CONTENTS Introduction Materials and Methods Results Discussion Conclusions Acknowledgments References Cited Common & Scientific Names of Species Discussed Acronyms Errata
NOAA Technical Memorandum NMFS-NE-180
Variability in Blood Chemistry of Yellowtail Flounder, Limanda ferruginea, with Regard to Sex, Season, and Geographic LocationRenee Mercaldo-Allen1,2, Margaret A. Dawson1, Catherine A. Kuropat1,3, and Diane Kapareiko1,4
National Marine Fisheries Serv., Milford Lab., 212 Rogers Ave., Milford, CT 06460
Web version posted May 4, 2004Citation: Mercaldo-Allen R, Dawson MA, Kuropat CA, Kapareiko D. 2003. Variability in blood chemistry of yellow flounder, Limanda ferruginea, with regard to sex, season, and geographic location. NOAA Tech Memo NMFS NE 108; 20 p.
Information Quality Act Compliance: In accordance with section 515 of Public Law 106-554, the Northeast Fisheries Science Center completed both technical and policy reviews for this report. These predissemination reviews are on file at the NEFSC Editorial Office.
ABSTRACT: Yellowtail flounder, Limanda ferruginea, were collected from six locations along coastal New England (i.e., Cape Cod Bay, Georges Bank, Massachusetts Bay, mouth of the Merrimack River, Mud Patch (south of Marthas Vineyard), and New York Bight), and were monitored for blood chemistry and hematology. Plasma osmolality, sodium, potassium, calcium, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration (MCHC) were measured.
Osmolality and sodium concentrations were frequently reduced during spring. Potassium was generally elevated during spring and/or summer, and reduced during fall and/or winter. Calcium was usually highest during fall. The hematological indices of hematocrit, hemoglobin, and MCHC showed an overall elevation during winter as compared to fall. Fish from the inshore locations of Cape Cod Bay, Massachusetts Bay, Merrimack River, Mud Patch, and New York Bight differed significantly from fish from the offshore reference location of Georges Bank for particular blood parameters during certain seasons. These data provide a baseline range for blood constituents of yellowtail flounder over an annual cycle and at key locations in the Northeast U.S. Shelf Ecosystem. These results present evidence of possible anthropogenic effects on the blood chemistry and hematology of yellowtail flounder collected from inshore areas. Variability in blood chemistry appears to be regulated by seasonally-induced physiological and/or environmental factors, and influenced by sex and capture location. This information may prove to be useful in monitoring the health of field-collected yellowtail flounder and in assessing the condition of aquacultured fish of this species.
Yellowtail flounder, Limanda ferruginea, is a commercially important flatfish inhabiting Northwest Atlantic waters ranging from the Gulf of St. Lawrence to Chesapeake Bay (Bigelow and Schroeder 1953; Collette and Klein-MacPhee 2002). Evidence suggests that yellowtail flounder from Cape Cod Bay, Georges Bank, and the region encompassing Southern New England to the Mid-Atlantic Bight represent three distinct stocks (Cadrin 2003; Cadrin and King 2003). Yellowtail flounder are fast growing, have high market value (Johnson et al. 1999), and have been identified as a candidate species for aquaculture (Rabe and Brown 2000). Despite the historical importance of yellowtail flounder as a commercial resource, relatively little information is available concerning the blood chemistry of this species.
Blood chemistry and hematological measurements can provide valuable tools for monitoring the health and condition of both wild and cultured fish. Physiological indices can offer critical feedback on rearing conditions and nutritional status, and can aid in the diagnosis of disease. These monitoring and feedback applications require an understanding of normal blood component levels specific to yellowtail flounder, as blood chemistry concentrations are known to vary among fish species (Larsson et al. 1976; Hardig and Hoglund 1983; Folmar 1993). Natural changes in environmental conditions associated with season can affect blood chemistry and hematology (Bridges et al. 1976; Warner and Williams 1977; Lane 1979; Dawson 1990; Folmar 1993; Houston 1997; Luskova 1998; O'Neill et al. 1998; Edsall 1999). Physiological variation in fish may be influenced by both internal and external cues, including reproductive stage, water temperature, dissolved oxygen, nutrient availability, and photoperiod, all of which undergo annual cycles (Sandstrom 1989; Dawson 1990). Seasonal changes may also be influenced by nonenvironmental factors such as diet, metabolic adaptations, and activity levels (Denton and Yousef 1975).
