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Trace Organic Analysis
Nitro musk adducts of rainbow trout hemoglobin: Dose-response and toxicokinetics determination by GC-NICI-MS for a sentinel species
Published in Am. Lab., 2004.
[note: minor content and formatting differences exist between this web version and the published
version]
M. A. Mottaleb, W. C. Brumley, L. R. Curtis†, G. W. Sovocool*
U.S. Environmental Protection Agency, National Exposure Research Laboratory, Environmental Sciences Division, P.O. Box 93478, Las Vegas, NV 89193-3478, USA.
†Department of Environmental and Molecular Toxicology, 1007 Agricultural and Life Sciences, Oregon State University, Corvallis, OR 97331-7301, USA.
*Corresponding author: G. Wayne Sovocool, U. S. EPA, P. O. Box 93478, Las Vegas, NV 89193-3478; Fax: (702) 798 2142; Email: sovocool.wayne@epa.gov
Abstract
Rainbow trout and other fish species can serve as "sentinel" species for the assessment of ecological status and the presence of certain environmental contaminants. As such they act as bioindicators of exposure. Here we present seminal data regarding dose-response and toxicokinetics of trout hemoglobin adduct formation from exposure to nitro musks which are frequently used as fragrance ingredients in formulations of personal care products. These hemoglobin adducts serve as biomarkers of exposure of the sentinel species as we have shown in previous studies of hemoglobin adducts formed in trout and environmental carp exposed to musk xylene (MX) and musk ketone (MK). Gas chromatography-electron capture negative ion chemical ionization-mass spectrometry (GC-NICI-MS) employing selected ion monitoring is used to measure 4-amino-MX (4-AMX), 2-amino-MX (2-AMX), and 2-amino-MK (2-AMK) released by alkaline hydrolysis from the sulfonamide adducts of hemoglobin. Dose-response and toxicokinetics were investigated using this sensitive method for analysis of these metabolites. In the dose-response investigation, the concentrations of 4-AMX and 2-AMX are observed to pass through a maximum at 0.10 mg/g. In the case of 2-AMK, the adduct concentration is almost the same at dosages in the range of 0.030 to 0.10 mg/g. For toxicokinetics, the concentration of the metabolites in the Hb reaches a maximum in the 3-day sample after administration of MX or MK. Further elimination of the metabolites exhibited kinetics with a presumed exponential decay and a half-life estimated to be 1-2 days. This suggests that a robust mechanism of elimination of the adducts exists in fish erythrocytes apparently analogous to that observed in mammals . Two sick fish were observed to yield from 5 to 24 times the amount of adducts of similarly exposed fish, suggesting that this elimination mechanism may have been impaired or lacking in susceptible individual fish. It appears that the adducts are destroyed in times far shorter than the expected life spans of the erythrocytes. This finding may have implications for the use of Hb biomarkers as integrative measures of exposure in some contexts. Additional conclusions from these preliminary data include the additive burden of exposure to multiple compounds and the increased susceptibility and direct observation of metabolic differences of individual members of the species completely independent of habitat and feeding habit variations.
Introduction
Musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobezene, MX) and musk ketone (1-tert-butyl-3,5-dimethyl-2,6-dinitro-4-acetylbenzene, MK), the most prevalent synthetic nitro musk compounds, are frequently used as fragrance and additive materials in personal care products and perfumed household products. They are used as a substitute for expensive natural musk, and their estimated annual production is about 1000 metric tons (1). Due to their persistence in the environment and high potential for bioaccumulation (2), MX and MK have been detected as contaminants in aquatic and terrestrial organisms (3,4), human tissues (5-7), North Sea, river and freshwater (1,8,9), sewage treatment effluent (10), Norwegian air samples (11), human adipose tissue and breast milk (2,12), developing and adult rats (13), and fish, mussels, and shrimp (14). Metabolites of MX and MK have been identified and quantified in samples of river waters, domestic and industrial sewage sludge (9,15), and homogenized whole fish tissues (16). Some studies have been reported on ecotoxicity of MX and its metabolites (17,18). Several studies suggested that MX is not genotoxic (20-22). MX and MK were identified as inducers of toxifying enzymes, cytochrome P450 1A1 and 1A2 in the rat liver (23).
