KS. Que 22ch. tug, Wa Cw lt TP 77-3 Sublethal Effects of Suspended Sediments on Estuarine Fish by J.M. O'Connor, D.A. Neumann, and J.A. Sherk, Jr. TECHNICAL PAPER NO. 77-3 FEBRUARY 1977 WHO? DOCUMENT } COLLECTION ? roved for public | Bie elas unlimited. Prepared for U.S. ARMY, CORPS OF ENGINEERS COASTAL ENGINEERING -, RESEARCH CENTER USO Kingman Building TY Fort Belvoir, Va. 22060 pad) 3 Reprint or republication of any of this material shall give appropriate credit to the U.S. Army Coastal Engineering Research Center. Limited free distribution within the United States of single copies of this publication has been made by this Center. Additional copies are available from: National Technical Information Service ATTN: Operations Division 5285 Port Royal Road Springfield, Virginia 22151 Contents of this report are not to be used for advertising, de names does not \e use of such UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered) READ INSTRUCTIONS REPORT DOCUMENTATION PAGE BRIREADINSTRUCTIONS UNS 1. REPORT NUMBER 2. GOVT ACCESSION NO.} 3. RECIPIENT’S CATALOG NUMBER TP 77-3 4. TITLE (and Subtitle) 5. TYPE OF REPORT & PERIOD COVERED SUBLETHAL EFFECTS OF SUSPENDED SEDIMENTS ON TechniiealuResert 7. AUTHOR(s) 8. CONTRACT OR GRANT NUMBER(s) J.M. O'Connor D.A. Neumann J.A. Sherk, Jr. DACW72-71-C-0003 9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT, PROJECT, TASK AREA & WORK UNIT NUMBERS Natural Resources Institute University of Maryland e Park, Maryland 20742 V04230 11. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE Department of the Army February 1977 Coastal Engineering Research Center (CERRE-CE) Kingman Building, Fort Belvoir, Virginia 22060 90 14. MONITORING AGENCY NAME & ADORESS(if different from Controlling Office) 15. SECURITY CLASS. (of this report) UNCLASSIFIED 15a. DECL ASSIFICATION/ DOWNGRADING SCHEDULE 16. DISTRIBUTION STATEMENT (of thie Report) Approved for public release, distribution unlimited. - DISTRIBUTION STATEMENT (of the abstract entered in Block 20, if different from Report) - SUPPLEMENTARY NOTES - KEY WORDS (Continue on reverse side if necessary and identify by block number) Estuarine fish Patuxent River, Maryland Mineral solids Sublethal effects Natural sediments Suspended solids . ABSTRACT (Continue on reverse side if necesaary and identify by block number) The objective of this study was to determine the effects, if any, of sublethal concentrations of suspended materials on the fish in estuarine systems. Experimental sediment suspensions reproduced the concentrations frequently found during flooding and at dredging sites and dredged-material disposal sites. The suspensions were of natural sediment, obtained from the Patuxent River estuary, Maryland, or commercially available fuller's earth. (Continued) DD en's, 1473 eprmion oF t Nov 65 1s OBSOLETE UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered) SC —_—_—__——————E SECURITY CLASSIFICATION OF THIS PAGE(When Data Entered) Fish were collected in the Patuxent River estuary and transported to the laboratory. The selected fish species inhabited ecologically different sections of the estuary; therefore, the overall reactions of each species were unique. Seven species of estuarine fish were exposed to fuller's earth and natural sediment suspensions for timed periods and hematological changes were noted. The effects of various concentrations of fuller's earth suspensions on white perch gill tissue were determined. Oxygen consumption rates of striped bass, white perch, and toadfish were measured in filtered Patuxent River water and compared to consumption rates in filtered river water suspensions of fuller's earth or Patuxent River sediment. Fish showed signs of stress in response to suspended sediments in most of the experiments. Results indicate that sublethal concentrations of suspended solids can affect estuarine fish. Additional experiments are discussed in Appendixes A to D. @ UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE(When Data Entered) PREFACE This report is published to provide coastal engineers with information on the sublethal effects of suspended sediments on estuarine organisms. The work reported is a part of a continuing program of research on the ecological effects of coastal engineering activities. The report presents the results of part of a 3-year laboratory study on the subject. The work was carried out under a contract originating in the Office, Chief of Engineers, which was monitored under the coastal ecology research program of the U.S. Army Coastal Engineering Research Center (CERC). The original contract report (CERC Contract No. DACW72-71-C-0003) was prepared by Dr. J.M. O'Connor, D.A. Neumann, and Dr. J.A. Sherk, Jr., while on the staff of the Natural Resources Institute, University of Maryland, College Park, Maryland. Special acknowledgment is given to A.M. Daley for her work, particularly as reported in Appendix C. A.K. Hurme and A.L. Meyer, CERC, technically reviewed, condensed, and revised that part of the original report pertaining to the sublethal effects of suspended solids on estuarine fish. Robert M. Yancey, Chief, Coastal Ecology Branch, was CERC contract monitor for the report, under the general supervision of R.P. Savage, Chief, Research Division. Comments on this publication are invited. Approved for publication in accordance with Public Law 166, 79th Congress, approved 31 July 1945, as supplemented by Public Law 172, 88th Congress, approved 7 November 1963. OHN H. COUSINS Colonel, Corps of Engineers Commander and Director II IV VI APPENDIX A CONTENTS CONVERSION FACTORS, U.S. CUSTOMARY TO METRIC (SI) INTRODUCTION TO SUBLETHAL EFFECTS OF SUSPENDED SOLIDS ON ESTUARINE FISH. SUBLETHAL EFFECTS OF SUSPENDED SOLIDS ON THE HEMATOLOGY OF ESTUARINE FISH . 1. Introduction. 2. Methods . : 3. Results and Interpretation. EFFECTS OF SUBLETHAL CONCENTRATIONS OF FULLER'S EARTH ON WHITE PERCH GILL TISSUE . 1. Introduction. 2. Methods . ¢ 3. Results and inaerameeal orl. EFFECTS OF SUBLETHAL CONCENTRATIONS OF FULLER'S EARTH ON CARBOHYDRATE METABOLISM IN THE HOGCHOKER. 1. Introduction. 2. Methods . . 3. Results and mecermerctarelion, EFFECTS OF SUSPENDED SOLIDS ON RESPIRATION OF ESTUARINE FISH. 1. imerodueriont 5 : 2. Materials and Methods 2 3. Results . 