Hematological measures, which can be affected by exposure to chemical pollutants (Heath 1995), are useful indicators of sublethal environmental stress in fish (Bridges et al. 1976; Warner and Williams 1977; Folmar 1993). Yellowtail flounder demonstrate strong site fidelity, are found both inshore and offshore (Royce et al. 1959; Lux 1964; Johnson et al. 1999; Cadrin 2003; Cadrin and King 2003), and live on or close to bottom sediments where contaminant loading occurs. Resident bottom-dwelling organisms, such as flatfish, often exhibit environmentally-induced disease (Ziskowski et al. 1987), making them potential indicator species for monitoring of anthropogenic effects. Before blood constituent data can be applied as a diagnostic tool, however, general patterns related to season, sex, and collection location need to be well documented and understood (Bridges et al. 1976; Courtois 1976; Hardig and Hoglund 1983; Folmar 1993).
The goal of this study was to determine baseline levels of blood parameters for yellowtail flounder. Blood chemistry and hematological measurements of osmolality, potassium, sodium, calcium, hematocrit, hemoglobin, and mean corpuscular hemoglobin (MCHC) were determined for fish collected from various locations in the Northeast U.S. Shelf Ecosystem. Blood measurements were compared with regard to fish sex, season, and collection location.
MATERIALS AND METHODS
Yellowtail flounder were collected from Northwest Atlantic waters between 1978 and 1985 aboard National Marine Fisheries Service, Northeast Fisheries Science Center (NEFSC) cruises. Nearly all fish were collected on cruises associated with the NEFSC's Ocean Pulse Program (OPP) and Northeast Monitoring Program (NEMP); a few fish were collected on cruises associated with the NEFSC's Bottom Trawl Survey Program. (The OPP and NEMP were the same fundamental program; the name changed from the former to the latter early in the effort, though.) The focus of NEMP was assessment of ecological, genetic, pathological, and physiological changes in coastal and shelf organisms of the Northeast U.S. Shelf Ecosystem, which had been potentially exposed to the effects of contaminant stress (Pearce 1998).
Yellowtail flounder were collected using a "¾ Yankee" (or "No. 36") otter trawl towed for a 30-min period (Survey Working Group, Northeast Fisheries Center 1988). Animals were obtained from five nearshore locations, including Cape Cod Bay, Massachusetts Bay, mouth of the Merrimack River, Mud Patch (south of Marthas Vineyard on the continental shelf), and New York Bight, as well as from an offshore location, Georges Bank (Figure 1). Table 1 shows the general coordinates (i.e., latitude and longitude) of the collection stations, as well as the number of adult yellowtail flounder collected at each station. For the Georges Bank, Mud Patch, and New York Bight locations, fish caught at adjacent stations within a given location were pooled to increase sample size for statistical analysis. Environmental conditions (e.g., depth, substrate, temperature) at these adjacent stations were fundamentally similar.
Fish were transferred to flowing seawater aboard ship and processed immediately to minimize stress-induced changes associated with capture (Larsson et al. 1976; Wedemeyer and Yasutake 1977; Dawson 1990). Changes in blood concentrations can occur within hours to days of collection (Umminger 1970; Wedemeyer and Yasutake 1977; Bourne 1986); therefore, it was critical that samples collected at sea be processed without delay. Blood was collected by cardiac puncture using a 3-ml plastic syringe with a 22-ga needle and transferred gently into an 8-ml glass vial coated with 150 units of dried ammonium heparin as an anticoagulant. Hemoglobin (g/100 ml) was determined by the cyanmethemoglobin method using Hycel reagents and a Bausch and Lomb Spectronic 20 spectrophotometer. (Note: Use of trade names does not imply endorsement by NMFS.) Hematocrit was determined by collecting blood in micro-hematocrit tubes, which were then centrifuged at 13,500 × g for 5 min and read. Hematrocrit was expressed as a percentage of the volume of the whole blood sample. The remainder of each blood sample was centrifuged at 12,000 × g, and the resulting plasma was frozen until further analyses could be conducted. The MCHC, the ratio between hemoglobin and hematocrit, was expressed as g/100 ml of packed red blood cells, and was calculated by the formula: MCHC = hemoglobin/hematocrit × 100. Plasma osmolality was determined using an Advanced 3L or 3C2 Cryomatic Osmometer and Advanced Instruments freezing-point calibration standards. Sodium, potassium, and calcium (mEq/L) were determined using a Perkin Elmer Coleman 51 Flame Photometer.
Sampling dates were assigned to a season according to calendar designations: winter (December 22 - March 19), spring (March 20 - June 20), summer (June 21 - September 22), and fall (September 23 - December 21). Some locations and seasons were sampled more frequently than others because of problems often associated with extensive field sampling at sea, such as equipment failure, inclement weather, and variation in fish availability. Occasionally, a sample was not obtained for a given season. Small sample sizes were included in a data set when information was available for other seasons at that location.