Fish serve as sentinel species for the assessment of ecological health and the presence of certain environmental contaminants (24). As such they function as bioindicators of exposure and thus provide information on the status of an entire ecosystem or watershed (25). The sentinel species in turn may have quantifiable biological responses to the exposure events. Exposure events consist of contact with agents or "stressors" in the environment that may consist of traditional contaminants such as pesticides or may be nontraditional substances such as certain endocrine disrupting compounds or pharmaceuticals and personal care products (26). Because of their aqueous habitat, fish experience continuous exposure to contaminants that perfuse into receiving waters. In addition, there is the factor of cumulative risk as a result of exposure to multiple stressors (27).
Hb-adducts have served as suitable biomarkers for exposure to carcinogenic aromatic amines and nitroarenes. The metabolites of nitro musks or other related nitroarenes, bound to Hb as biomarkers of exposure, can be used potentially to integrate continuous exposures over a longer time range (potentially over the life time of red blood cells), and thus, may be better suited for risk assessment than quantitation of urinary metabolites (28,29). Nitroarenes are subject to enzymatic reduction, and their reactive intermediate, nitrosoarenes, react with the SH group of cysteine in Hb to form an acid/base labile sulfonamide that hydrolyzes to aromatic amines in the presence of aqueous base (30). This process is shown schematically in Fig. 1.
Figure 1. The metabolic pathway of cysteine Hb adduct formation with nitro musk compound using 4-AMX as the example. (click figure to view larger image)
The formation of Hb adducts is often associated with the formation of DNA adducts as well. Recently, we detected a 4-AMX metabolite from carp Hb for the purpose of ecological assessment of MX exposures (31). In the course of our earlier studies of exposed trout to MX and MK a trout Hb adduct of a 4-AMX was found, suggesting that nitro-reduction of MX may occur in fish (32, 33) as well as in humans (34). In this work, the 4-AMX, 2-AMX, and 2-AMK metabolites bound to Hb, formed by enzymatic reduction of MX and MK, are detected and quantified by gas chromatography-electron capture negative ion chemical ionization-mass spectrometry (GC-NICI-MS) using selected ion monitoring (SIM). This was found to be an improvement in sensitivity over our earlier method of analysis (31,35). The present investigation is, to our knowledge, the first report on dose-response and toxicokinetics of nitro musk Hb adducts from the sentinel species of rainbow trout exposed to MX and MK and, as such, is the first report on Hb biomarkers of a sentinel species thus exposed.
Experimental
Standards, chemicals, and solvents
Sodium dodecyl sulfate (SDS), sodium hydroxide pellets, and n-hexane (HPLC grade) were purchased from the Sigma-Aldrich, Fisher Scientific, and J. T. Baker, respectively. The internal standard (I.S.), 2,3,4,5,6-pentafluorobenzophenone (purity 99%) was obtained from Aldrich, USA. The standards of MX and MK were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Standards of 4-AMX, 2-AMX, and 2-AMK metabolites were synthesized by Dr. L. I. Osemwengie, U.S. Environmental Protection Agency (EPA), Las Vegas, Nevada (10). Purities of greater than 98% were provided. Tricane methane sulfonate (MS 222) was obtained from Sigma-Aldrich. De-ionized water was used for all preparations.
Exposure of trout to nitro musk compounds
Trout exposure experiments were conducted at the Department of Environmental & Molecular Toxicology, Oregon State University (OSU), Oregon for the sampling periods of 24 h (1-day), 72 h (3-day), and 168 h (7-day). A series of standard test solutions containing 10, 30, 100, and 300 mg/mL MX or MK were prepared in salmon oil as the vehicle (Pharmaceutical grade, Yukon Nutritional Company, USA) for trout exposure to MX and MK. At the highest intended concentration, neither the MX nor the MK dissolved completely in the oil, but instead, formed an emulsion. Well-shaken standard solutions were injected intraperitoneally into fish that were anaesthetized in an aqueous MS 222 solution containing 75 mg/L in a 15-L tank. The anaesthetized trout were weighed before injecting the standard solutions into the fish. For the dose-response study, 24 trout were exposed to MX or MK solutions, three trout for each level. For the toxicokinetic investigations, twelve more trout were exposed to 30 mg/mL MX or MK, six with each standard solution. For control work, nine fish were exposed to the vehicle (no MX or MK) for the same sampling period. After exposure, fish were returned to labeled tanks with circulating water at 13oC.