4. Discussion. SUMMARY AND CONCLUSIONS . LITERATURE CITED. LIVER GLYCOGEN CONCENTRATIONS IN FOUR ESTUARINE FISH AND LIVER GLYCOGEN DEPLETION RATES IN WHITE PERCH . HEMATOLOGICAL CORRELATIONS IN ESTUARINE FISH. PRELIMINARY OBSERVATION ON THROUGH-GUT TRANSPORT OF SUSPENDED SOLIDS BY ESTUARINE FISH. ANALYSIS OF SEDIMENTS . Page 59 62 67 80 10 11 12 13 14 Experimental hematocrit, perch. Experimental CONTENTS TABLES and control values of red blood cell count, micro- hemoglobin concentration, and osmolality of white and control values of red blood cell count and microhematocrit of hogchokers. Experimental killifish. Experimental mummichog. Experimental hematocrit, Experimental hematocrit, and control microhematocrit values of striped and control microhematocrit values of and control values of red blood cell count, micro- and hemoglobin concentration of spot . and control values of red blood cell count, micro- hemoglobin concentration, and osmolality of striped bass . Experimental and control values of microhematocrit and osmolality of striped bass. Experimental hematocrit, toadfish . Experimental hematocrit, spot . Experimental hogchokers . and control values of red blood cell count, micro- hemoglobin concentration, and osmolality of and control values of red blood cell count, micro- hemoglobin concentration, and osmolality of and control liver glycogen concentrations of Covariance analysis of oxygen consumption and live weight regressions for striped bass . Covariance analysis of oxygen consumption and live weight regressions of male and female striped bass. Covariance analysis of oxygen consumption and live weight regressions of striped bass. Covariance analysis of oxygen consumption and live weight regressions of striped bass. Page 11 13 13 14 14 LS 15 7 18 28 33 34 37 40 NS 16 CONTENTS TABLES-Continued Covariance analysis of oxygen consumption regressions of striped bass. Covariance analysis of oxygen consumption regressions of white perch . Covariance analysis of oxygen consumption TSK OSSUONS Ose WNGWES jXSKEN og 6!5 6 0 6 Covariance analysis of oxygen consumption regressions of male and female toadfish. Covariance analysis of oxygen consumption regressions of toadfish. FIGURES Gill section from white perch held 5 days Gill section from white perch held 5 days Gill section from white perch exposed for acWULILEIENS CEECEN 5 5616 06, 6 Gill section from white perch held 5 days and iin lean weight weight e ° ° ° ° weight weight weight water. in clean water. 5 cays 0 0.65 @ it~" in clean water. Gill section from white perch exposed for 5 days to 0.65 g 17! fuller's earth . Secondary lamellae from white perch held for 5 fuller's earth . Oxygen consumption of striped bass swimming at Oxygen consumption of striped bass swimming at Oxygen consumption of striped bass swimming at Oxygen consumption of striped bass swimming at Oxygen consumption of striped bass swimming at days 0.28 1.02 Le 5S 1.05 Ike Sx) i O65 @ 17" FiG// Sic sre So PeE/ So fite/iSi- fatayaSte Oxygen consumption of white perch swimming at 0.28 ft/s . Oxygen consumption of white perch swimming at 1.02 ft/s . Oxygen consumption of white perch swimming at 0.39 ft/s . Oxygen consumption of white perch swimming at 1.02 ft/s... . Page 42 44 50 50 51 20 ZA 23 24 25 26 35 36 38 41 43 45 46 47 49 CONVERSION FACTORS, U.S. CUSTOMARY TO METRIC (SI) UNITS OF MEASUREMENT U.S. customary units of measurement used in this report can be converted to metric (SI) units as follows: Multiply by To obtain inches 25.4 millimeters — 2.94 centimeters square inches 6.452 square centimeters cubic inches 16.39 cubic centimeters feet 30.48 centimeters 0.3048 meters square feet 0.0929 square meters cubic feet 0.0283 cubic meters yards 0.9144. meters square yards 0.836 square meters cubic yards 0.7646 cubic meters miles 1.6093 kilometers square miles 259.0 hectares knots 1.8532 kilometers per hour acres 0.4047 hectares foot-pounds 1.3558 newton meters miullibars 1.0197 X 10-7 kilograms per square centimeter ounces 28.35 grams pounds 453.6 grams 0.4536 kilograms ton, long 1.0160 metric tons ton, short 0.9072 metric tons degrees (angle) 0.1745 radians Fahrenheit degrees 3/9 Celsius degrees or Kelvins’ ‘To obtain Celsius (C) temperature readings from Fahrenheit (F) readings, use formula: C = (5/9) (F — 32). To obtain Kelvin (K) readings, use formula: K = (5/9) (F — 32) + 273.15. i batit i 1 nl) SUBLETHAL EFFECTS OF SUSPENDED SEDIMENTS ON ESTUARINE FISH by J.M. O'Connor, D.A. Neumann, and J.A. Sherk, Jr. I. INTRODUCTION TO SUBLETHAL EFFECTS OF SUSPENDED SOLIDS ON ESTUARINE FISH The lethal effects of a variety of solids are documented for numerous freshwater fish (Ellis, 1936, 1937; Wallen, 1951; Wilson, 1956; Cordone and Kelley, 1961; Herbert, et al., 1961; Herbert and Merkens, 1961) and for some estuarine species (Rogers, 1969; Sherk and O'Connor, 1971; O'Connor, Neumann, and Sherk, 1976). However, the sublethal effects are only dealt with in histological studies of fish gill tissues (Southgate, 1962; Herbert, et al., 1961; Herbert and Merkens, 1961; Ritchie, 1970). The physiological impact of sublethal concentrations has not been studied previously. This part of a 3-year laboratory study (Sherk, O'Connor, and Neumann, 1976; O'Connor, Neumann, and Sherk, 1976) presents the results of histological and physiological studies of the sublethal effects of suspended solids on estuarine fish. Seven estuarine fish species (white perch, Morone americana; striped bass, Morone saxatilts; hogchoker, Trinectes maculatus; spot, Letostomus xanthurus; mummichog, Fundulus heteroclitus; striped killifish, Fundulus majalts; and oyster toadfish, Opsanus tau) were placed in fuller's earth and natural sediment suspensions, and hematological changes were noted during timed exposures. The effects of fuller's earth suspensions on gill tissue in white perch and on carbohydrate metabolism in hogchoker were determined at various concentrations. Oxygen consumption rates of striped bass, white perch, and toadfish were measured in filtered water from the Patuxent River, Maryland, and compared to consumption rates in filtered river water suspensions of fuller's earth or Patuxent River sediment. A knowledge of the sublethal effects of suspended materials is impor- tant in evaluating the effects of dredging or of disposal of dredged materials. This report provides base-line data which can be combined with knowledge of local conditions in preproject consideration of the effects of dredging activities. II. SUBLETHAL EFFECTS OF SUSPENDED SOLIDS ON THE HEMATOLOGY OF ESTUARINE FISH 1. Introduction. This section presents an assessment of the effects of suspensions of fuller's earth and natural sediments on several basic hematoiogical param- eters in fish: Microhematocrit (packed red blood cell volume), red blood cell count, hemoglobin concentration, and osmolality (ionic concentration of the blood). 2. Methods. Hematological studies of the seven fish species were conducted in both an experimental tank and a control tank. Each species was exposed to a concentration of fuller's earth or natural Patuxent River sediment which had caused less than 10-percent mortality, and was no greater than the previously determined 24-hour lethal concentration for 10-percent mor- tality (LCj9) for each species (O'Connor, Neumann, and Sherk, 1976). A quantity of fuller's earth sufficient to maintain the desired concentra- tion was placed in the experimental tank and mixed by submersible pumps for 24 hours before an experiment. The control tank did not contain fuller's earth. Mineral solids were maintained in suspension throughout the experiment in the two tanks by continuous pumping and aeration. Twelve or 15 fish were placed in each of the tanks during a test. Blood samples were taken from at least 10 individuals selected at random from the tanks after the exposure period. The samples were obtained from white perch and striped bass by severing the second branchial artery on the right side (McErlean and Brinkley, 1971), and from hogchokers, spot, and killifish by severing the caudal peduncle with a heparinized blade.: Blood was collected in heparinized pipets and, when possible, was mixed before samples were removed for analysis. Microhematocrit was determined according to methods outlined by Hesser (1960). Hemoglobin concentration was estimated by the cyanmethemoglobin method with modifications as suggested by Larsen and Snieszko (1961). Red blood cells were counted at X 100 on an improved Neubauer hemacytometer, using a modified Hayme's solution as the dilution medium (Heinle and Morgan, 1972). Whole blood osmolality was measured with a freezing-point depression osmometer. 