A Pearson product moment correlation was used to determine whether hydrographic characteristics such as temperature, salinity, and dissolved oxygen are associated with blood chemistry values. A multivariate analysis of variance was conducted to determine effects of sex, location, and season on osmolality, sodium, potassium, calcium, hemoglobin, hematocrit, and MCHC. These seven blood parameters served as the dependent variables. Independent variables for the model included sex (males and females), location (Cape Cod Bay, Georges Bank, Massachusetts Bay, Merrimack River, Mud Patch, and New York Bight), season (winter, spring, summer, and fall), sex × location, sex × season, and sex × location × season. Fish length was used as a covariate. Post-hoc tests consisted of multiple pairwise comparisons of least square means for significant effects (P<0.05). Statistical comparisons were limited to seasons and locations containing a sample size of N>5. The significant differences which have been reported, but based on small sample sizes, should be interpreted conservatively. All statistical analyses were performed using PC-SAS Software Version 8.3 (SAS Institute 1989).
Two of the three hydrographic parameters correlated highly with blood indices. Salinity correlated positively with MCHC (Table 2). Dissolved oxygen correlated positively with hemoglobin and MCHC, and negatively with osmolality and sodium. There were also relationships between certain blood parameters. Sodium correlated positively with osmolality, and negatively with MCHC. Hemoglobin correlated positively with hematocrit and MCHC. A significant difference was observed for sex × location × season for all seven blood parameters (Table 3). Following are results for differences related to sex, season, and location. Location-related differences are limited to a comparison of results from the five inshore locations versus those from Georges Bank, the offshore reference location.
SEX-RELATED DIFFERENCES IN BLOOD CHEMISTRY
Significant differences in blood parameters were observed between males and females within the same season at Georges Bank (8 instances), New York Bight (5), Mud Patch (5), and Merrimack River (2), and are shown underlined in Tables 4-10. These differences were observed most frequently during spring (8), but were also observed during summer (6), winter (4), and fall (2). Male and female blood chemistry concentrations differed significantly from one another for hemoglobin (6), osmolality (4), hematocrit (4), calcium (3), potassium (2), and MCHC (1).
Mud Patch males had significantly greater hematocrit during winter, and hematocrit, hemoglobin, and osmolality during spring, than females. Mud Patch females had significantly greater hemoglobin during summer than males. Merrimack River females had significantly greater calcium during spring and summer than males. New York Bight males had significantly greater hematocrit and osmolality during winter, hemoglobin and osmolality during spring, and potassium during fall, than females. Georges Bank males had significantly greater hemoglobin during winter, hematocrit and hemoglobin during spring, hemoglobin, potassium, and MCHC during summer, and osmolality during fall, than females; however, females had significantly greater calcium during summer than males.
SEASONAL DIFFERENCES IN BLOOD CHEMISTRY
Overall, osmolality showed few significant differences among seasons, although there was a minor trend toward reduced osmolality during spring, as observed in both males and females from Georges Bank (Table 4). Fall osmolality in Mud Patch fish was significantly greater than summer osmolality in males, and spring osmolality in females. Osmolality in New York Bight males was significantly elevated during winter and spring, and reduced during fall.
Generally, sodium concentrations were significantly lower during spring, as observed in males from Georges Bank, Merrimack River, Mud Patch, and New York Bight, and in females from Georges Bank and Mud Patch (Table 5).
Potassium concentrations were often significantly higher during spring or summer, and significantly lower during fall or winter (Table 6). Potassium showed significantly higher levels during spring in males from Merrimack River, and in females from Georges Bank and Mud Patch. Summer levels were significantly higher in fish of both sexes from Georges Bank and Mud Patch. Potassium was significantly lower during fall than spring and/or summer in males from Georges Bank, Merrimack River, and Mud Patch, and in females from Georges Bank. Winter potassium was significantly lower than summer for females from Georges Bank and Mud Patch.
Calcium concentrations were generally higher during fall, as observed in fish of both sexes from George Bank and Mud Patch, and in females from New York Bight (Table 7). A highly elevated calcium concentration during spring was documented in females from Merrimack River.
Yellowtail flounder demonstrated a trend toward significantly higher hematocrit during spring, as compared with fall (Table 8). Hematocrit was significantly greater during spring than fall in fish of both sexes from Merrimack River, Mud Patch, and New York Bight, and in males from Georges Bank. Winter hematocrit concentration was significantly greater than fall concentration in females from Georges Bank.
Hemoglobin showed a similar pattern to that observed for hematocrit, but the results were less consistent (Table 9). Hemoglobin tended toward significantly greater concentrations during winter and/or spring than fall. Hemoglobin in fish of both sexes from Georges Bank and Merrimack River was significantly higher during winter than fall, while spring levels were significantly higher than fall levels in males and females from Merrimack River, Mud Patch, and New York Bight, and in males from Georges Bank.
