmo gairdneri. Environ. Health Perspect. 1986, 65, 133–139. (39) Yilmaz, M.; Gu ¨ l, A.; Karako¨se, E. Investigation of acute toxicity and the effect of cadmium chloride (CdCl2 · H2O) metal salt on behavior of the guppy (Poecilia reticulata). Chemosphere 2004, 56, 375–380. (40) Calabrese, A.; Thurberg, F. P.; Dawson, M. A. ; Wensloff, D. R. Sublethal physiological stress induced by cadmium and mercury in the winter flounder, Pseudopleuronectes americanus. In Sublethal Effects of Toxic Chemicals on Aquatic Animals; Koeman, J. H., Strik, J. J. T. W. A., Eds.; Elsevier Publishing Company: Amsterdam, 1975; Vol. 1, pp 5-21.. (41) Espina, S.; Saliban, A.; Dı´az, F. Influence of cadmium on the respiratory function of the grass carp (Ctenopharygodon idella). Water Soil Air Pollut. 2000, 119, 1–10. (42) Dawson, M. A.; Gould, E.; Thurber, F. P.; Calabrese, A. Physiological response of juvenile striped bass Morone saxatilis,

(43) (44)

(45)

(46)

(47) (48)

(49) (50) (51)

(52)

(53)

(54)

(55)

(56) (57)

(58)

to low levels of cadmium and mercury. Chesapeake Sci. 1977, 18, 353–359. Couture, P.; Kumar, P. R. Impairment of metabolic capacities in copper and cadmium contaminated wild yellow perch (Perca flavescens). Aquat. Toxicol. 2003, 64, 107–120. Gonzales, P.; Baudrimont, M.; Boudou, A.; Bourdineaud, J. P. Comparative effects of direct cadmium contamination on gene expression in gills, liver, skeletal muscles and brain of the zebrafish (Danio rerio). BioMetals 2006, 19, 225–235. Gargiulo, G.; Arcamone, N.; de Girolamo, P.; Andreozzi, G.; Antonucci, R.; Esposito, V.; Ferrara, L.; Battaglini, P. Histochemical study of the effects of cadmium uptake on oxidative enzymes of intermediary metabolism in kidney of goldfish (Carassius aruatus). Comp. Biochem. Physiol. 1996, 113C, 177– 183. Achard-Joris, M.; Gonzales, P.; Marie, V.; Baudrimont, M.; Bourdineaud, J. P. Cytochrome c oxydase subunit I gene is upregulated by cadmium in freshwater and marine bivalves. BioMetals 2006, 19, 237–244. Wang, Y.; Fang, J.; Leonard, S. S.; Rao, K. M. K. Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radical Biol. Med. 2004, 36, 1434–1443. Oh, S. H.; Lim, S. C. A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteinemediated catalase upregulation. Toxicol. Appl. Pharmacol. 2006, 212, 212–223. Ruckdeschel, G.; Renner, G. Effects of pentachlorophenol and some of its known and possible metabolites on fungi. Appl. Environ. Microbiol. 1986, 51, 1370–1372. Weinbach, E. C. The effect of pentachlorophenol on oxidative phosphorylation. J. Biol. Chem. 1954, 210, 545–550. Johansen, P. H.; Mathers, R. A.; Brown, J. A. Effect of exposure to several pentachlorophenol concentrations on growth of young-of-year largemouth bass, Micropterus salmoides, with comparisons to other indicators of toxicity. Bull. Environ. Contam. Toxicol. 1987, 39, 379–384. Brown, J. A.; Johansen, P. H.; Colgan, P. W.; Mathers, R. A. Impairment of early feeding behavior of largemouth bass by pentachlorophenol exposure: a preliminary assessment. Trans. Am. Fish. Soc. 1987, 116, 71–78. Nagler, J. J.; Aysola, P.; Ruby, S. M. Effect of sublethal pentachlorophenol on early oogenesis in maturing female rainbow trout (Salmo gairdneri). Arch. Environ. Contam. Toxicol. 1986, 15, 549–555. Farrell, A.; Gamperl, A. K.; Birtwell, I. K. Prolonged swimming, recovery and repeat swimming performance of mature sockeye salmon Oncorhynchus nerka exposed to moderate hypoxia and pentachlorophenol. J. Exp. Biol. 1998, 201, 2183–2193. Kim, W. S.; Jeon, J. K.; Lee, S. H.; Huh, H. T. Effects of pentachlorophenol (PCP) on the oxygen consumption rate of the river puffer fish Takifugu obscurus. Mar. Ecol.: Prog. Ser. 1996, 143, 9–14. Marrs, T. C. Organophosphate poisoning. Parmacol. Ther. 1993, 53, 51–66. Beauvais, S. L.; Jones, S. B.; Brewer, S. K.; Little, E. E. Physiological measures of neurotoxicity of diazinon and malathion to larval rainbow trout (Oncorhynchus mykiss) and their correlation with behavioral measures. Environ. Toxiocol. Chem. 2000, 19, 1875– 1880. Pavlov, D. D.; Chuiko, G. M.; Gerasimov, Y. V.; Tonkopiy, V. D. Feeding behavior and brain acetylcholinesterase activity in bream (Abramis brama L.) as affected by DDVP, an organo-