3. Results and Interpretation. Hematological characteristics of white perch, hogchokers, and striped killifish changed in response to sublethal concentrations of suspended solids. The effects of these sublethal concentrations were analyzed extensively for white perch (Table 1). Exposure of white perch to 0.65 gram per liter (g 17!) fuller's earth for 5 days resulted in Significant increases in microhematocrit, hemoglobin concentration, and red blood cell count. The ionic concentration of the blood, estimated by whole blood osmolality, did not change. There was a relatively greater increase in red blood cell counts than in microhematocrits and hemoglobin concentrations. The increase in red blood cell count for experimental groups was 30 percent greater than the increase for control groups. Hemoglobin concentrations increased by 15 percent; microhematocrit values exceeded those of control fish by 17 percent. “Aytttqeqord = d, ‘uotzetndod oy. FO 1eY WOLF uvoW pojeUTIsS oY} FO UOTISTASP 9Y. ‘,,2 S,IUEPNIS, = I¢ “STENPLATPUL JO LoquNN, “uOTIETASP PLepueyS + poessozdxe ote SONTBA uBON, (OT) If‘ OTF 10° LZ (OT) L8°8F 19° 182 (,-34 wsow) (;-8 001 3) (euntoa [1e9 peyoed y9d) | (,_ww JOT xX STT99) uoTIeLIUIIUOD 2UnNOD AYTTeTouso UT GOT SOUS 1 TIIOLCWSYOIITW STI99 Ppootq poy Tor4Uo) [equoutsodxg , Upree S$, LeTINF pT 8 S9°Q 03 SAdep S 10} pesodxe yoroed a3tymM Jo AATTeTOWSO pUe ‘UOTeI]USDU0D UTGOTSOWeY ‘ZTLIOJeWSYyOIOTW ‘UNOS [199 pooTq pet FO SeNTeA [oOI}UOD pue TeJUOUTIedxg ‘“{T OTqGe] Hogchokers exposed for 5 days to 1.24 g 17! of fuller's earth increased red blood cell counts from 1.58 to 2.08 cells X 10® mm™3 (millions of cells per cubic millimeter) and increased microhematocrit from 15.62 to 19.93 percent (Table 2). The red cell count increase for hogchokers was proportionately the same as the increase in microhematocrit (30.4 and 27.6 percent, respectively); for white perch the proportional increase in red cells was much greater than the increase in microhematocrit and hemoglobin concentration. Striped killifish were exposed to 0.96 g 17! fuller's earth for 5 days (Table 3). Their microhematocrit value rose from 24.99 to 32.29 percent (probability” (p))<"0201))) a zelative increase of (29-7 percent tor athe experimental group over the control group. Experiments with white perch, striped killifish, and hogchokers demon- strated that significant hematological changes occur after exposure to sublethal concentrations of fuller's earth. Although these species show similar responses to sublethal concentrations of suspended solids, they differ markedly in response to lethal concentrations of the same material (O'Connor, Neumann, and Sherk, 1976). The hogchoker and the striped killifish were difficult to kill. An LC-response curve could not be gen- erated for the hogchoker, which may be due to hogchokers' high tolerance for suspended solids. The killifish showed a high 24-hour LCs59 of 38.18 g 17! fuller's earth, about the same as the mummichog value of 39 g lea fuller's earth. However, white perch were classified as a sensitive species because their 24-hour LCs5 9 values were below 10 g AE sap iexe! earth (O'Connor, Neumann, and Sherk, 1976). Low concentrations of sus- pended solids may induce sublethal effects, such as hematological altera- tion, even in relatively tolerant species. The highly sediment-tolerant hogchoker showed a significant increase in energy utilization during a 5-day exposure to 1.24 g 17! fuller's earth (see Section IV). Sublethal hematological effects of 1.6 g 17! fuller's earth suspensions were determined for the common mummichog at 4-, 7-, and 12-day intervals (Table 4). The mean microhematocrit values of experimental fish were sig- nificantly different from those of control fish at each interval. There was an increase in the mean value of the experimental group at 12 days. Spot were studied after a 5-day exposure to 1.27 g 1-1 fuller's earth, a concentration below the 24-hour LCj9 value of 13 g 171 (O'Connor, Neumann, and Sherk, 1976). There were no significant differences between the hematological values from experimental and control groups (Table 5). The data for striped bass were not directly comparable to data for other species because the bass were exposed for 11 and 14 days (Tables 6 and 7). After 11 days' exposure to 0.60 g 17! fuller's earth, there were no detectable differences in red blood cell count, microhematocrit, hemo- globin concentration, or osmolality of experimental and control groups. Striped bass exposed to 1.5 g 17! fuller's earth for 14 days showed an increase in microhematocrit (p < 0.01) over control fish. However, these Table 2. Experimental and control values of red blood cell count and microhematocrit of hogchokers exposed for 5 days to 1.24 g 17! fuller's earth. Group Individuals |Red blood cell count Microhematocrit (No. od) (cells x 10° mn= 3) (pct packed cell volume) jexpenimental 2. 082 19.933 +0.35 £4.32 Control 1.58 15.62 +0.26 +4.06 Mean TAS are expressed + eee Revacions re < 0.01 (t = 3.480, degrees of freedom (d.f.) = 18). 3p < 0.05 (t = 2.299, d.f. = 18), Table 3. Experimental and control microhematocrit values of striped killifish exposed for 5 days to 0.96 g 1! fuller's earth.! Individuals Microhematocrit (No. ) (pet packed cell volume) Experimental jMean values are expressed + standard deviation. “5 SOON: Table 4. Experimental and control microhematocrit values of mummichog exposed for 4, 7, and 12 days to 1.62 g 171 fuller's earth.! Experimental 33.084 20R522 BA iar +5.74 +443 +4 28 Control 24.14 +6.54 IMean values are expressed + standard deviation. 9) << OoOl (2 S S.2408, Glozty S 8). Sn) < O,02 (eS QE87S)° Gite Sr WSs ) < O00 Ges ALSRBA, dye, S 16). iT] I Table 5. Experimental and control values of red blood cell count, microhematocrit, and hemoglobin concentration of spot exposed for 5 days to 1.27 g 17! fuller's earth.! Red blood cell count Microhematocrit Hemoglobin concentration (cells X 10 mm-3) (pet packed cell volume) (g 100 g7}) ‘Experimental 1.542 | J zen18. a tee oe Control 1.46 =) eegn 6.69) orn a aaa aed a lMean values are expressed + standard deviation. 2p > 0.50. 3p > 0.10. Table 6. Experimental and control values of red blood cell count, microhematocrit, hemoglobin concentration, and osmolality of striped bass exposed for 11 days to 0.6 g 17! fuller's earth.! Red blood cell count Microhematocrit Hemoglobin Osmolality concentration (cells X 10© mm73),. (pet packed cell volume) a+ ss — Experimental Control IMean values are expressed + standard deviation. 2Number of individuals. Table 7. Experimental and control values of microhematocrit and osmolality of striped bass exposed for 14 days to 1.5 g 17! fuller's earth.! Experimental Microhematocrit Osmolality (pet packed cell volume) (m¢@sm kg!) Control IMean values are expressed + standard deviation. 