chapter thirty Spectral models for assessing exposure of fish to contaminants Donald C. Malins, Virginia M. Green, Naomi K. Gilman, and Katie M. Anderson Pacific Northwest Research Institute John J. Stegeman Woods Hole Oceanographic Institution Contents Introduction Materials required Tissues Supplies and equipment for DNA extraction Supplies and equipment for FT-IR spectral analysis Procedures DNA extraction FT-IR spectroscopy Other considerations Statistical analyses FT-IR mean spectra Principal components analysis DNA damage index Results and discussion Comparison of mean DNA spectra Principal components analysis DNA damage index Acknowledgments References Introduction Fourier transform-infrared (FT-IR) spectroscopy is capable of identifying a wide variety of chemical structures on the basis of their unique vibrational and rotational properties. 1–3 DNA has a characteristic ‘‘signature’’ spectrum, the peaks, shoulders, and other spectral properties of which have been identified by spectroscopists as corresponding to specific structures in the DNA molecule. 1 In addition, statistical models of FT-IR spectra have the Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 537 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis remarkable ability to reveal subtle changes in complex cellular structures resulting from various biological and chemical stresses. 3,4 Recent examples include the ability to discriminate, with high sensitivity and specificity, between the DNA of healthy and cancerous prostate tissues, thus providing a basis for predicting the probability of pros- tate cancer. 5 Furthermore, the unique ability of the FT-IR statistical models to differentiate between diverse groups of tissues was evident when it was shown that primary prostate tumors could be readily distinguished from metastasizing primary tumors. This achieve- ment was the basis for statistical models for predicting which tumors are most likely metastasizing without having to wait for metastatic cells to be detected at distant sites in the body (e.g., the groin), at which point treatment options are limited. 5 We recognized that the FT-IR statistical models had the potential for studying the effects of toxic chemicals on fish. In 1997, we showed that liver DNA of English sole (Parophrys vetulus) from the chemically contaminated Duwamish River (DR) in Seattle, WA was structurally different from that of English sole from the relatively clean, rural environment of Quartermaster Harbor (QMH) in Puget Sound, WA. 4 A subsequent study, conducted in October 2000, 6 showed that the DNA from the gills of English sole from the DR could be readily distinguished from the gill DNA of the same species from QMH. The FT-IR spectral differences between groups were consistent with a marked increase in the sediment contamination (e.g., concentra- tions of polychlorobiphenyls [PCBs] and aromatic hydrocarbons) and the degree of CYP1A expression in the gills. A logistic regression analysis of the spectral data sets resulted in the development of a DNA damage index with high sensitivity and specificity. The present report illustrates the application of the FT-IR statistics technology to assess differences in the DNA structure of various fish tissues between reference and contaminated environments. The resulting data can be used for assessing the quality of marine environments, toxic effects on fish, and the effectiveness of remediation protocols. Overall, the FT-IR statistics technology is best used in conjunction with other markers of exposure or toxicity, such as CYP1A expression 7–9 and various histological 10 and histo- chemical indices. 7,11 Although initially applied to fish, this technology has the potential for application to various other aquatic organisms, in addition to a variety of human diseases. Materials required Tissues Groups of fish (preferably sex-matched and not differing significantly in size and mass) are obtained from contaminated and essentially non-contaminated reference sites. Females should be restricted to those with quiescent gonads to minimize the effects of reproductive stage (e.g., suppression of CYP1A by estradiol) on the bio- marker data. Each fish should be given a unique identification and carefully weighed and measured. In field studies, fish are kept alive until sacrificed via decapitation aboard the vessel. The desired tissue (e.g., gill, liver) is removed and immediately frozen in liquid nitrogen. Tissues should be maintained in a À808C freezer until DNA extraction. Prior to freezing, a few milligrams of the tissue are preserved in neutral formalin for histological examination or histochemical determin- ations or both (e.g., CYP1A). 