MCHC was generally higher during winter than spring, as observed at Georges Bank and Merrimack River for males, and at Merrimack River and New York Bight for females (Table 10). Winter also tended to have higher MCHC than fall, as seen at Georges Bank and Merrimack River for males, and at Merrimack River and Mud Patch for females.
LOCATION-RELATED DIFFERENCES IN BLOOD CHEMISTRY AS COMPARED TO GEORGES BANK
Blood parameters in fish from the inshore locations were compared to such parameters in fish from Georges Bank, the reference location. During winter, osmolality in female yellowtail flounder from Merrimack River was significantly lower. During spring, osmolality in both sexes from New York Bight and in males from Cape Cod Bay was significantly higher. During summer, osmolality in both sexes from Merrimack River and in males from Mud Patch was significantly lower. During fall, osmolality in males from Merrimack River, Mud Patch, and New York Bight was significantly lower (Table 4).
During winter, Merrimack River, Mud Patch, and New York Bight sodium concentrations in females were significantly reduced. During spring, sodium in females from Massachusetts Bay, males from Cape Cod Bay, and both sexes from New York Bight was significantly elevated. Fall sodium concentrations were reduced in both sexes from Merrimack River (Table 5).
Reduced potassium was observed during spring in males from Cape Cod Bay and in females from Massachusetts Bay and New York Bight. Merrimack River showed elevated potassium during spring in males. Significantly reduced potassium was measured in Merrimack River males during summer. Females from Merrimack River and Mud Patch and both sexes from New York Bight had elevated fall potassium (Table 6).
Females from Merrimack River and Massachusetts Bay had significantly higher calcium during spring. Summer calcium was reduced in female fish from Mud Patch. Calcium concentrations during fall were significantly lower in both sexes from Merrimack River and in males only from Mud Patch (Table 7).
During winter, hematocrit was significantly lower in males from Cape Cod Bay and in females from Mud Patch. During spring, hematocrit was significantly higher in both sexes from Merrimack River, and in females from New York Bight. During summer, hematocrit was significantly higher in females from New York Bight (Table 8).
During winter, hemoglobin in males from Cape Cod Bay and New York Bight, and in both sexes from Mud Patch, was significantly reduced. Merrimack River females had greater winter hemoglobin. During spring, hemoglobin was reduced in female fish from Massachusetts Bay and elevated in females from Merrimack River. Summer hemoglobin was reduced in Mud Patch males. Fall hemoglobin in New York Bight and Mud Patch males was significantly higher (Table 9).
Males from Cape Cod Bay and New York Bight had significantly lower winter MCHC, while in females from Merrimack River, MCHC was significantly greater. During summer, males from Merrimack River and females from New York Bight had significantly reduced MCHC. Male fish from Merrimack River, Mud Patch, and New York Bight had significantly higher MCHC during fall (Table 10).
SEASONAL AND SEX-RELATED DIFFERENCES IN BLOOD CHEMISTRY
Blood chemistry and hematological measurements are significantly affected by the wide range of natural environmental conditions experienced by fish over the course of a year (Bridges et al. 1976; Warner and Williams 1977; Lane 1979; Dawson 1990; Folmar 1993; Houston 1997; Luskova 1998; O'Neill et al. 1998; Edsall 1999). Changes in temperature (Powers 1980) and season (Shell 1961) are known to affect oxygen metabolism and hematology. Variations in blood constituents may be related to natural physiological cycles, environmental stimuli, or both (Bridges et al. 1976; Luskova 1998). Metabolic fluctuations in fish such as growth, activity, and feeding are strongly influenced by environmental cues (Barnhart 1969; Fletcher 1977). Reproductive cycles, associated with season, can place fish under extremes of metabolism and osmoregulation (Courtois 1976), and may alter the content of fish blood (Nagler et al. 1987; Sandstrom 1989; Bjornsson et al. 1998; Luskova 1998).
Yellowtail flounder experienced only minor seasonal fluctuations. In several cases, osmolality values were reduced during spring. Windowpane (Scophthalmus aquosus) also experience significantly lower osmolality during spring than summer and fall (Dawson 1990). In a laboratory study, however, no significant differences were observed in the serum osmolality of winter flounder (Pseudopleuronectes americanus) held at 1° and 15°C (Umminger 1970).
The significantly elevated osmolality observed during winter and spring in males from New York Bight in our study has some adaptive significance. Pearcy (1961) suggests that elevated osmotic pressure in flounder during cooler weather may protect against freezing in fishes inhabiting cold shallow waters. Serum osmolality was also highest in winter flounder during the winter months, just prior to spawning (Pearcy 1961; Umminger and Mahoney 1972). Osmolality in mummichog (Fundulus heteroclitus) was found to increase significantly at colder water temperatures (Umminger 1969).