2p) HOH p 6 Oe 3 < 0,05. “Number of individuals. fish also showed a significant increase in osmolality during the same time period. The increased microhematocrit may reflect a concentration of blood components due to loss of body water (Hall, Grdy, and Lepkovsky, 1926; Forster and Berglund, 1956). Toadfish held in 14.6 g 17! of suspended natural sediment for 72 hours exhibited no significant differences from a control group in hematological values (Table 8). Mean hemoglobin concentrations for control and experi- mental fish were 3.67 and 3.73 g 100 g7!, respectively. Red blood cell count and mean microhematocrit for experimental fish were 19.90 X 10° mm and 21.67 percent, respectively. Values for control fish were 17.78 X 10© mm? and 20.10 percent, respectively. Osmolality was 246.63 milliosmoles per kilogram (mOsm kg~!) for experimental fish and 251.69 mdsm kg~! for control fish. The hematological parameters were measured in spot exposed to 14.68 to 16.96 g 17! resuspended natural sediment over 7 days at 1-, 3-, and 7-day intervals. No significant changes in hematology were observed (Table 9). A time-dependent study was conducted on white perch exposed to 2 g 1o¥ resuspended natural muds for 4-, 6-, and 14-day intervals. The mean values of red blood cell count, microhematocrit, hemoglobin concentration, and osmolality for experimental fish were greater than for control fish after 4 days of exposure, but the differences were not statistically significant (0.07 > p > 0.05). Red blood cell count, microhematocrit, and hemoglobin concentration of experimental fish increased after 6 days (0.05 > p > 0.01). Blood osmolality did not change (p > 0.5). Red blood cell count, microhematocrit, hemoglobin concentration, and osmolality (0.5 > p > Q.1) of the two groups were again similar after 14 days of exposure. Replicate experiments assessed the sublethal effects of resuspended natural muds on striped bass. Studies were conducted at an arbitrary con- centration because LC}g, LCs5g, and LCgg responses for this species were not consistent. Hematological analysis revealed that exposure of striped bass to concentrations of 1.5 to 6 g 17! of natural muds for 6 days caused no detectable differences between experimental and control groups. In Sees! bass, a comparison of the effect of concentrations of 1.5 to 6 g 17! and 6 to 8 g 17! natural mud suggested that a threshold level may exist between 6 and 8 g 17!. Below 6 g 17! survival is essentially 100 percent; no sublethal hematological effects occurred over a period of 6 days at 2 to 6 g 17!. Above 6 g 17!, bass suffer mortality during 6 days of exposure. Fish exposed to sublethal concentrations of suspended solids showed the same basic hematological responses as fish deprived of sufficient oxygen--increased red blood cell count, increased hematocrit, and increased hemoglobin concentration in peripheral blood. The hematological responses to sublethal concentrations of suspended solids seen in white perch, hogchokers, and striped killifish were similar to responses observed in “JUBITFTUSTS ION, “STENPTATPUT FO Toquinn, “UOTIETAOP plepueys F posserIdxe oie sonTeA UBoW{ “S°N 81 Zvyt"o (6) GS” Wt 69° 1SZ [o1}uU0) (6) 99° STF £9°9b7 Tequowtszedxg (,-34 wsou) (,-3 001 3) (eunTOA TTe9 peysed 39d) (-_wu 401 X STT29) uoT}eL}UEDU0D AYTTe Lous uTGOTSOUSH ATILIOJeWSYOLITp junod [199 pootq pey , }USUTpes Aienjse JeAtYy JUexnjzeq popuedsnser ,_1 3 9°pT 09 sinoy ZZ 103 pesodxe ystypeo} Fo AATTeTOUSO puke ‘UOTIJeIZUSDUOD UTqGOTSOWEY ‘1TLOOJEWAYOLTOTU ‘UNOS [TED POOTq pet FO son,TeA J[OTJUOD pue [eqUeWTIedxy °*g 9TqQeL 17 Table 9. Experimental and control values of ‘red blood cell count, microhematocrit, hemoglobin concentration, and osmolality of spot exposed for 1-, 3-, and 7-day intervals to a range of 14.68 to 16.96 g 1” 1 resuspended natural sediment. } Exposure Red blood cell count Microhematocrit Hemoglobin Osmolality concentration (cells X 10° mm73) (pet packed cell volume) (g 100 g~4) (mOsm kg7}) One_day Experimental Control Three days Experimental Control t = 1.3866 t = 0.63 p> 0.1 p> 0.5 Seven days Experimental ° . 8.00 +0.97 (10) Control ° 8.31 +1.07 (10) t = 0.688 p>o.s lMean values are expressed + standard deviation. 2Number of individuals. goldfish and trout exposed to extremely low concentrations of dissolved oxygen for periods of 4 to 25 days (Phyllips, 1947; Prosser, et al., 1957; Ostroumova, 1964). If sublethal concentrations of suspended solids reduce the oxygen available at the gill, then it must be determined if suspended solids can affect gas transport across the respiratory epithelium, inducing a de facto hypoxia. Section III presents histological evidence that, in white perch, the primary site of respiratory Bes exchange, the secondary lamellae, was damaged by exposure to 0.65 g 1” fuller's earth. It appears that exposure to sublethal concentrations of fuller's earth can reduce a fish's ability to obtain oxygen by disrupting the gill surface and rendering the tissue partially dysfunctional. III. EFFECTS OF SUBLETHAL CONCENTRATIONS OF FULLER'S EARTH ON WHITE PERCH GILL TISSUE tay initaoductalone The gills are the primary site of respiratory gas exchange in most fish. The fish's blood is brought in close contact with the surrounding water at the gill surface. A membrane composed of two layers of cells separates the blood from the water; the gas exchange occurs through this membrane. Oxygen is absorbed from the water by the hemoglobin of the red blood cells, while carbon dioxide and other excretory products, such as ammonia, are released into the water. This system provides little barrier to gas transfer, but leaves the gill vulnerable to toxic or abrasive materials. This section presents the results of a histological study of gill tissue in white perch exposed for 5 days to fuller's earth suspensions. The study was designed to determine the damaging effects, if any, of sus- pended mineral solids on the gills of white perch. 2. Methods. White perch were exposed for 5 days to concentrations of 0.65 g ies fuller's earth. After exposure the fish were removed from the experimental and control tanks and killed. The first gill arch on the right side was removed from each fish and fixed in Bouin's solution. The tissue was embedded in paraffin, and 6-micrometer-thick serial sections were cut. The sectioning plane was dorsoventral, moving serially from the distal to the proximal end of the gill filaments. This made the mucus goblet cells located on the margins of the gill filaments visible; individual secondary lamellae were also clearly visible. Slides containing six to eight serial sections were stained alternately with iron-hematoxylin and Gomori's tri- chrome technique. 