7,11,12 Otoliths may be removed for subsequent age determinations. 13 Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 538 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis Supplies and equipment for DNA extraction . Scalpels, forceps, spatulas . Mortars and pestles . Liquid nitrogen . Qiagen Genomic DNA Buffer Set, #19060 . Qiagen Genomic-tip 100/G, #10043 . Falcon 15-ml graduated polypropylene tubes (conical bottom), #352096 . Osmonics Cameo 30N syringe filter, nylon, 5.0 m, 30 mm, #DDR50T3050 . Roche RNAse A (1 mg ml À1 ), #109169 . Worthington Proteinase K (20 mg ml À1 ), #LS004222 . Isopropanol . Ethanol 70% (ice cold) . Microcentrifuge tubes 2 ml, polypropylene . Transfer pipettes 1.5 and 3 ml, disposable, polyethylene . 508C water bath . Refrigerated (48C) centrifuge . Microcentrifuge at 48C . Optima grade water (Fisher), #W74LC Supplies and equipment for FT-IR spectral analysis . FT-IR microscope spectrometer (System 2000, Perkin-Elmer) . BaF 2 plate, 38.5 mm  19.5 mm  2 mm (International Crystal Laboratories, Gar- field, NJ) . Aluminum BaF 2 plate holder (Custom-made for the Pacific Northwest Research Institute, Seattle, WA; See Figure 30.1) . 0.1–2.5 ml pipetter with tips . Dissecting microscope A B 76 mm 25 mm 39 mm 37 mm 20 mm 3 mm 1.5 mm 18 mm 1 mm Figure 30.1 (A) Diagram of custom-made aluminum BaF 2 plate holder and (B) cross-section schematic. Dimensions are given; images are not to scale. Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 539 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis Procedures DNA extraction Frozen tissue ($100 mg; À808C) is ground to powder with a mortar and pestle while submerged in liquid nitrogen. DNA ($50 mg) is then extracted from each sample with Qiagen 100/G Genomic-tips (Qiagen, Chatsworth, CA) using the standard Qiagen extrac- tion protocol with the following modification: after elution, the DNA solution (eluate) is passed through a 5.0-m Cameo 30N filter (Osmonics, Minnetonka, MN) to remove re- sidual resin from the Qiagen Genomic-tip prior to precipitation. After filtration, the Qiagen protocol is resumed. In preparation for FT-IR spectral analysis, the DNA pellet is dissolved in 10–40 ml (depending on size) of optima grade water (Fisher Scientific). The DNA is allowed to dissolve overnight at 48C. The Qiagen procedure is an ion-exchange system and does not constitute a source for artifactual oxidation of purines during extraction. FT-IR spectroscopy A 0.2-ml aliquot of the DNA solution is spotted directly on a BaF 2 plate and allowed to spread, forming an outer ring that contains the DNA. Two separate spots are created for each DNA sample. The spots are allowed to dry. Spotting is repeated until the ring is at least 100 m wide, the width of the aperture of the System 2000 microscope spectrometer (Perkin-Elmer). The plate is then placed in a lyophilizer for 1 h to completely dry the DNA. Initially, a background energy reading (percent transmittance) is determined from a blank area of the BaF 2 plate. Energy readings are then taken at various points around the ring (Figure 30.2), and the points for spectral determinations are selected where the energy readings are 15–25% less than the background energy (optimally close to 15% less). Ten spectral determinations are made around each of the two rings per sample and the percent transmittance values are converted (Fourier-transformed) into absorbance values (Figure 30.2). The spectral data obtained are saved in a database for subsequent statistical analysis. Using MS Excel, each spectrum is baselined by taking the mean absorbance across 11 wave numbers, centered at the minimum absorbance value between 2000 and 1700 cm À1 , and subtracting this value from the total absorbance at each wave- number. Each spectrum is then normalized by dividing the entire baselined absorbance values by the mean absorbance between 1750 and 1550 cm À1 . Baselining and spectral normalization adjust for the optical characteristics of each sample (e.g., related to film thickness). The mean absorbance value of the 20 spectral determinations for each sample is then calculated at each wavenumber between 1750 and 1275 cm À1 . Other considerations To avoid any batch effects, it is recommended that samples from reference and contam- inated sites be randomized during DNA extraction and FT-IR spectroscopy. Tissues may be ground and stored at À808C prior to DNA extraction. One technician can extract 6–12 samples, using the modified Qiagen protocol, in about 6 h, including a 2-h incubation time. FT-IR analysis should be performed on the extracted samples within a day or two. For long-term storage, we do not recommend that the DNA be kept in water, but rather in the dry state at À808C. Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 540 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis Statistical analyses FT-IR mean spectra A mean spectrum is determined for each fish group (i.e., from contaminated and refer- ence sites; Figure 30.3A). A t test is then performed at each wavenumber to establish statistical differences (P values) between the mean spectra (Figure 30.3B). Over the wavenumbers used, spectral regions with P < 0.05 are likely to represent real structural differences between groups when they comprise 5% of the spectral range. 3 These struc- tural differences represent alterations in various aspects of the DNA molecule (e.g., as illustrated in Figure 30.2). Principal components analysis Statistical model development is accomplished by first conducting principal components analysis (PCA) on the mean spectrum of each individual DNA sample (S-Plus 2000 Professional Release 1, Mathsoft Engineering & Education, Cambridge, MA). PCA entails nearly 1  10 6 correlations between $1000 independent variables relating to the absorb- ance, wavenumbers, and other properties of the spectrum. 14 PCA results in 10 principal 0 0.5 1 1.5 2 1750 1650 1550 1450 1350 Wavenumber (cm −1 ) Absorbance a b c d e f g h a, 1688 cm −1 NH 2 scissoring vibrations of cytosine b, 1655 cm −1 c, 1602 cm −1 d, 1576 cm −1 Thymine ring vibrations; adenine NH 2 bending and C N stretching vibrations e, 1525 cm −1 Cytosine residue; alteration in N7−C8 stretching vibration of imidazole rings f, 1487 cm −1 g, 1418 cm −1 h, 1369 cm −1 In-plane vibrations of base residues for N−H and C−H deformation modes In-plane ring vibrations of cytosine x x x x x x x x x x Figure 30.2 A DNA ring with 10 spectral determination points (x), the subsequent DNA spectra obtained, and the wavenumber and structural assignments for designated peaks. Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 541 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis component (PC) scores for each sample. Significant differences (P 0.05) in each PC score between groups are determined using t tests. The PC scores showing the most significant differences between the groups are used to construct two- or three-dimensional PC plots (Figure 30.4). Those PC scores representing fish with similar DNA structures will cluster together and be separated from other clusters reflecting a different DNA structure. DNA damage index Logistic regression analysis is performed (SPSS statistical package 10.0, SPSS, Chicago, IL) using a single, significant (P 0.05) PC score to establish a DNA damage index. Using a 1750 1650 1550 1450 1350 Wave number (cm −1 ) Wave number (cm −1 ) Absorbance 1.5 0.5 0 A B 1.0 A 2.0 DR QMH 1 2 3 4 5 0.01 0.05 1 P value B 1750 1650 1550 1450 1350 Figure 30.3 (A) Comparison of mean FT-IR spectra of gill DNA from DR and QMH fish; (B) P values from a t test comparing mean spectra at each wave number. The peak wavenumber designations are as follows: 1, 1688 cm À1 ; 2, 1655 cm À1 ; 3, 1602 cm À1 ; 4, 1525 cm À1 ; 5, 1487 cm À1 . (Modified and reprinted from Malins, D.C., Stegeman, J.J., Anderson, J.W., Johnson, P.M., Gold, J. and Anderson, K.M., Environ. Health Perspect. (Perspect., 2004, 112: 511–515.) Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 542 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis scale of 1–10, this index is based on the different spectral and structural properties of DNA from each fish group (Figure 30.5) and is a measure of DNA damage that provides a means to discriminate between fish from reference and contaminated sites. 6 Results and discussion Comparison of mean DNA spectra As an example, comparison of the mean FT-IR spectra for the DNA from the DR (n ¼11) and QMH (n ¼11) fish is given in Figure 30.3A. Some of the differences in the mean PC5 −0.06 −0.04 −0.02 0.0 0.02 −0.12 0.0 0.12 0.24 0.06 PC9 0.0 −0.06 PC10 Figure 30.4 Three-dimensional separation of PC scores from the FT-IR spectra of gill DNA from the DR ( . ; n ¼ 11) and QMH ( ; n ¼ 11) fish. Dotted drop lines represent the distance from the PC9 baseline level of 0. (Reprinted with permission from Malins, D.C., Stegeman, J.J., Anderson, J.W., Johnson, P.M., Gold, J. and Anderson, K.M., Environ. Health Perspect. (Perspect., 2004, 112: 511–515.) 