Observation of trout during the sampling period
In total, 45 trout were exposed to MX or MK solutions, and vehicle, followed by sampling periods of 1-day, 3-day, and 7-day. Following exposure, no food was given to the fish, which were closely monitored in the labeled circulating water tank. Two trout exposed to the 30 mg/mL MX solution was observed to be sick (moribund) in the tank on day-1 and day-7. One fish exposed to 10 mg/mL MK died on day-1 and was not included in the study.
Collection of trout blood and separation of Hb
Before drawing the blood samples, trout were anaesthetized with MS 222 (250 mg/L). This concentration of the MS 222 solution was also fatal to the trout. Blood samples were drawn from the trout into heparinized individual syringes from the caudal vein and placed into heparinized individual sterile interior Vacutainers (Becton Dickson, NJ). After 1-day exposure, twenty-three blood samples were drawn from 23 trout that were exposed to 10, 30, 100, and 300 mg/mL MX or MK. Three days and and 7-days after exposure, 12 blood samples, six on each exposure day were collected from 12 trout that were exposed to 30 mg/mL MX or MK solutions. Nine control blood samples were also drawn from nine trout for 1-day, 3-day, and 7-day after exposure. All blood samples were kept on ice immediately after collection, and the fish were sacrificed. Erythrocytes or red blood cells were separated from plasma by centrifuging at 3500 x g for 10 min at 4oC and were washed twice with equal volumes of 0.9% saline and re-centrifuged. The red blood cells were lysed by adding 2 volumes of distilled water. The Hb solutions were solidified in a freezer at -24oC. The solid Hb solutions were shipped by overnight delivery from OSU to the National Exposure Research Laboratory, EPA, Las Vegas, Nevada in an insulated box packed with dry ice. Upon receipt, the water was eliminated from the solid Hb solutions by a freeze-drying procedure using a Sentry Microprocessor Control, Freezemobile and Benchtop Freeze-dryer (The VirTis Company, Inc., NY). The dried Hb was then placed in a freezer at -24oC for subsequent analysis of the nitro musk metabolites.
Liberation of bound amine metabolites from the Hb
Recently, we reported the alkaline hydrolysis, extraction, and preconcentration procedures for liberation of the bound amino metabolites from the carp Hb ( ). The same procedures were used in this study. The dried extract (about 45 mL) was concentrated under a stream of nitrogen at 45oC to a volume of about 50-65 mL, to which 10 mL of 100 pg/mL of I.S. was added. The solution was sealed in GC-vials and analyzed by GC-NICI-MS using the SIM mode.
Two non-hydrolyzed Hb control experiments were also performed to investigate the possible presence of unbound amino metabolites in the Hb samples. In the experiments, all chemicals and solvents except for the NaOH were added to the Hb (about 50 mg), and the same extraction and preconcentration procedures were followed as described in the alkaline hydrolysis work. A laboratory control experiment was carried out by using the same amounts of solvents, chemicals, and reagents used for the hydrolysis, except no Hb was used.