Seasonal differences in osmotic pressure may be caused by osmotic imbalance or other factors (Pearcy 1961). An increase in serum constituents other than the major ions, such as a buildup of organic and inorganic materials, can elevate osmolality during cold weather (Pearcy 1961; Umminger 1969; Umminger and Mahoney 1972). An increase in serum electrolytes may be a compensatory mechanism used by fish to maintain the total number of osmotically active particles in the plasma (Shell 1961). In several instances, males showed significantly greater osmolality than females during winter and/or spring. Perhaps females are undergoing osmotic changes related to preparations for late spring spawning.
In our study, osmolality and sodium were highly positively correlated. This high positive correlation would be expected, as sodium is an important osmotic constituent. In our study, both of these parameters had similar patterns of reductions during spring. Also in our study, dissolved oxygen and osmolality were highly negatively correlated, which suggests that the reduced osmolality during spring may be associated with the elevated dissolved oxygen levels following spring turnover.
Marine fish exposed to low seawater temperatures often experience increased sodium (Umminger 1969; Murphy and Houston 1977; Sandstrom 1989). Increases in freezing point depression of winter flounder blood serum during the winter months can be attributed in part to elevated plasma sodium (Fletcher 1981). Winter flounder sampled in Newfoundland had significantly higher sodium during winter than summer (Fletcher 1977), while sodium in winter flounder from New England was also higher during late winter (Pearcy 1961; Fletcher 1977). Winter-collected windowpane had significantly higher sodium than summer and autumn fish (Dawson 1990). Interestingly, Umminger and Mahoney (1972) noted no seasonal change in sodium among winter flounder collected from New Jersey.
In measurements on freshwater fish, sodium levels in the Eurasian perch (Perca fluviatilis) reached their maximum during mid-winter (Sandstrom 1989), and in rainbow trout (Oncorhynchus mykiss) were higher during both fall and winter than during summer (Houston et al. 1968). In our study, dissolved oxygen concentrations were correlated negatively with both sodium and osmolality. This correlation suggests a possible disruption of osmoregulatory function at reduced oxygen levels.
The significantly reduced potassium levels during cold weather, which were observed in our study, have also been described in winter flounder, and may be related to a decline in metabolism and feeding at lower temperatures (Umminger and Mahoney 1972; Bentinck-Smith et al. 1987). Reduced blood potassium concentrations during winter may also be attributed to a loss of potassium from the cells of body tissues in response to the cold (Umminger 1969). Potassium in the windowpane was also found to be higher during summer than fall and winter (Dawson 1990).
Changes in blood potassium levels have also been observed in freshwater fish. Rainbow trout collected during fall-winter had reduced levels as compared to those fish collected during summer (Houston et al. 1968). Elevated potassium levels were also observed at high water temperatures in smallmouth bass (Micropterus dolomieu) (Shell 1961), rainbow trout (Murphy and Houston 1977), channel catfish (Ictalurus punctatus) (Ellsaesser and Clem 1987), Eurasian perch (Sandstrom 1989), and madai (Pagrus major) (Woo 1990).
Increased plasma potassium may result from disruption of potassium regulatory ability at either the external level (i.e., between the fish and seawater) or internal level (i.e., between the intracellular and extracellular fluid), with a resulting release of potassium into the blood. Serum potassium levels may increase in response to elevated tissue catabolism, or due to osmotic adjustment when compensating for a decline in other serum components (Shell 1961).
Calcium plays an important role in osmoregulation (Shell 1961), and is a critical component in the reproductive processes of fish. In our study, calcium concentrations were generally higher during fall and lower during spring. This trend toward calcium being elevated during fall and depressed during spring may reflect the spring and summer spawning pattern of yellowtail flounder, which peaks during late May (Bigelow and Schroeder 1953; Royce et al. 1959; Fahay 1983; Collette and Klein-MacPhee 2002; Cadrin and King 2003). Also in our study, a contrasting pattern was observed in females from Merrimack River; they showed significantly elevated calcium during spring which may be related to anthropogenic, rather than seasonal, influences.
In our study, female fish from Georges Bank and Merrimack River had significantly elevated spring and/or summer calcium levels as compared to males. These elevated calcium levels in females are most likely related to hormonal changes linked to spawning activity. In freshwater fish also, calcium concentrations in females during the spawning period were significantly higher than in males for lake trout (Salvelinus namaycush)(Edsall 1999), brook trout (Salvelinus fontinalis)(Booke 1964), and rock bass (Ambloplites rupestris)(Bidwell and Heath 1993).