9 7 5 2 1 −1 −0.06 −0.04 −0.02 0.0 0.02 0.04 DNA damage index 1 2 3 4 5 6 3 4 6 10 8 0 PC10 Figure 30.5 DNA damage index for gill DNA from the DR ( . ; n ¼ 11) and QMH ( ; n ¼ 11) fish. Overlapping points: 1, two QMH; 2, one DR and one QMH; 3, one DR and five QMH; 4, four DR; 5, one DR and one QMH; 6, four DR. (Reprinted with permission from Malins, D.C., Stegeman, J.J., Anderson, J.W., Johnson, P.M., Gold, J. and Anderson, K.M., Environ. Health Perspect. (Perspect., 2004, 112: 511–515.) Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 543 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis spectra may appear to be almost imperceptible; however the P values at each wave- number shown in Figure 30.3B indicate that significant differences (P 0.05) were found for the five peaks identified in Figure 30.3A. The peak differences occurred at the following wave numbers: 1688, 1655, 1602, 1525, and 1487 cm À1 . The structural assign- ments for these peaks are given in Figure 30.2 and represent an array of differences in the structures of the nucleotide bases between the two DNA groups. Significant differences between the mean DNA spectra for the two fish groups represented 26.5% of the spectral range (differences <5% may occur by chance). 3 Although a broader range of wave- numbers can be used (i.e., from 1750 to 700 cm À1 ), 4 we have found that the narrower range used here (i.e., from 1750 to 1275 cm À1 ) has given the most reliable results with studies of fish DNA. This spectral range primarily represents vibrations of the nucleotide bases, thus spectral differences between groups of DNA would include structural changes associated with the genome. Comparison of mean spectra is a procedure for identifying the nature and extent of DNA structural differences (Figure 30.2) in fish from reference and contaminated environments. These comparisons also allow for initial evaluation of whether sufficient differences exist between the group mean spectra to justify further statistical analyses. However, the technique of PCA described in the following paragraph has the ability to discriminate between groups, even when the spectral means show few, or even no differences. Principal components analysis PCA is a powerful means of discriminating subtle differences in DNA structures between groups of fish from different environments. Groups of DNA samples representing fish from contaminated and reference environments will cluster in different areas of the PC plots by virtue of their different spectral and structural properties. An example of this discrimination is a three-dimensional projection of PC scores (PC10, PC5, and PC9) representing the gill DNA from the DR (contaminated) and QMH (reference) fish (Figure 30.4). Despite the high degree of separation, the relatively small number of overlapping points between the groups may reflect fish migrations or other aberrations known to exist in natural fish populations. DNA damage index In this example with gill DNA, logistic regression analysis was conducted based on PC10, selected for its significance (P < 0.01). The resulting sigmoid-like curve shows a distinct separation between the DR and QMH fish groups with 9/11 DR scores falling above $5.0 on the index and 10/11 of the QMH scores falling below this value. This indicates that the DR fish may have more damage to their gill DNA than the fish from QMH, which is further substantiated by the sediment chemistry and CYP1A data. 6 This results in an 82% probability of correctly identifying a DR sample and a 92% probability of correctly identifying a QMH sample. The DNA damage index provides a means of quantifying the DNA damage between fish from reference and contaminated environments. This index can be determined for any two fish populations to assess environmentally induced DNA damage using a variety of tissues (e.g., gill, liver, gonads, and kidney). The example described employing fish gill 6 has the added advantage of non-lethality if tissues are obtained via punch biopsies. 