Gas chromatography and mass spectrometry
In our earlier studies we used EIMS, but also performed a comparison study and changed to GCCINIMS. An Agilent Technologies HP 6890 series GC system equipped with a HP 5973 mass selective detector (MSD) connected to a Agilent 7683 auto sampler and Agilent 6890 GC were used in this investigation. The helium carrier gas was passed through a DB-5 (J&W Scientific, Agilent Technologies, CA) capillary column (40 m long, 0.18-mm i.d., and 0.18-mm film thickness) at a constant flow rate of 0.50 mL/min (average linear velocity 22 cm/sec) using the pulsed splitless mode. The auto sampler injected a 2-mL volume of sample extract or standard solution into the GC with an oven gradient temperature starting at 60oC for 1 min, 150oC at 10oC min-1, 250oC at 8oC min-1, and 300oC at 10oC min-1, and holding the final temperature for 6 min. The injector and transfer line temperatures were 250 and 280oC, respectively. The ion source temperature 150oC, quadrupole analyzer temperature 106oC, filament emission current 49.4 mA, and 1800 or 2000 V on the electron multiplier were used with a methane flow rate of 0. 76 mL/min. The mass spectral acquisitions were performed with dwell times of 25 msec/ion using the GC/MSD Agilent ChemStation software, version B.02.05. Ions monitored included the I.S. (pentafluorobenzophenone) m/z 272 molecular anion; m/z 267 molecular anion) and m/z 268 (isotopic molecular anion) for 4-AMX and 2-AMX; and m/z 264 (molecular anion) and 265(isotopic molecular anion) for 2-AMK.
Quality assurance/quality control (QA/QC)
Calibration curve
A regression analysis was carried out on the ratio of areas (analyte area divided by internal standard area) versus the ratio of 2-AMX, 2-AMK, and 4-AMX concentration to internal standard concentration resulting in a 6-point calibration curve. Unweighted regression was considered and resulted in coefficient of determination (R2) of 0.996 for 4-AMX, 0.997 for 2-AMX, and 0.997 for 2-AMK for the resulting equation of the line that was used to calculate the concentrations of the 4-AMX, 2-AMX, and 2-AMK. Each group of samples to be analyzed was bracketed before and after by a representative standard/internal standard QC sample to establish adherence to the calibration curve equation and agreement with the retention time of the standard. Deviations from the calibration curve greater than ±10% would cause rerunning of standards, construction of a new calibration curve, or replacement of the capillary GC injector tubing. Contaminated tubing resulted in poor peak shape (tailing), which affected quantitation. Retention time variations were generally less than 6 sec., and peak widths at half-height were about 3 sec. Standards run on the day of analysis were followed by a solvent blank run, followed by the extract. No carryover of the 4-AMX, 2-AMX, and 2-AMK was observed.
Results and discussion
All 44 sample extracts obtained from the alkaline hydrolysis of the trout Hb, injected with the MX, MK, and the salmon oil (control samples), were subjected to GC-MS analysis. The GC-NICI-MS determined the 4-AMX, 2-AMX, and 2-AMK metabolites in 35 samples that were exposed to MX or MK for the exposure periods of 1-day, 3-days, and 7-days. In the case of the 9 control samples, two non-hydrolyzed extracts, and one reagent blank sample, no 2-AMX, 2-AMK, and 4-AMX metabolites were observed.
Detection and identification of MX and MK adduct metabolites in the Hb
Retention times and spectral properties were identical for standards and samples. Figure 2A illustrates the chromatographic plot of traces of typical selected ions (summed for the four masses measured). The correlation of retention time of standards and analyte responses from samples was excellent. The mass spectral responses for the 4-AMX and 2-AMX were virtually identical and distinction between these two rested on the GC retention times of the respective standards. The mass spectra afforded by a standard for 4-AMX and that detected by GC-NICI-MS in a Hb sample extract were virtually identical and is shown in Fig. 2B. The ratio of the (M+1) -./ M-. abundances generally agreed between standard and sample by + or- 12% of the respective values.
Figure 2. A: GC-NICI-MS selected ion chromatograms summed for the 4 masses measured for a mixture (100 pg/mL each) of standard solution of 2-AMX, 2-AMK, and 4-AMX metabolites labeled with retention time and sample responses, respectively, containing 1 ng of I.S. (not shown), (B) extract from the sample after 1-day exposure to 6.3 mg MK (Table 1, S24) containing 1 ng of I.S. (not shown) and (C) extract from the sample after 1-day exposure to 5.4 mg MX (Table 1, S4) containing 1 ng of I.S. (not shown). GC-MS conditions are given in the experimental section. B: Typical electron capture NICI mass spectrum using 4-AMX as the example. (click figure to view larger image)
The limit of detection (LOD) for the metabolites was calculated based on a signal-to- noise ratio of 3:1. The LOD for 4-AMX, 2-AMX, and 2-AMK corresponded to the approximately 1.7, 1.4, and 0.30 ppb (based on 50 mg of Hb per 50 mL final volume of extract). Recoveries of 82% were obtained from spiked fish.
