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General Experimental Procedures injury list (Appendix A) were eliminated due to lack of occurrence (i.e., embolisms were removed). A few Details of experimental procedures are pre- other injuries were combined into one score because sented in Appendix G. In brief, two Chinook salmon they indicated the same injury (i.e., external sign of were transferred into the HICI-FT's acrylic chamber pericardial hemorrhage was combined with internal and allowed to swim freely in the chamber for a 20- scoring of pericardial hemorrhage). Examination of min acclimation period. After the 20 min, the fish the injury panel showed that not all injuries had the were scored to be negatively, neutrally, or positively same physiological significance for the health of the buoyant. Fish were allowed to enter the tube, and fish following exposure. then they were closed in by lowering and locking the The physiological cost or effects of trauma and top shaker onto the tube. Control fish were put barotrauma are poorly understood in fish, thus a through the same process as treatment fish but with- novel model was developed to qualitatively assess out the pile driving sound. When the exposure was barotrauma and was applied to this study and to a completed, fish were transferred to the barotrauma concurrent study considering the effects of explo- injury assessment. sive sound from underwater rock blasting (Carlson The fish were exposed to one of eleven pile driv- et al., 2011). The physiological significance of ing treatments (see Chapter 3 Results). These treat- each injury was determined using available literature ments varied in SELcum, SELss, and number of strikes, whether fisheries or mammalian-based (Husum which affected the duration of the exposure. For and Strada, 2002; Oyetunji et al., 2010) and pro- example, for fish exposed to 1,920 strikes, the expo- posed energetic costs based on an understanding of sure duration was 48 minutes, while for 960 strikes each type of injury (Woodley and Halvorsen, per- it was 24 minutes. sonal observations; Gaspin et al., 1975; Iwama et al., 1997). Barotrauma Analysis and Characterization Physiological significance of each observed injury was assessed and given a physiological rank Following exposure in the HICI-FT, fish were and a weight (Table 1). Observed injuries were examined to determine if physical injuries were assigned to trauma categories based on the physio- associated with sound exposure. Prior to examina- logical significance for each observed injury, indi- tion, fish were euthanized in a buffered MS-222 vidually. The injuries were then separated into three solution of 100 mg/L. The fish were examined for trauma categories: Mortal, Moderate, and Mild. external and internal signs of barotrauma. All poten- The Mortal trauma category included observed tial injuries, and a fuller treatment of this analysis, injuries that were severe enough to lead to death. are provided in Appendix A. The Moderate trauma category included observed The design for the assessment of barotrauma fol- injuries likely to adversely impact fish health, but lowed the procedures developed by co-investigator which, when considered individually, were likely Carlson's group at the Pacific Northwest National recoverable under ideal conditions (i.e., no addi- Laboratory (PNNL) for their study on rapid decom- tional stressors) without being Mortal (Casper et al., pression in salmonids (Stephenson et al., 2010). in prep.). Finally, Mild trauma category refers to That study created a framework that assessed baro- observed injuries that had minimal to no physiolog- trauma and the effects of fish physiological condi- ical cost to fish, which quickly recovered under ideal tions on barotrauma injuries, and then statistically conditions (Casper et al., in prep.). analyzed and modeled those barotrauma injury A mathematical weighting was applied to the observations. trauma categories to underscore the contribution of the observed injury to the response weighted index (RWI, see below). Mortal injuries (injuries catego- Response Variable Derivation rized under Mortal trauma) were assigned a weight The barotrauma data set was based on binary vari- of 5, Moderate injuries (injuries categorized under ables (0 or 1) that denoted the presence or absence of Moderate trauma) weighted as 3, and Mild injuries observed external and internal barotrauma injuries. (injuries categorized under Mild trauma) weighted After thorough review of the entire injury regime, as 1 (Krischer, 1979; Chawda et al., 2004). The many injury indices from the original barotrauma weight assignments to the trauma categories were 10

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Table 1 Observed barotrauma injuries by mathematical weight, category, injury, physiological rank, and brief biological significance statement. Trauma Physiol. Wt Category Injury Description Rank Biological Significance of Injury 5 Mortal Dead within 1 hr 1 Dead 5 Mortal Pericardial (heart) hemorrhage 2 Discrete organ, main body blood pump, bleed- ing from heart; decreased blood pressure 5 Mortal Hepatic (liver) hemorrhage 3 Discrete organ; bleeding from liver; decreased blood pressure 5 Mortal Renal (kidney) hemorrhage 4 Non-discrete spongy organ, held in place with membrane, bleeding; decreased blood pressure 5 Mortal Ruptured swim bladder 5 Lost ability to maintain buoyancy, sank to bottom; may affect hearing 3 Moderate Intestinal hemorrhage 6 Blood filling the abdominal cavity; decreasing blood pressure 3 Moderate Burst capillaries along body 7 Decreased ability to get blood to muscle; wall decreased blood pressure 3 Moderate Pericardial (heart) hematoma 8 Could decrease efficacy of heart 3 Moderate Intestinal hematoma 9 Major portal system, decreased amount of blood flow to the rest of body. 3 Moderate Renal (kidney) hematoma 10 Large amount of blood pooling in more severe cases 3 Moderate Body muscles hematoma 11 Could affect swimming ability 3 Moderate Swim bladder hematoma 12 Could affect ability to regulate buoyancy; could potentially affect hearing 3 Moderate Fat hematoma 13 Related to swim bladder, caused from swim bladder 3 Moderate Ovaries/testes hematoma 14 Potential short-term damage but potential long- term consequences for reproductive success 1 Mild Blood spots on vent 15 Dilated capillaries near skin, respiratory acidosis, stress with a predisposition, or severe damage 1 Mild Dorsal fin hematoma 16 Dilated capillaries near skin, respiratory acidosis, stress with a predisposition, or severe damage 1 Mild Caudal fin hematoma 17 Dilated capillaries near skin, respiratory acidosis, or stress with a predisposition, or severe damage 1 Mild Pelvic fin hematoma 18 Fin is near intestinal portal system 1 Mild Pectoral fin hematoma 19 Fin is near the heart portal system 1 Mild Anal fin hematoma 20 Dilated capillaries near skin, caused by respira- tory acidosis, stress with a predisposition, or severe damage 1 Mild Fully deflated swim bladder 21 Negatively buoyant, which could be beneficial (no ruptures) for less barotrauma, quick recovery by surface air gulp 1 Mild Partially deflated swim bladder 22 Negatively buoyant, which could be beneficial (no ruptures) for less barotrauma, quick recovery by surface air gulp 11