15 One attractive application of the FT-IR statistics technology would be to determine the Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 544 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis effects of remediation on chemically contaminated aquatic environments. After remedia- tion, DNA samples would be evaluated using the damage index developed for that specific environment, species, and tissue type to determine whether the spectral and structural characteristics had improved to closely match the index values established for the reference site. The usefulness of the DNA damage index has so far been limited to the study of English sole in Puget Sound. 6 We look forward to the application of the FT-IR statistics technology, including the DNA damage index, to other fish and aquatic species, as well as to other environments having different contaminant profiles. Acknowledgments We thank Robert Spies and Jordan Gold of Applied Marine Sciences, Inc., 4749 Bennett Dr., Livermore, CA 94550, for fish collections and Nhan Vo for technical assistance. This publication was made possible by the National Institute of Environmental Health Sci- ences, NIH, grant number P42 ES04696. References 1. Tsuboi, M., Application of infrared spectroscopy to structure studies of nucleic acids, Appl. Spectrosc. Rev., 3, 45–90, 1969. 2. Parker, F.S., Applications of Infrared, Raman, and Resonance Raman Spectroscopy in Biochemistry, Plenum Press, New York, 1983. 3. Malins, D.C., Polissar, N.L., Ostrander, G.K. and Vinson, M.A., Single 8-oxo-guanine and 8-oxo-adenine lesions induce marked changes in the backbone structure of a 25-base DNA strand, Proc. Natl. Acad. Sci. USA, 97, 12442–12445, 2000. 4. Malins, D.C., Polissar, N.L. and Gunselman, S.J., Infrared spectral models demonstrate that exposure to environmental chemicals leads to new forms of DNA, Proc. Natl. Acad. Sci. USA, 94, 3611–3615, 1997. 5. Malins, D.C., Johnson, P.M., Barker, E.A., Polissar, N.L., Wheeler, T.M. and Anderson, K.M., Cancer-related changes in prostate DNA as men age and early identification of metastasis in primary prostate tumors, Proc. Natl. Acad. Sci. USA, 100, 5401–5406, 2003. 6. Malins, D.C., Stegeman, J.J., Anderson, J.W., Johnson, P.M., Gold, J. and Anderson, K.M., Structural changes in gill DNA reveal the effects of contaminants on Puget Sound fish, Environ. Health Perspect. Perspect., 112, 511–515, 2004. 7. Woodin, B.R., Smolowitz, R.M. and Stegeman, J.J., Induction of cytochrome P450 1A in the intertidal fish Anoplarchus purpurescens by Prudhoe Bay crude oil and environmental induction in fish from Prince William Sound, Environ. Sci. Technol., 31, 1198–1205, 1997. 8. Stegeman, J.J., Schlezinger, J.J., Craddock, J.E. and Tillitt, D.E., Cytochrome P450 1A expression in midwater fishes: potential effects of chemical contaminants in remote oceanic zones, En- viron. Sci. Technol., 35, 54–62, 2001. 9. Miller, K., Addison, R. and Bandiera, S., Hepatic CYP1A levels and EROD activity in English sole: biomonitoring of marine contaminants in Vancouver Harbour, Mar. Environ. Res., 57, 37–54, 2004. 10. Moore, M.J. and Myers, M.S., Pathobiology of chemical-associated neoplasia in fish, in Aquatic Toxicology: Molecular, Biochemical and Cellular Perspectives, Malins, D.C. and Ostrander, G.K., Eds., Lewis Publishers, Boca Raton, FL, 1994, pp. 327–386. 11. Smolowitz, R., Hahn, M. and Stegeman, J., Immunohistochemical localization of cytochrome P-450IA1 induced by 3,3’,4,4’-tetrachlorobiphenyl and by 2,3,7,8-tetrachlorodibenzoafuran in liver and extrahepatic tissues of the teleost Stenotomus chrysops (scup), Drug Metab. Dispos., 19, 113–123, 1991. Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 545 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis 12. Van Veld, P.A., Vogelbein, W.K., Cochran, M.K., Goksoyr, A. and Stegeman, J.J., Route-specific cellular expression of cytochrome P4501A (CYP1A) in fish (Fundulus heteroclitus) following exposure to aqueous and dietary benzo[a]pyrene, Toxicol. Appl. Pharmacol., 142, 348–359, 1997. 13. Secor, D.H., Manual for Otolith Removal and Preparation for Micro Structural Examination, Electric Power Research Institute, Palo Alto, 1991. 14. Timm, N.H., Ed., Multivariate Analysis, Brooks/Cole, Monterey, CA, 1975, pp. 528–570. 15. McCormick, S.D., Methods for non-lethal gill biopsy and measurement of Na þ ,K þ -ATPase activity, Can. J. Fish Aquat. Sci., 50, 656–658, 1993. Ostrander / Techniques in Aquatic Toxicology L1664_c030 Final Proof page 546 16.12.2004 7:23am Copyright © 2005 by Taylor & Francis [...]