Blood calcium has been shown to follow spawning patterns in numerous fish including brook trout (Booke 1964), rainbow trout (Nagler et al. 1987), English sole (Parophrys vetula)(Johnson et al. 1991), striped mullet (Mugil cephalus), pinfish (Lagodon rhomboides) (Folmar et al. 1992), rock bass (Bidwell and Heath 1993), and Atlantic halibut (Hippoglossus hippoglossus) (Bjornsson et al. 1998). Blood calcium in rainbow trout rises in connection with yolk synthesis and reaches maximum levels just prior to spawning (Hille 1982). High spring calcium levels observed in windowpane (Dawson 1990) appear to correspond with a late spring and summer spawning season (Bigelow and Schroder 1953). Calcium varied irregularly in the Eurasian perch, with fluctuations increasing sharply before and after spring spawning (Sandstrom 1989). Variations in blood calcium of fish appear to be tied more closely to reproductive changes than to seasonality or hematopoiesis (i.e., blood formation) (Luskova 1998).
Hematocrit, Hemoglobin, and MCHC
Hematocrit and Hemoglobin
Hematocrit provides a measurement of red blood cells (erythrocytes) in whole blood, while the hemoglobin within those erythrocytes is the main transport mechanism for oxygen and carbon dioxide in the blood. Alterations in blood oxygen capacity reflect seasonal adjustment in oxygen transport (Cameron 1970; Anderson et al. 1985). Elevations in spring hematocrit and hemoglobin are likely due, in part, to an increase in oxygen consumption and metabolic rates corresponding to a rise in water temperatures (Powers 1980; Dwyer et al. 1983; Zanuy and Carrillo 1985; Martinez et al. 1994). Spring hemoconcentration of the blood may also contribute to elevated hematological levels (Preston 1960).
In addition to these seasonal rhythms, sex also appears to play a role in hematocrit and hemoglobin levels. In our study, males sometimes showed higher hematocrit and hemoglobin levels than females during winter and spring. Lane (1979) also observed significantly higher hematocrit and hemoglobin levels in male versus female rainbow trout. These differences in hematology with regard to males and females may be related to differential oxygen demand by sex, which in turn may be related to reproductive activity. Elevations in hematocrit and hemoglobin of rock bass seemed to occur in relation to the onset of spawning (Bidwell and Heath 1993).
The fall reduction in yellowtail flounder hematocrit and hemoglobin levels observed in our study may reflect a corresponding hemodilution of the blood which results in decreased hematological parameters and/or a reduction in hemopoetic capacity (Preston 1960). Rhythms in hematocrit and hemoglobin concentration may also result from changes in plasma volume or erythrocyte volume (Sandstrom 1989), for example, an expansion of plasma volume reduces the density of circulating red blood cells, thereby decreasing hematocrit (Courtois 1976). Changes in metabolism and hormonal activity, triggered by cooler water temperatures and declining photoperiod during fall, may result in anemia (Lane 1979) and reduced erythropoetic production (Lane 1979; Zanuy and Carrillo 1985). Physiological stresses associated with fasting and spawning can trigger a decline in fish condition (Bridges et al. 1976; Sano 1960ab; Lane 1979; Zanuy and Carrillo 1985).
Seasonal hematological patterns vary among species. In plaice, Pleuronectes platessa, hematocrit was lowest during winter, increased through the spring into summer, and declined again into fall. Hemoglobin levels in that species were high at the end of winter, were lowest during spring, were again high during the summer, and had a further decline during late fall (Preston 1960). In windowpane, Dawson (1990) found significantly lower winter hematocrit. Winter flounder from Maine had the lowest levels of hematocrit and hemoglobin during winter and early spring (Bridges et al. 1976), while winter flounder from New Jersey had lower levels of hemoglobin during late winter (Umminger and Mahoney 1972). [ERRATUM (posted May 10, 2004): A more comprehensive analysis of Hudson-Raritan Estuary winter flounder hematological variables (Mahoney and McNulty 1992) showed hemoglobins and hematocrit values to be high from October to April and low from June through September; values in May were transitional.] Tun and Houston (1986) found increased hematocrit and hemoglobin in rainbow trout exposed to summer versus winter conditions. An overall trend toward increased hemoglobin was observed in pinfish and striped mullet held at high temperatures (Cameron 1970). Hematocrit and hemoglobin in striped bass (Morone saxatilis) were highest during fall and winter and lowest during summer (Lochmiller et al. 1989). Haider (1969) found hemoglobin in rainbow trout to be highest during winter and lowest during fall.
Changes in the hemoglobin content of the blood in response to the environment might come about either by a change in the number of erythrocytes or by a change in the hemoglobin concentration of the individual cells (Anthony 1961). MCHC, the amount of hemoglobin in a given number of red blood cells, was generally higher during winter than spring and fall. Since MCHC values are directly related to erythrocyte maturation, the relatively high proportion of newly proliferated young erythrocytes during late spring -- when fish are in a state of rapid growth -- is associated with relatively low MCHC values (Denton and Yousef 1975; Hardig and Hoglund 1983). MCHC corresponded with hematocrit and hemoglobin in striped bass, being highest during fall and winter and lowest during summer (Lochmiller et al. 1989). MCHC in the largescale blackfish (Girella punctata), increased during spring and decreased during fall (Kakuno and Koyama 1994). Dawson (1990) observed no seasonal or location differences in MCHC among windowpane collected from various locations in Long Island Sound. [ERRATUM (posted May 10, 2004): Mean MCHC in winter flounder from the Hudson-Raritan Estuary was generally highter during the period October through April as compared to May through September (Mahoney and McNulty 1992).]