Dose-response and concentration of nitro musk adducts in the Hb
The trend of adduct formation as a function of concentration of nitro musks has been investigated in the trout Hb by taking samples, at 24 h (1-day) after exposure, with increasing initial exposure concentrations of MX and MK. Table 2 includes data for all three metabolites and Fig. 3 graphs that dose-response relationship data for 4-AMX metabolite formation in Hb. The initial dose-response is approximately linear but falls off at the higher dose.
Figure 3. Dose-response relationship plots for concentration of 4-AMX observed in Hb at 1-day exposure versus dose of MX given to trout. The initial dose/response was approximately linear as shown but fell off at higher doses (click figure to view larger image).
It can be seen from Table 2 that, in general, the binding of MX or MK as adducts to the Hb increases in a dose-dependent but nonlinear manner for 4-AMX and 2-AMX with maximum formation at dose 0.10 mg/g, beyond which it decreases. For MK, adduct formation as 2-AMK also increases with dose. Maximum formation was observed at doses in the range of 0.030 to 0.10 mg/g. The average concentrations of 4-AMX, 2-AMX, and 2-AMK metabolites at dose level 0.10 mg/g were found to be 700, 7.4, and 2.2 ng/g, respectively. In Table 2, the higher concentrations of 4-AMX and 2-AMX in the Hb were observed over that of the 2-AMK indicating that the MX may more easily be reduced by enzyme activity. We note that the rate of formation of 4-AMX adduct is almost 100-fold greater than the rate of the other two. A drop in the adduct formation was seen at dose 0.30 mg/g for both MX and MK. The attempt to prepare a concentration (300 mg/mL of salmon oil) of MX or MK, which would have yielded a dose of 0.30 mg/g fish, did not result in a true solution but in a thick emulsion that did not dissolve all of the solutes. The rates of formation of adducts may have experienced a drop due to the suspensions of the MX or MK and the limitations of the resultant absorption. Therefore, the formation of metabolites may have been affected absorption kinetics such that the rates of formation resemble those at the 0.03 mg/g dosing level. The relatively greater reduction in the rate of formation from 4-AMX relative to other two metabolites (2-AMX, and 2-AMK) may be a consequence of the observation that the other two are not linearly increasing with increase dose beyond the 0.030 mg/g dosing level.
Table 2 summarizes the concentration of 2-AMX, 2-AMK, and 4-AMX metabolites formed in the individual trout Hb for the exposure period of 1-day, 3-days, and 7-days with MX and MK. (As such these data are preliminary in nature because more data would be needed to establish more detailed correlation with toxicokinetic models and the shape of the observed response.) A considerable individual variation of metabolite concentrations was found among the trout. As mentioned, two trout injected with the 30 mg/mL MX solution were found to be sick when the blood samples were collected from them on 1-day or 7-day after exposure. For the fish sampled on 1-day after exposure, the concentrations of 2-AMX and 4-AMX were 31 and 1150 ng/g, respectively. These values were 5- to 9- fold higher than other fish exposed during the same period. In the case of the fish sampled on 7-day after exposure, the concentrations of 2-AMX and 4-AMX were 28 and 865 ng/g, respectively, and were 10-28 times higher than that observed for other fish over the same time frame. These results may suggest that some additional metabolic activity of the sick fish promotes increased formation of the Hb adducts, but we favor the view that the removal process for Hb adducts may be impaired in the sick fish resulting in continual increase of observed adducts as would be expected from an integrative model of Hb adduct formation.