3. Results and Interpretation. Gill sections from control fish showed the typical structure for teleost fish (Figs. 1 and 2). Control fish had moderate concentrations of mucus goblet cells, particularly on the anterior margin of each gill filament (Fig. 2). There were concentrations of one to several mucus cells in each “OSc X 7B Use 030Ud -(Az042e [113 = e838 ‘S[[9e9 pooTq pet = Jo ‘s{jeo ae{td = od ‘umttoeyaytde = de) eanzonzys [Teo seytd ayz 03 ATIYy3TI pottdde st ‘untzTeyytde pue ‘peqinqstpun st 94njonizs Ie, Towe, ALepuoses "Io1eM UvdTO UT SABP gS PToY Yyorod 92TYM WOLF UOTIDES TITD 20 °00r X 38 USYe. OJO0YUg ° (AT [IIS Fo o8e[IIeD = AIeD ‘fl0jIJe [[TS = e8 *ST{[9D YeTGos snonw = Bu) ST[99 IeTqGo3 snonu 9}OU }USWeTTF oy. FO UTSIeW IOTI9}Ue BY. Sapn,ToUT UOT IES *ro}eM UBsTS UT Skep g¢ pley Yyosed 93TYyM WOLF UOTIDES TITD °Z oan3sty 2l serial section, although a single cell rarely occurred in more than one section. This concentration varied little over the length of a given filament. Individual mucus cells appeared to be less than 6 micrometers in diameter. Many mucus goblet cells appeared on the gills of white perch exposed to fuller's earth concentrations (Fig. 3). In some cases mucus cells were the only visible cellular component of the tissue at the anterior margin of the filaments. The mucus cells were confined to the margins of the filaments, particularly to the anterior margin which is the first to come in contact with the water. Lattle; at any; imcrease: in) mucus) cell icon- centration was observed elsewhere in the gill. Examination of serial sections revealed no increase in the size of individual mucus cells. High mucus cell concentrations made identification of individual cells difficult. The secondary lamellae on the gill sections of white perch consisted of a supportive tube of pilar cells with red blood cells present inside the tube. A single,thin layer of epithelium covered the lamellae (Fig. 4). The integrated structure of the secondary lamellae provides for maximum respiratory gas exchange efficiency by maintaining a minimum distance between the hemoglobin-rich red cells and the oxygen-rich water. The secondary lamellae of white perch exposed to 0.65 g 17! fuller's earth were swollen. The epithelium was separated from the pilar cell tube and the epithelial cells were enlarged, forming a thick covering (compare Figs. 4 and 6). Pilar cell structure usually remained intact (Fig. 5), although it was occasionally disrupted (Fig. 6). The effects of fuller's earth suspensions on gill tissues of white perch were similar to the effects of diatomaceous earth on rainbow trout gills (Southgate, 1962) and the effects of china-clay mining waste on brown trout gills at high concentrations (Slanina, 1964) and low concen- trations (Herbert, et al., 1961; Herbert and Merkens, 1961). The gills of fish exposed to suspended solids showed separation of the epithelium from the lamellar structure, thickening of the epithelium, and occasional disruption of the pilar cell structure of the lamellae (Herbert and Merkens, 1961; Herbert, et al., 1961; Southgate, 1962; Slanina, 1964). These effects were induced using concentrations of suspended solids between 0.40 and 0.81 g 17!, with a high percentage of particles in the silt-clay range. The effect of particle size on gill tissue has not been fully evaluated. However, based on available data, a definite concentration effect is associated with silt-clay-sized particles. Concentrations of fuller's earth below the 24-hour LC, 9 value may adversely affect the gill tissue structure of the white perch in a 5-day period. Gill damage caused by suspended solids has not been positively iden- tified as harmful to fish in terms of overall survival. Ritchie (1970) pointed out that the type of gill damage caused by particles in suspension effectively reduces the respiratory surface area. He stated that a reduced 22 "091 X 2B UdyeI 004d ° (eT [owe], ATepuodas &@ [S “ST[eo JeT[qGos snonl = BW “Aer [[13s FO OdeTLRIeD = FAed ‘Xki9jLe [TBs = e3) JUOWeT[TF TITS oy. FO ULsLeW TOTIO,Ue SY UO ST[99 191TG03 snonu Fo uoTJeLeFTTOId paytew smoyS “YyyIeS S, JOT [NF j-1 3 S9°0 09 skep s Loz posodxe yozed 931YyM WOLF UOTIIES [ITO 23 smoys "O0r X Fe uoyei oJ0Yq * (UMTToYITde doa ‘Az90,1e 0} uoTJ.OUUOD ATeT{[Tded = deo ‘sT[9o settd = dd ‘s[T[e9 poo[Tq ped = 10) oAnjonI3s JeTTowe, ATepuodes Tedtddy ‘198M UvOTD UT Shep Gg PTOYy Yyorted 94TYM WOTF UOTIIES [TTD *p omn3sty 24 JO0v Xue uayed o0yg * (AI T[T3 Jo oBeTTWed = 41ed ‘aqnz [Teo Ie{td = od ‘umt~Teyjztde = do) pesny oq 03 seodde se, [oue, Fo oseq oy. 3e seory ‘umtroyjytde uetTToms pue s{Teo ate,{td usemzeq adeds ZutAeeT ‘peqeredes sey oeT[owe, AZepuodes Fo umtpoy;tdyg “yee s,LOT [NF ;-1 3 $9°0 03 skep ¢ tof pasodxe yored 93TYM WOLF UOTIDES [ITN °g oan3sTy 25 ‘00v X 2 UsyeI OJOYUG ‘*(STIE99 pootq pez = Ie ‘untpTouqztde = do) el [owe], UST [OMS 9Y} SPTSUT 972eT[NIATI 0} STT99 pooTq perl Butseotor ‘poqdnastp useq sey oinjoONA}S [[99 Ie[ tq “se [oweT usemMieq pue UO posiep[US o1e ST[9O [eTTeYyITdY “YIP S,TOT [NF {-I 3 S9°0 UT skep ¢ 1OF PTY yotod 93TYM WOLF seTpTowe, ATepuodes °9 oansTYy 26 gill surface may debilitate fish, but no supporting data were given. Many species of freshwater fish can survive for several weeks in highly turbid conditions (European Inland Fisheries Advisory Commission, 1964), indica- ting that compensatory reactions may enable fish to survive despite gill damage. Randall (1970) pointed out that shunt mechanisms are commonly used by fish so that not all of the gill surface is used for respiration. By using the "reserve" surface area, fish may have sufficient functional, but damaged, gas exchange surface to survive prolonged exposure to sus- pended solids. The functional decrease in gill surface area caused by suspended solids also may be offset by compensatory increases in the gas exchange capacity of the blood (Section II). IV. EFFECTS OF SUBLETHAL CONCENTRATIONS OF FULLER'S EARTH ON CARBOHYDRATE METABOLISM IN THE HOGCHOKER 1, Wimterxoalorereshorn Fish livers contain large quantities of carbohydrate stored as animal starch or glycogen. During periods of starvation or stress increased metabolic demands for energy are met by breaking down liver glycogen into glucose and releasing it into the blood. This section presents the results of experiments determining the rate of glycogen utilization in the hogchoker during exposure to sublethal concentrations of fuller's earth. 2. Methods. The glycogen content in liver samples from hogchokers was determined after the fish had been held for 5 days in either control conditions or in suspensions of 1.24 g 17! fuller's earth. Glycogen was extracted from liver tissue by boiling the tissue in 30-percent potassium hydroxide (KOH), followed by precipitation with 95-percent ethanol (Good, Kramer, and Somogyi, 1933). Quantitative estimates of glycogen concentration were derived colorimetrically using the phenolsulfuric acid technique (Montgomery, 1957). Liver glycogen concentrations were expressed as milli- grams per 100 milligrams (mg 100 mg~!) of liver tissue. The results were analyzed statistically using "Student's" t-distribution (Snedecor and Cochran, 1967). 3. Results and Interpretation. Liver glycogen content from freshly caught hogchokers was about 15 to 17 mg 100 mg 7} (Sherk, O'Connor, and Neumann, 1972). Mean glycogen content in hogchoker livers decreased to 15.17 + 3.6 mg 100 mg™! (Table 10) after 5 days in control conditions. Fish held in a suspension of 1.24 g 17! fuller's earth had a liver glycogen content of 10.77 + 3.2 mg 100 mg-!, Significantly less than the value determined for control fish (p < 0.01, Table 10). Similar studies conducted with white perch and striped bass provided no useful data (App. A). Glycogen mobilization rates in these species were so high that the final liver glycogen concentrations in experimental and control fish were below the limits of the analytical procedure. AU Table 10. Experimental and control liver glycogen concentrations of hogchokers SPOEes for S days in 1.24 g 1"! fuller's earth.2 Group Individuals = oHeset (No. ) (mg 100 mg™ Experimental LO 77 225} 2 Control IWS) 6 IL7/ +3.6 sMean values are expressed + standard deviation. a) < O01 (2 & 2.880, dof, = 18). Rates of glycogen mobilization in fish