As hemoglobin is a measure of the amount of oxygen carried in the blood, the strong positive correlation observed in our study between dissolved oxygen and two hematological indices, hemoglobin and MCHC, would be expected. Similarly, Kakuno and Koyama (1994) observed a strong positive correlation between hematological parameters and dissolved oxygen in teleost fishes. Also in our study, MCHC correlated positively with salinity and negatively with sodium concentration. These results suggest that hematological indices may be affected by seasonal changes in hydrographic conditions.
LOCATION-RELATED DIFFERENCES IN BLOOD CHEMISTRY AS COMPARED TO GEORGES BANK
One of the most important potential uses of blood chemistry data from a toxicological perspective is to assess environmental quality by comparing animals from different locations (i.e., reference versus contaminated locations) (Folmar 1993). Physiological biomonitoring, using hematological and metabolic indices to assess stress, could provide an alternative to traditional population-based approaches (Lochmiller et al. 1989). A knowledge of normal physiological values is necessary to evaluate the health of fish with respect to their physiological responses to a stimulus or stress which affects homeostasis (Luskova 1998). A variety of environmental pollutants, such as certain chlorinated hydrocarbons and heavy metals, have been shown to affect osmotic and ionic regulation in fish (Haux 1979; Heath 1995).
Differences in blood parameters between yellowtail flounder from inshore locations and those from Georges Bank, the offshore location, suggest a possible anthropogenic effect at several inshore locations. Blood constituents that are significantly elevated or reduced in inshore fish as compared to Georges Bank fish may indicate a physiologically stressed condition (Reid et al. 1987). By virtue of location, the Georges Bank location experiences minimal input from anthropogenic sources, whereas the inshore locations are known to contain contaminated sediments (Boehm 1983). In a summary of the NEMP, Reid et al. (1987) documented elevated trace metals in the sediments from New York Bight, Mud Patch, and Massachusetts Bay, as well at the mouths of coastal estuaries. New York Bight had the highest measurements of polychlorinated biphenyls, while the incidence of fin rot at that location was found to be significantly greater than at other Northwest Atlantic locations (Ziskowski et al. 1987).
Osmolality and Sodium
Significantly lower osmolality levels in Merrimack River, Mud Patch, and New York Bight male yellowtail flounder during fall, and significantly lower sodium levels in Merrimack River males and females during the same season, suggest reduced osmoregulatory capacity, and may indicate an anthropogenic effect, as chronic low osmolality levels are thought to result from exposure to contaminants (Wedemeyer and Yasutake 1977). Dawson (1990) documented significantly reduced osmolality in windowpane from a polluted harbor in Long Island Sound as compared to fish from other, less impacted areas. European flounder (Platichthys flesus) exposed to titanium dioxide industrial effluent experienced reductions in osmolality and blood sodium (Larsson et al. 1980). Reduced blood sodium was documented in rainbow trout exposed to chlorine (Zeitoun et al. 1977). Leakage of sodium and other ions from extracellular fluids, or decreased uptake of these ions through the gills, can contribute to impaired osmoregulatory ability (Pearcy 1961; Larsson et al. 1980).
The significantly elevated spring osmolality and blood sodium levels observed in New York Bight male and female yellowtail flounder may indicate a pollutant effect. Windowpane exposed to mercury in the laboratory showed elevated sodium concentrations (Dawson 1990). A marked increase in blood sodium was observed in seawater-acclimated rainbow trout exposed to bunker C oil, most likely resulting from direct interference of the oil with the sodium transport system of the gills (McKeown and March 1978).
Significantly elevated potassium levels were observed during fall in yellowtail flounder males and females from New York Bight, and in females from Merrimack River and Mud Patch. These elevated levels may result from impaired uptake of potassium ions through the gills, disruption of food absorption in the intestinal mucosa, or defective renal function and impaired reabsorption of potassium ions in the renal tubes (Larsson et al. 1981).
Elevated potassium is also known to result from chlorine exposure in rainbow trout (Zeitoun et al. 1977). European flounder exposed to sublethal cadmium concentrations showed a dose-dependent reduction in potassium concentration (Larsson et al. 1981).