Toxicokinetics of the nitro musk metabolites in the trout Hb
Table 3 contains data for sampling times versus the natural logarithm of average concentration of the metabolites. The concentrations of 2-AMX and 4-AMX metabolites obtained from the sick trout were not considered in the kinetics study. The time course of the adduct formation for each of the metabolites shows a maximum at about 3-days after exposure, although the frequency of the sampling precludes definitive treatment of the data and definition of the maximum reached. The kinetics of the removal of the Hb-adduct metabolites is presumably non-linear and occurs significantly faster than expectations based on human or rat erythrocyte elimination. The lifetime of trout erythrocytes is unknown. We estimate 1-2 days half-life of the metabolites in the trout Hb, based on assuming first-order kinetics.
Figure 4 shows the three data points for 4-AMX and a theoretical curve derived from estimates of initial and declining portions of the data together with a single compartment model from pharmacokinetics (36,37) involving a rate of metabolite formation (km) and a rate of elimination (kel) [conc = K [e-kel * t - e-km * t], Where K = 1491 (ave of the 3 values calculated for the three data points) and km = 0.90 and kel = 0.45 and t is the time in days.
Figure 4. Plot of the equation [1491 * (exp(-0.45t) - exp(-0.9t))] versus days (t) and the three data points for 4-AMX (caveat that an additional known value of 0 at time 0 applies). (click figure to view larger image)
It is obvious from the calculated curve that this type model is not a good fit to the data, but it provides a visual comparison of the overall shape of such a model relative to the data and to an expected exponential fall off of the adduct when an elimination process is present. The model itself assumes an instantaneous distribution of dosed compound and the entire organism as one compartment. Obviously, these assumptions may be far from reality. In particular, the time course of absorption may have a large effect on the overall response we observe. Unfortunately, the lack of more sampling points precludes a more definitive analysis of the elimination kinetics and does not warrant an exhaustive search of potential models.
Table 3 depicts the estimated individual values of the elimination rate constants and half-lives of 2-AMX, 2-AMK, and 4-AMX metabolites found in the trout Hb if first order kinetics of elimination were followed. These values are comparable to the reported half-life for MX of less than a few days in the rat (23). This half life (rate of elimination) does not seem to correlate with the Hb erythrocyte life span we assume in fish to be at least the same order of magnitude of 60 days reported for the rat (38). It has long been assumed that hemoglobin adducts as biomarkers are integrative measures for the life span of the erythrocytes. This does not appear to be the case for these fish in our work and rat species in the work of others with these kinds of adducts. Such rapid destruction of the adducts suggests an enzymatic cleavage process to release the metabolites. Free amines were not found associated with the isolated hemoglobin, suggesting that the metabolites are rapidly removed from the erythrocytes. For comparison, the biotransformation and toxicokinetics of MX in human blood plasma were investigated, and an average half-life of 70 days for a 4-AMX metabolite was reported (39). This is also less than the 120 day human erythrocyte life span by as factor of 0.58. The existence of the hemoglobin adducts and the existence of a repair or elimination process attest to the appropriateness and strengthen the use of fish as a sentinel species for ecological status and the presence of contaminants.
Conclusion
The 4-AMX, 2-AMX, and 2-AMK were identified and quantified as metabolites bound to trout Hb, released by alkaline hydrolysis of the extracts of 35 trout samples, and measured by GC-NICI-MS with SIM. No metabolites were observed in the control samples, non-hydrolyzed Hb samples, or reagent blank extracts, establishing that the adducts had to be bound to the Hb, consistent with the accepted mechanism. Assuming first order kinetics for the elimination process, an estimate of 1-2 days may be calculated for the half-life of the hemoglobin adducts, although more data are needed for a definitive assessment of the rate of elimination. The existence of an elimination mechanism in this sentinel species is analogous to observations of similar exposure in mammals. This finding draws into question the use of hemoglobin adducts as integrated exposure assessments (i.e., over the lifetime of erythrocytes), at least in the context of aromatic amines. This is, to our knowledge, the first report of toxicokinetics and dose-response studies of trout Hb adducts with nitro musks and the first such report for Hb biomarkers of a sentinel species. Although preliminary, the data point to considerable variation among individual fish, with susceptible individuals exhibiting as much as a 10-fold or greater level of adduct formation. This suggests that differences found in environmental sampling may not all be due to differences in habitat or feeding habits but may be part of the normal variation among individuals of the species. The existence of specific rates of formation and elimination of the MX and MK adducts suggests that at least an additive effect for multiple compound exposure can be anticipated in accordance with the concomitant rates of formation and elimination of the individual compounds. These data as such, can be used as a guide to design more definitive toxicokinetic experiments, and help validate the use of sentinel species as indicators of ecological status as well as contaminant levels.