may be used to estimate the energy utilization rate during starvation (Prosser and Brown, 1961; Kamra, 1966; Beamish, 1968; Swallow and Fleming, 1969). Thus, one interpretation of the rapid glycogen utilization in hogchokers exposed to suspended sedi- ments is that the sediment stress resulted in an increased energy require- ment. Several observations support this hypothesis. Hogchokers have a daily activity rhythm that persists in the laboratory (O'Connor, 1972). Those exposed to fuller's earth did not restrict their activity to specific parts of the daily cycle as did control fish. Therefore, an increase in locomotor activity may account for an increase in energy utilization during exposure to fuller's earth suspensions. Fish in fuller's earth suspensions may use more reserve energy for compensatory hematological responses. Hogchokers exposed to suspended solids showed evidence of significant alterations in basic hematological parameters (Section II), indicating an increase in the oxygen exchange capacity of the blood. Compensatory physiological alterations demand energy which must come from existing internal storage during starvation. V. EFFECTS OF SUSPENDED SOLIDS ON RESPIRATION OF ESTUARINE FISH 1. Introduction. The gills of fish are in constant contact with water. Water flowing across the gill surface helps supply the oxygen necessary for metabolism. Any materials dissolved or suspended in the water may come in contact with the gill surfaces. This section assesses the effects of suspended solids on oxygen con- sumption of estuarine fish. Fuller's earth suspensions were used to test the particle effects of clean clay. Patuxent River sediment suspensions were used to test the effects of naturally occurring particulate matter and associated substances on fish respiration. 28 Oxygen consumption rates were determined for striped bass, white perch, and toadfish in filtered Patuxent River water (base line) and in filtered river water suspensions of fuller's earth or Patuxent River sediment. Several methods are commonly used to measure fish oxygen consumption (Fry, 1971). Brett (1962) described the following three levels of fish respiration, in terms of activity: (a) Standard oxygen consumption, required to support tissue metabolism during periods of inactivity; (b) routine oxygen consumption, required during periods of random activity; and (c) active oxygen consumption, required during periods of swimming at moderate to maximum speeds. Respiration rates of pelagic fish in this report were determined under conditions of moderate activity. Values reported for demersal fish are measures of routine oxygen consumption. 2. Material and Methods. a. Equipment. A tunnel-type respirometer (Brett, 1964), which main- tained suspensions of fine particles and provided the variety of flow rates used to control swimming speeds of fish, was used for this project. A prototype respirometer (72-liter capacity), similar to that described by Farmer and Beamish (1969), was also constructed (Sherk and O'Connor, 1971). The respirometer loop and centrifugal pump were type-316 stainless steel. A cast acrylic chamber with plastic grids at each end was installed in the lower section of the loop. An oval section cut from the top of the chamber permitted insertion and removal of fish from the respirometer. Rubber gaskets and hose clamps sealed the access port during experiments. Straightening vanes upstream from the chamber ensured laminar flow. The centrifugal pump was driven by a variable-speed electric motor. An orifice plate in the upper part of the loop measured flow rates. Two needle valves and a fill pipe on the upper side of the loop and two neoprene stoppered openings in the chamber provided access to the water. These access points were used extensively during experiments to bleed the respirometer of trapped air, and to sample suspensions. At two points on the respirometer loop, 20-meter copper coils controlled temperature via counter-current heat exchange with water pumped from a constant temperature bath. Four inverted versions of the prototype respirometer (62-liter capacity) were used for these experiments. Flow rates were measured by annular flow sensors. A water jacket around the outside of the lower part of each loop controlled temperature via counter-current heat exchange with water pumped from a constant temperature bath. 29 All respirometers were wrapped with standard fiberglass insulation to Maintain constant temperatures. Plywood enclosures were placed around each chamber during experiments to isolate fish from laboratory activity. Each enclosure contained a 15-watt cool white fluorescent lamp, 42 centi- meters above the chamber to provide constant illumination during experi- ments. Fish were observed through a viewing slit in each enclosure. Surfaces immediately below each chamber were blackened. Changes in dis- solved oxygen concentrations were monitored by Yellow Springs Instrument Co., Inc. Model 54RC oxygen meters. Oxygen electrode leads were passed through neoprene stoppers that sealed the fill pipes into which the elec- trodes were inserted. b. Fish. Fish were collected by otter trawl from the Patuxent River estuary. Actual collection sites ranged from the Lower Marlboro area to the vicinity of Drum Point, Maryland, depending upon species and time of year. Fish were kept on the collecting vessel in 80-liter plastic trash cans. A constant flow of ambient river water was maintained through the cans until the vessel returned to the laboratory. The laboratory holding facilities consisted of 208-liter polyethylene tanks immersed in controlled-temperature water baths. Water in the tanks continually passed through an inline protein-skimmer filtration system. Patuxent River water, which passed through a 5-micrometer mesh nylon filter, was usually used to supply the tanks. During the summer months of 1972 a commercial marine salt mix dissolved in laboratory well water was used to supply the holding tanks. Salinity of water used in the laboratory was about 5 parts per thousand. Holding tank and experimental temperatures were adjusted to approximate seasonal changes. The fish were placed in the laboratory holding tanks, where care was taken to avoid overcrowding. Unhealthy or dead fish were removed imme- diately. Supplemental aeration was provided when large numbers of fish were held. The fish were under continuous fluorescent illumination. They were not fed following capture because active digestion increases standard and routine oxygen consumption (Beamish, 1964; Glass, 1968). Fish were held a minimum of 3 to 5 days before oxygen consumption rates were deter- mined. c. Measurement of Oxygen Consumption. Respirometers were filled with water from the holding tanks (Fry, 1971) during experiments. As soon as the water in the apparatus could completely cover the fish, each fish was transferred in a bucket of water from the holding tank to the respirometer. When the respirometers were full, water was circulated at 0.28 to 0.39 foot per second (ft/s) to force out entrained air which was replaced simultane- ously by holding tank water. In addition, flow rate was increased by 0.18 ft/s at 4- or S5-minute intervals to drive out trapped air. Maximum flow attained during this procedure was 2.5 to 4 times the minimum experimental rate, depending on species. Flow was reduced to the minimum experimental exposure rate and all access points were closed. The plywood enclosure was placed around the chamber. 