(59) (60)

(61)

(62)

(63)

(64) (65) (66)

(67)

(68)

(69)

(70)

(71)

(72)

(73)

(74)

(75) (76)

phosphorous insecticide. Comp. Biochem. Physiol. 1992, 103C, 563–568. Bhattacharga, S. Target and non-target effects of anticholinesterase pesticides in fish. Sci. Total Environ. 1993, 859–866. Sahib, I. K. A.; Rao, K. S. J.; Rao, K. V. R. Effect of malathion exposure on some physical parameters of whole body and on tissue cations of teleost Tilapia mossmbica (Peters). J. Biosci. 1981, 3, 17–21. Basha, S. M.; Rao, K. S. P.; Rao, K. R. S. S.; Rao, K. V. R. Respiratory potentials of the fish (Tilapia mossambica) under malathion, carbaryl and lindane intoxication. Bull. Environ. Contam. Toxicol. 1984, 32, 570–574. Alvarez, M. C.; Fuiman, L. A. Ecological performance of red drum (Sciaenops ocellatus) larvae exposed to environmental levels of the insecticide malathion. Environ. Toxicol. Chem. 2006, 25, 1426–1432. McKim, J. M.; Schmieder, P. K.; Niemi, G. J.; Carlson, R. W.; Henry, T. R. Use of respiratory-cardiovascular responses of rainbow trout (Salmo gairdneri) in identifying acute toxicity syndromes in fish: Part 2, malathion, carbaryl, acrolein, and benzaldehyde. Environ. Toxicol. Chem. 1987, 6, 313–328. Haubenstricker, M. E.; Holodnick, S. E.; Mancy, K. H.; Brabec, M. J. Rapid toxicity testing based on mitochondrial respiratory activity. Bull. Environ. Contam. Toxicol. 1990, 44, 675–680. Casarett, L. J.; Doull, J. Toxicology, the Basic Science of Poisons, 5th ed.; Macmillan: New York, 1996. Nguyen, L. T. H.; Janssen, C. R.; Volckaert, A. M. Susceptibility of embryonic and larval African catfish (Clarias gariepinus) to toxicants. Bull. Environ. Contam. Toxicol. 1999, 62, 230–237. Sawyer, P. L.; Heath, A. G. Cardiac, ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide. Fish Physiol. Biochem. 1988, 4, 203–219. LeDuc, G. Cyanides in water: toxicological significance. In Aquatic Toxicology, Vol. 2; Weber, L. J., Ed.; Raven Press: New York, 1984; Vol. 15; pp 3-224.. U.S. Environmental Protection Agency. Ambient Water Quality Criteria for Cyanides; Report 440/5-80-037; Washington, DC, 1980. Hanawa, M.; Harris, L.; Graham, M.; Farrell, A. P.; BendallYoung, L. I. Effects of cyanide exposure on Dascyllus aruanus, a tropical marine fish species: lethality, anaesthesia and physiological effects. Aquar. Sci. Cons. 1998, 2, 21–34. Holcomb, M.; Cloud, J. G.; Woolsey, J.; Ingermann, R. L. Oxygen consumption in unfertilized salmonid eggs: an indicator of egg quality. Comp. Biochem. Physiol. 2004, 138A, 349–354. Smith, L. L., Jr.; Broderius, S. J.; Oseid, D. M.; Kimball, G. L.; Koenst, W. M.; Lind, D. T. Acute and Chronic Toxicity of HCN to Fish and Invertebrates; Report 600/3-79-009; U.S. Environmental Protection Agency: Washington, DC, 1979. U.S. EPA. Ambient Aquatic Life Water Quality Criteria for Atrazine - Revised Draft; 822-R-03-023; U.S. EPA Office of Water: Washington, DC, 2003. U.S. EPA. Update of Ambient Water Quality Criteria for Cadmium; 822-R-01-001; U.S. EPA Office of Water: Washington, DC, 2001. U.S. EPA. Drinking Water Standards and Health Advisories; 822R-06-013; U.S. EPA Office of Water: Washington, DC, 2006. U.S. EPA. National Recommended Water Quality Criteria; U.S. EPA Office of Water: Washington, DC, 2006.

ES702879T

VOL. 42, NO. 18, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

7017