Acknowledgments
This work was performed while the author (MAM) held a National Research Council Research Associateship Award at the National Exposure Research Laboratory, U.S. Environmental Protection Agency (EPA), Las Vegas, Nevada. MAM would like to thank the Department of Chemistry, University of Rajshahi, Bangladesh for granting study leave to perform the research in the EPA laboratory. The authors would like to thank Dr. L. I. Osemwengie for providing the MX and MK and synthesizing the 4-AMX, 2-AMX, and 2-AMK metabolite standards.
Notice: The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded and performed the research described. This manuscript has been subjected to the EPA's peer and administrative review and has been approved for publication. Mention of trade names or commercial products in the manuscript does not constitute endorsement or recommendation by EPA for use.
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31. Mottaleb, M. A., Brumley, W. C., Sovocool, G. W. Nitro musk metabolites bound to carp hemoglobin: determination by eims versus electron capture negative ion ms. 2004; Int. J. Environ. Anal. Chem. (in press).
31. Mottaleb, M. A., Zhao, X., Curtis, L. R., Sovocool, G. W. Formation of nitro musk adducts of rainbow trout hemoglobin for potential use as biomarkers of exposure. 2004; Aquat. Toxicol. 67: 315-324.
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Table 1. Test materials and dosing schedule: in vivo trout exposure with nitro musk compounds, and with a vehicle (salmon oil).
| Exposure time, Day | MX exposure |
MK exposure |
|||||||
MX conc., mg/ mL |
MX conc., mg/ mL |
MX dose per trout, mg | Av. dosing level, mg/g | MK conc., mg/ mL | Fish wet weight (sample number), g | MK dose per trout, mg | Av. dosing level, mg/g | ||
| Day-1 | 10 | 202 (S1) | 2.0 | 0.01 | 10 | 257 (S19) | 2.6 | 0.01 | |
| 256 (S2) | 2.5 | 237 (S20) | 2.4 | ||||||
| 165 (S3) | 1.6 | 222 (S21)† | 2.2 | ||||||
| 30 | 180 (S4) | 5.4 | 0.03 | 30 | 199 (S22) | 6.0 | 0.03 | ||
| 256 (S5) | 7.5 | 230 (S23) | 6.9 | ||||||
| 280 (S6)* | 8.4 | 212 (S24) | 6.3 | ||||||
| 100 | 236 (S7) | 24.0 | 0.10 | 100 | 272 (S25) | 27.0 | 0.10 | ||
| 264 (S8) | 26.0 | 271 (S26) | 27.0 | ||||||
| 204 (S9) | 20.0 | 197 (S27) | 20.0 | ||||||
| 300 | 250 (S10) | 75.0 | 0.30 | 300 | 190 (S28) | 57.0 | 0.30 | ||
| 310 (S11) | 90.0 | 270 (S29) | 81.0 | ||||||
| 227 (S12) | 69.0 | 250 (S30) | 75.0 | ||||||
| Control | 206 (C1) | 0.20 mL, salmon oil injected only |
|||||||
| 304 (C2) | 0.30 mL, salmon oil injected only |
||||||||
| 184 (C3) | 0.18 mL, salmon oil injected only |
||||||||
| Day-3 | 30 | 208 (S13) | 6.3 | 0.03 | 30 | 278 (S31) | 8.4 | 0.03 | |
| 244 (S14) | 7.2 | 156 (S32) | 4.5 | ||||||
| 193 (S15) | 6.0 | 196 (S33) | 6.0 | ||||||
| Control | 253 (C4) | 0.25 mL, salmon oil injected only |
|||||||
| 272 (C5) | 0.27 mL, salmon oil injected only |
||||||||
| 233 (C6) | 0.23 mL, salmon oil injected only |
||||||||
| Day-7 | 30 | 212 (S16)* | 6.3 | 0.03 | 30 | 121 (S34) | 3.6 | 0.03 | |
| 230 (S17) | 6.9 | 241 (S35) | 7.2 | ||||||
| 204 (S18) | 6.0 | 167 (S36) | 5.1 | ||||||
| Control | 273 (C7) | 0.27 mL, salmon oil injected only |
|||||||
| 305 (C8) | 0.30 mL, salmon oil injected only |
||||||||
| 250 (C9) | 0.25 mL, salmon oil injected only |
||||||||
†Indicated that trout was found dead and blood sample was not collected from the dead fish. *Represented that trout was found sick during collection of blood for the respective exposure periods.