30 Oxygen electrodes were calibrated and inserted through the fill pipes. Time, temperature, dissolved oxygen concentration, and flow rate were recorded for each respirometer as soon as it was set up and at hourly intervals thereafter. Preliminary studies demonstrated that at a constant flow of 0.28 ft/s, the hourly oxygen consumption decreased until the third hour, after which rates were relatively constant. Data from the third hour were used in all analyses. Several species were tested at various swimming speeds. The above procedure was used until the third-hour data had been recorded. Flow rates were then increased about 0.18 ft/s at 5-minute intervals until the desired speed was achieved. The same parameters were recorded 5 minutes after this speed was attained, and again 1 hour later. If information was re- quired at higher levels of activity this procedure was repeated. All] parameters were monitored for 1 hour at each of the increased flow rates. At the end of each experiment the respirometers were drained and the fish were removed for weighing, length measurement, and sex determination. Respirometers were flushed with tapwater, then refilled with tapwater until used again. Terramycin (oxytetracycline hydrochloride, 15-milligram activity per liter) was added to the water when the respirometers would not be used for longtime periods. Predetermined volumes of solids were added to about 16 liters of water in 80-liter plastic trash cans. The slurries were aerated continuously, and a submersible electric pump mixed the material. Slurries were prepared 18 hours before use, and were pumped into the respirometers as the units were filled with holding tank water. Respirometers were washed several times with tapwater at the end of experiments to prevent accumulation of materials in the units. Concentrations of suspended materials were determined by the dry weight difference between three 5-milliliter replicate samples drawn from each respirometer at the beginning of an experiment, and three similar samples drawn from the holding tank (no suspended material added) at the same time. The oxygen demand of natural sediment suspensions was determined by measur- ing the oxygen uptake of slurries. Slurries were pumped into the respirom- eters as described above. Respirometers were set up as before but without fish. Mean third-hour oxygen consumption values of the slurries were used to check the oxygen demand of the sediment during experiments. The equipment used for long-term exposure of fish to suspensions of solids is described in O'Connor, Neumann, and Sherk (1976) for bioassay €xperiments or for sublethal hematological studies (see Section II). d. Data Analyses. Oxygen consumption rates were plotted against live weight on double logarithmic grids. Curves were fitted to the data by least squares linear regression analysis (Snedecor and Cochran, 1967). Correlation coefficients were determined for each group of data (Simpson, Roe, and Lewontin, 1960). 3! Group comparisons were made by covariance analysis of log-transformed data (Snedecor and Cochran, 1967). Sex influence on respiration rates was tested by covariancej analysis for each species when possible. Data from males and females were combined for comparisons of base-line and experimental values. 3. Results. a. Striped Bass. Fish were held at approximately 15° Celsius and 5 parts per thousand salinity for a minimum of 3 days before respiration rates were determined. Oxygen consumption rates were determined at three Swimming speeds--0.28, 1.02, and 1.58 ft/s. In filtered water, a 50-gram fish swimming at 0.28 ft/s consumed 19.0 milligrams oxygen per hour (mg 0) h7!) and a 150-gram fish used 31.4 mg O07 h7!. At a speed of 1.02 ft/s, a 50- and a 150-gram fish used 24.3 and 41.3 mg Op h7!, respectively. At speeds of 1.58 ft/s, oxygen consump- tion rates increased to 33.7 mg Op h=! for a 50-gram fish, and 63.7 mg O27 h7! for a 150-gram fish. A significant increase in respiration rates was observed between measurements made at 0.28 and 1.02 ft/s and between 1.02 and 1.58 ft/s (Table 11). ‘Covariance analysis showed that oxygen consumption rates of male and female fish did not differ at either swimming speed (Table 12). Covariance analysis was not made for swimming speeds of 1.58 ft/s due to the small number of females tested. A 50-gram fish consumed 19.2 mg Oj h7! and a 150-gram fish consumed 37.8 mg 0p h7! at a swimming speed of 0.28 ft/s during exposure to 0.79 g 1! fuller's earth. A 50- and a 150-gram fish swimming at 1.02 ft/s under these conditions consumed 24.1 and 41.2 mg Op h7!, respectively (Figs. 7 and 8; Table 13). Striped bass swimming at 1.58 ft/s in fuller's earth suspensions consumed less oxygen than fish swimming at that speed under base-line conditions (Fig. 9; Table 13). Oxygen consumption was uniformly depressed by about 25 percent throughout the weight range studied. A graph of the oxygen consumption rates of striped bass swimming at 0.28 and 1.02 ft/s during exposure to 0.79 g 17! fuller's earth showed different slopes. Respiration rates at swimming speeds of 1.02 and 1.58 ft/s during this exposure were not different, Rates for fish swimming at 0.28 and 1.58 ft/s were different at the l-percent level (Table 11). Oxy- gen consumption rates of male and female striped bass swimming at 0.28 and 1.02 ft/s in fuller's earth suspensions were not different (Table 12). Comparisons of male and female oxygen consumption rates at 1.58 ft/s could not be made because too few females were studied. Striped bass held at 22.5° Celsius and 9 parts per thousand salinity were tested at swimming speeds of 1.05 and 1.58 ft/s under base-line con- ditions and during exposure to natural sediment suspensions. The experi- mental procedure was modified because increased temperature, salinity, and sediment-oxygen demand reduced dissolved oxygen. The 3-hour acclimation period at a swimming speed of 0.28 ft/s was changed to 3 hours at 1.05 ft/s 32 *qUBSTFIUSTS ION, *SNTST9) ,ST anoqe qe (oUuTT 9aseq) 10oqeEM IOATY auemard pertey{[Tz ut pus) lsze° S,L9TINZ ;_1 8 OL°O UT RE 8s‘T pus ‘Z0°L ‘8z°0 go speeds 2 SuTWUTMS OR aUT[ eseg 8S'T 8Z°0 8s'T Z0°T Z0°T 82°0 yaee storing AyTTTqeqord | at 1tqeqosd | “Aytttqeqoid | me 1 (eae | oe Sedge! OOS perenbs uray eos ory, perenbs ueay uosti1eduo,) Jo saei390q JO Saaizaq JO soaidaq *,s8Seq podtazs 10J suotssarZa1 yy8tem eAT] pue uot ydunsuod uaBXxo zo stsk[eue AIUETIEACD ‘IT 2TQeL 33 *JUBITFTUBTS ON, *sntsta9 oSI 3oge 2 SUOTITPUOD BUTT-eSeq opun puke YyILee S,JOTINF ;_I 3 6L°0 02 eimsodxe Butznp eee ZO°T pue gz‘o jo spoads BuTuITMS IV, Aqytttqeqoid katt tqeqoad | | dat ttqeqosd | TERE 4s (8/23) -wopose1y -wopes.1s -wopesrz JO seetdaq | pazenbs ueaw | jo sd0180q | pozenbs ueaw | so saez3aq | parenbs ueop poeds Zutumtms uoTIBASTY SdUeTIEA [eENPtTsoy *,ssBq pedtzqs oTeuaz pue afew jo suotssor3e1 yYyStem aATT pue uotydmnsuod uaskxo Jo stsk[eue sdUeTIeAOD soTeuoy soT ey soTeuey soTew OUT, oseg so[ ewes soTen sol Buoy SOTBW yzzee s,laT[ny uostieduo5 “tT 8TdeL 34 80 60 A a log Y = 0.616 log x + 0.237 ) Oxygen Consumption (mg 02 h7! as (0) 20 40 60 80 100 200 400 Live Weight (9) Figure 9. Oxygen consumption of striped bass swimming at 1.58 ft/s during exposure to 0.79 g 17!. fuller's earth (dashline) and under control conditions (solid line). 