Calcium was significantly elevated in Massachusetts Bay and Merrimack River females during spring, while levels were significantly reduced in females from Mud Patch during summer, and in fish of both sexes from Merrimack River and males from Mud Patch during fall. These values differed significantly from Georges Bank, perhaps symptomatic of impaired physiological processes. Larsson et al. (1981) speculates that a disturbance in calcium metabolism of fish may be caused by defective calcium absorption or impaired calcium reabsorption in the renal tubes, or a combination of these effects.
In laboratory exposures, windowpane exposed to mercury (Dawson 1990) and European flounder exposed to cadmium experienced reduced calcium concentration (Larsson et al. 1981).
Hematocrit, Hemoglobin, and MCHC
With only one exception, yellowtail flounder from Cape Cod Bay had the lowest levels of hematocrit, hemoglobin, and MCHC by sex during winter. These levels for Cape Cod Bay fish were also among the lowest levels over all seasons and locations in our study. Mud Patch females had the lowest level of winter hematocrit; they also had the next-to-lowest level of winter hemoglobin. Similarly, anemic summer flounder (Paralichthys dentatus), winter flounder, and windowpane were also collected from Cape Cod Bay and Mud Patch during NEMP cruises (Reid et al. 1987). Reduced hematocrit and/or hemoglobin can indicate physiological stresses such as anemia, dehydration, and hemodilution resulting from either gill damage (Wedemeyer and Yasutake 1977) or increased red blood cell breakdown in the spleen (Larsson et al. 1980). Reduced MCHC suggests disruption of the hemoglobin-forming process as opposed to the rate of blood cell production (Heath 1995). Reduced hematocrit, hemoglobin, and MCHC were documented in redear sunfish (Lepomis microlophus) collected next to a selenium discharge site; the selenium appeared to interfere with normal hemoglobin formation, evidenced by the disproportionate reduction in hemoglobin as compared to red blood cells (Sorensen and Bauer 1983). European flounder exposed to cadmium in the laboratory experienced significant reductions in hematocrit and hemoglobin with no associated change in MCHC. In this case, the cadmium-induced anemic response may be due to increased blood plasma volume, accelerated loss or destruction of erythrocytes, and/or decreased rate of erythrocyte production (Johansson-Sjobeck and Larsson 1978).
During spring, elevated hematocrit was noted in yellowtail flounder males and females from Merrimack River, and females from New York Bight; it likely represents a swelling of cells in response either to high blood levels of carbon dioxide or to low pH rather than an increase in the number of circulating erythrocytes (Heath 1995), as no significant difference was noted in MCHC. Hematocrit may increase in response to stress at a different rate than that of the hemoglobin contained in the erythrocytes (Wells et al. 1986).
During fall, males from Mud Patch and New York Bight showed elevated hemoglobin, possibly a response to anthropogenic factors. Abnormally elevated hematocrit or hemoglobin may be related to hemoconcentration, dehydration stress, or polycythemia (Wedemeyer and Yasutake 1977), or to osmoregulatory dysfunction (Heath 1995). Also during fall, significantly elevated MCHC was measured in males from Merrimack River, Mud Patch, and New York Bight, and may indicate a greater amount of hemoglobin per unit of red blood cells and possibly swollen erythrocytes. Windowpane from a polluted harbor in Long Island Sound had significantly higher hematocrit and hemoglobin than other less-impacted areas, suggesting increased hematopoiesis (Dawson 1990). Larsson et al. (1980) also saw increased hematocrit and hemoglobin in female European flounder exposed to titanium dioxide industrial effluent in the field, and noted that flounder may try to compensate for impaired oxygen uptake by a release of erythrocytes from the spleen. Increased hematocrit was observed in rainbow trout stressed by chlorine exposure (Zeitoun et al. 1977).
Seasonally-induced variability was observed in yellowtail flounder blood constituent levels. This variability suggests that blood chemistry values are best defined in terms of a physiological reference range (Luskova 1998).
Seasonal patterns were not always consistent among the study locations and may reflect natural variability among locations (Bidwell and Heath 1993). This variability may result from inherent differences in regional oceanographic conditions, such as temperature and salinity, but may also be influenced by handling stress, and/or stock-related variations or a combination of these factors (McCarthy et al. 1973; Hille 1982).
Our results present some evidence for possible anthropogenic effects on certain blood chemistry parameters of yellowtail flounder collected from inshore locations. Perhaps the most valuable aspect of this study, though, is that it presents a benchmark for changes due to environmental factors or anthropogenic influences over an annual cycle at key locations in the Northeast U.S. Shelf Ecosystem.
The authors thank Anthony Calabrese, Ronald Goldberg, and Frederick Thurberg for editorial assistance, Suellen Fromm for providing the NEMP hydrographic data, Christine Zetlin for designing Figure 1, and Jose Pereira and Dee Tucker for technical assistance.
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