Table 2. Concentrations of 2-AMX, 4-AMX, and 2-AMK metabolites in the trout Hb.
| Exposure period, Day | Exposure level (dose), mg/g | Individual sample numbers | Concentration of 2-AMX and 4-AMX metabolites, ng/g | Individual sample numbers | Concentration of 2-AMK metabolite, ng/g | |
| - | - | - | 2-AMX | 4-AMX | - | - |
| Day-1 | 0.01 | S1 | 5.45 | 97.60 | S19 | 0.63 |
| - | - | S2 | 3.10 | 105.53 | S20 | 0.54 |
| - | - | S3 | 2.10 | 65.82 | S21† | - |
| - | 0.03 | S4 | 6.64 | 170.54 | S22 | 1.94 |
| - | - | S5 | 4.97 | 136.38 | S23 | 2.10 |
| - | - | S6* | 30.94 | 1147.64 | S24 | 2.55 |
| - | 0.10 | S7 | 4.91 | 675.94 | S25 | 1.92 |
| - | - | S8 | 10.99 | 894.14 | S26 | 2.34 |
| - | - | S9 | 6.26 | 517.37 | S27 | 2.42 |
| - | 0.30 | S10 | 5.03 | 169.53 | S28 | 1.11 |
| - | - | S11 | 6.01 | 217.14 | S29 | 1.38 |
| - | - | S12 | 6.18 | 211.59 | S30 | 1.03 |
| - | Control | C1 | ND-------------------- | |||
| - | - | C2 | ND-------------------- | |||
| - | - | C3 | ND-------------------- | |||
| Day-3 | 0.03 | S13 | 16.80 | 555.08 | S31 | 33.37 |
| - | - | S14 | 12.82 | 528.54 | S32 | 30.44 |
| - | - | S15 | 17.88 | 595.98 | S33 | 32.74 |
| - | Control | C4 | ND-------------------- | |||
| - | - | C5 | ND-------------------- | |||
| - | - | C6 | ND-------------------- | |||
| Day-7 | 0.03 | S16* | 28.10 | 864.99 | S34 | 6.91 |
| - | - | S17 | 2.86 | 39.19 | S35 | 4.73 |
| - | - | S18 | 1.45 | 30.68 | S36 | 5.59 |
| - | Control | C7 | ND-------------------- | |||
| - | - | C8 | ND-------------------- | |||
| - | - | C9 | ND-------------------- |
†Indicated that trout was found dead and blood sample was not collected from the dead fish. *Represented that trout was found sick during collection of blood for the respective exposure periods, and the concentrations observed from 2-AMX and 4-AMX were not used in plotting the dose-response and the toxicokinetics curve. ND represents not detected.
Table 3. Elimination rate constant (k) and half-life (T1/2) of the bound metabolites in trout Hb based on first order kinetics.
| Metabolites found in the trout Hb | log of average concentration of the metabolites over day-3 and day-7 exposure period, (ng/g) | Elimination rate constant (k), (Day-1) | Half-life (T1/2), Day | |
| - | Day-3 | Day-7 | - | 2-AMX |
| 2.76 | 0.77 | 0.50 | 1.4 | 4-AMX |
| 6.33 | 3.55 | 0.695 | 1.0 | 2-AMK |
| 3.47 | 1.75 | 0.43 | 1.6 | - |