... headstanding (46 ) (herpes-like) (39, 47 ) Edwardsiella ictaluri Fish hang listlessly at surface in a head-up-tail down posture, sometimes swimming rapidly in circles; corkscrew spiral swimming, depression (48 ) Epizootic epitheliotro- Sporadic flashing, corkscrew swimming pic disease (herpes virus-like) Eubacterium tarantellus Erratic swimming, loss of (49 ) equilibrium, spiral swimming; floating at surface... challenging Recent improvements in computer and video automation have made possible significant progress in the ease, utility, and affordability of obtaining, interpreting, and applying behavioral endpoints in a variety of applications from water quality monitoring to use in toxicity identification evaluation (TIE).1–9 Consequently, behavioral endpoints in aquatic toxicology are shifting from being met... creatinine kinase (CK), aspartate aminotransferase (AST), glucose, and total protein were performed by the Clinical Pathology Laboratory at the NCSU College of Veterinary Medicine Blood samples for clinical pathology were taken in heparin-coated syringes, kept on ice, and sent directly for plasma chemistry analysis Copyright © 2005 by Taylor & Francis Ostrander / Techniques in Aquatic Toxicology L16 64_ c031... and flow rating of the protein skimmer, or foam fractionator, employed and species-specific requirements Our system includes 260-l fiberglass circular tanks for the 1- and 2-h acute exposures and 855-l dark blue, circular polyethylene tanks for the long-term exposures Copyright © 2005 by Taylor & Francis Ostrander / Techniques in Aquatic Toxicology L16 64_ c031 Final Proof page 549 16.12.20 04 7:27am Each... by Taylor & Francis Ostrander / Techniques in Aquatic Toxicology L16 64_ c031 Final Proof page 556 16.12.20 04 7:27am This precisely controlled hypoxia system has proven to be useful for the experimental induction of hypoxic responses in fish in our laboratory With ever increasing influences of anthropogenic inputs into the nation’s watersheds, particularly those resulting in eutrophication, this system... Ostrander / Techniques in Aquatic Toxicology L16 64_ c032 Final Proof page 567 21.12.20 04 6:19am Table 32.1 Continued Host species Stressor Mosquito fish Goldfish OPs Parathion Mummichog Pb Rainbow trout fingerlings Minnow Phenol Herring Rainbow trout Roach Three-spined stickleback Brook trout Trout Carp Largemouth bass and mosquito fish Fish Fish Fish Cold freshwater fish Bluegill Phenol and p-chlorophenol... during a preexposure period Copyright © 2005 by Taylor & Francis Ostrander / Techniques in Aquatic Toxicology L16 64_ c032 Final Proof page 571 21.12.20 04 6:19am Intra- and interspecific interactions For hazard assessment and environmental regulation, it is important to show a causal linkage with the population in order to provide a predictive index of population-level effects Recently, behavioral toxicology. .. Ostrander / Techniques in Aquatic Toxicology L16 64_ c032 Final Proof page 566 21.12.20 04 6:19am Table 32.1 Continued Host species Stressor Rainbow trout Cd Zebra fish Bluegill Green sunfish Cd Cd, Cr, Zn Chlordane Rainbow trout Co Rainbow trout Copper sulfate, dalapon, acrolein, dimethylamine salt of 2 ,4 d, xylene Crude oil Cu Cu Cu Behavior/movement comments References Altered dominance, feeding and aggression... Ostrander / Techniques in Aquatic Toxicology L16 64_ c031 Final Proof page 5 54 16.12.20 04 7:27am sharply as the fish passed from a mild to a severe stress state with reduced oxygen tensions Partial pressure of CO2 in the blood fell as oxygen saturation decreased in the exposure tank, indicating that O2 availability is coordinately falling (Figure 31 .4) Blood ion concentrations likely shift in response... gained popularity as animal models in aquatic toxicology as recent advances have increased our knowledge of normal physiologic conditions and responses to various stressors.20 Fish models are also increasingly being used in research leading to information regarding human diseases and genetic and reproductive responses.21 In this chapter, we describe a laboratory system for examining the response of aquatic . model Any PVC pipe 1.5 in. rigid piping Any PVC tubing 1 in. flexible tubing Any Ostrander / Techniques in Aquatic Toxicology L16 64_ c031 Final Proof page 550 16.12.20 04 7:27am Copyright © 2005. 8-oxo-guanine and 8-oxo-adenine lesions induce marked changes in the backbone structure of a 25-base DNA strand, Proc. Natl. Acad. Sci. USA, 97, 1 244 2–1 244 5, 2000. 4. Malins, D.C., Polissar, N.L used on this system depending on the capacity and volume rating of the skimmer used. Ostrander / Techniques in Aquatic Toxicology L16 64_ c031 Final Proof page 549 16.12.20 04 7:27am Copyright © 2005