38 with continuous aeration. Respirometers were closed at the end of the third hour and oxygen concentrations were monitored for 1 hour. Flow rates were gradually increased to 1.58 ft/s. Base-line oxygen consumption rates of a 50- and a 150-gram fish swim- ming at 1.05 ft/s were 23.2 and 55.0 mg O09 h-1, respectively. At a swim- ming speed of 1.58 ft/s, a 50-gram fish consumed 23.2 mg Oo Ago aelsO= gram fish consumed 48.1 mg 09 h-!. Respiration rates at the two swimming speeds were not significantly different, and sex influence on oxygen consumption rates was not apparent at either speed (Table 14). Respiration rates of striped bass swimming at 1.05 and 1.58 ft/s during exposure to 1.31 and 1.33 g 17! natural sediment, respectively, were reduced by 30 to 40 percent of the base-line values. At a swimming speed of 1.05 ft/s, a 50-gram fish used 14.2 mg O05 h-!; a 150-gram fish used 34.4 mg Op h7! (Fig. 10; Table 15). Similar weight fish swimming at 1.58 ft/s consumed 14.1 and 34.9 mg Op h7! (Fig. 11; Table 15). Respira- tion rates at the two swimming speeds were not significantly different, ‘and oxygen consumption rates of males and females during exposure to nat- ural sediment were not different at either swimming speed (Table 14). b. White Perch. Fish wer¢ maintained at about 15° Celsius and 5 parts per thousand salinity for a minimum of 3 days. Oxygen consumption rates were determined at swimming speeds of 0.28 and 1.02 ft/s under base-line conditions. At a swimming speed of 0.28 ft/s, a 50-gram fish used 13.3 mg 0) h7!; a 150-gram fish used 27.1 mg Oo hale Ralsihiofmthie Same weights swimming at 1.02 ft/s consumed 24.4 and 44.6 mg Oo he, Tespec= tively. Respiration rates were greater at swimming speeds of 1.02 ft/s than 0.28 ft/s (Table 16); male and female respiration rates did not differ aemeutcherEspeed m(MabilerslG)))s Oxygen consumption rates were determined for white perch during exposure to 1.09 g 1~! fuller's earth suspension at swimming speeds of 0.28 (Fig. 12) and 1.02 (Fig. 13) ft/s. The data were dispersed at both swimming speeds--correlation coefficient, r = 0.017 (not significant) at 0.28 ft/s and r = 0.201 (not significant) at 1.02 ft/s. ‘Covariance analyses were not attempted because of poor data correlation. Similar results were observed when oxygen consumption was determined for white perch swimming at 0.39 and 1.05 ft/s during exposure to 2.12 g 1~! natural sediment suspensions. Low correlation coefficients, r = 0.143 (mot significant) at 0.39 ft/s and r = 0.017 (mot significant) at l02) £t/s, prevented further statistical treatment of these data- White perch were held in suspensions of 2.58 g 17! natural sediment for 72 hours. Oxygen consumption rates were measured in filtered river water at swimming speeds of 0.39 and 1.05 ft/s. Data for fish swimming at 0.39 ft/s after a 72-hour exposure were too scattered, r = 0.508 (not Significant), to permit further analysis (Fig. 14). After a 72-hour expo- sure to natural sediment the oxygen consumption rates of a 50- and a 39 *QUBITJIUSTS ION, “snTST9) ,S°2Z 28 SUOTITPUOD SUTT-aSeq Japun pue JUoWTpas [eInzeU |_T 8 ZE*T 02 ainsodxa Zutinp s/3J gs*I pue So°T Jo speeds 3utmmtms qWy —— Ioc00s0mtn kas =— = nn ENG 700 LON === en GOO a soTemod vt ‘T 1900°0 9L10°0 saTen Svl0°O so[euay s$v00°0 SOTeN ¥800°0 sa[eula4y salen y9Z0°0 so[emo4 £60T ‘0 66S0°0 SOTEN Z000°0 ATT tqeqoid -wopaa1z -wopedrs -wopaarz jo saaidaq JO saois09q jo soaidaq 15524 pedtiis Jo suotssaiZe1 4Yy3tam aALT[ pue uotidunsuod uaskxo Jo stsd[eue BIURTIEAOD ‘pl STqeL 40 80 A 7 60 log Y= 0.786 log x + 0.031 a n=2l r=0.832 p<0.00I Z oa 40 7 7 he 10) 7 A Ae log Y=0.8034 logx -0O.2110 y n=22 r=0.8042 p<0O.00I Oxygen Consumption (mg O2 h7! (e°) as 0) 20 40 60 80 100 200 400 Live Weight (gq) Figure 10. Oxygen consumption of striped bass swimming at 1.05 ft/s during exposure to 1.31 g 1-1 natural sediment (dashline) and under base-line conditions (solid line). 4| “QUBITFTIUSTS ION, “SNTSTAD ,S°ZZ 3B SUOTITPUOD SUTT-aSeq Jopun pue qUaUTpes [eInjeU ;_[ 3 ¢¢°{T 03 einsodxa Butinp S/F 8S°T 3 pue SUOTITPUOD SUT[-asSeq LapuN puke JUSWTpes [eInjeU ,;_[ 8 [¢*]T 02 einsodxe Butinp s/3}F so*T Jo speeds Butumtms Wy 0982 ‘0 p8I0°0 = se ~ auTT oseg 61Z0°0 1ZZ0°0 zZ£0°0 [equoutsedxq s00'0 > d 999¢ °0 s000°0 £610°0 euTT eseg Or ‘T £S20°0 6S20°0 e220 °0 Tequowtsedxg Ayr tqeqord Aat{TqGeqoad | 4atttqeqosd -wopaarz -WOopadry -wopedrj JO saeidaq | perenbs ueay | Fo saertZaq | pasenbs uvaw | go seei8eq | petenbs ueay | stenptatpuy | paeds SUTUUTMS uostieduo9 *sseBq pedtz15 fo SuotSsarZe1 YyItOM BATT pue uot dunsuod ua8kxo Fo stsA[eue sdUeTIeAOD ‘ST eTqeL 42 80 7 60 7 A log Y=0.643 log x+0.273 40 n=!I7 r=0.859 p<0O.00I 7 7 7 7 —20 7 7 7 7 7 A 7 log Y=0.8222 log x -0.2469 10 n=20 r=0.8108 p<0O.00I Oxygen Consumption (mg Oo h'! jee) S O 20 40 60 80 |00 200 400 Live Weight (gq) Figure 11. Oxygen consumption of striped bass swimming at 1.58 ft/s during exposure to 1.33 g 17! natural sediment (dashline) and under base-line conditions’ (solid line). 43 *QUBDTFIUSTS ION, *SUOTZTPUOD SUTT-aseq Lepun s/3FZ ZO'T pue gz°O JO Speeds Butuutms ty, sal euey solew Saxes poutquioy uoT39aTAS WopuBYy sol eule4 -wopaely -wopaelz JO soai30q -wopaer5 JO sa0i390q JO saoi89q uot eAe Ty SDUBTIVA [TeNPTsoy pueted aaTym Jo suotsseiZe1 yYyZteM aAT{ pue uotydunsuod uaskxo Fo stsA[eUe BdUeTIEAOD “OT ATQEL 44 80 60 A 40 log Y= 0.435 log x + 0.485 n=13 r=0.555 p<0.05 = A0) A log Y=O.0II logx + 1.237 n=13 r=0.017 not significant o Oo (op) Oxygen Consumption (mg Oz h'! 4 0) 20 40 60 80 100 200 400 Live Weight (q) Figure 12. Oxygen consumption of white perch swimming at 0.28 ft/s during exposure to 1.09 g 1°* fuller's earth (dashline) and under base-line conditions (solid line). 45 80 60 A log Y=0.548 log x + 0.457 NG n=Il r=0.678 p<0.05 A log Y=0.164 log x + 1.001 vA n=9 r=0.20I not significant 8 fop) Oxygen Consumption (mg 0, h7! ASS 0 20 40 60 80 100 200 400 Live Weight (gq) Figure 13. Oxygen consumption of white peach swimming at 1.02 ft/s during exposure to 1.09 g 1~* fuller's earth (dashline) and under base-line conditions (solid line). 46 80 60 40 ine) [e) Oxygen Consumption (mg 0, h!) Figure 14. A log Y= 0.760 log x -0.136 yer n=l! r=0.508 not significant 7 A 7 log Y = 0.435 log x + 0.485 7 n=13 r=0.555 p<0.05 20 40 60 80 100 200 400 Live Weight (g) Oxygen consumption of white perch swimming at 0.39 ft/s in clean water following 72-hour exposure to 2.58 g 17! resus- pended natural sediment (dashline) and at 0.28 ft/s under base-line conditions (solid line). 47 150-gram fish swimming at 1.58 ft/s in filtered river water were 10.8 and 55.3 mg Op h7!, respectively (Fig. 15). Elevations of the lines describing these data and hase-line data at 1.58 ft/s were different at the 5-percent level (Table 17). c. Toadfish. Fish were held at least 5 days at 18° to 20° Celsius and 5 parts per thousand salinity. Oxygen consumption rates were deter- mined at a flow rate of 0.39 ft/s. Toadfish did not consistently swim into the current. In filtered river water flowing at 0.39 ft/s,a 50-gram fish used 5 mg Op h7! and a 150-gram fish used 10.1 mg 02 h-!, Sex influence on oxygen consumption rates was not apparent from these data (Table 18). Respiration rates of toadfish during exposure to 2.20 g 1-! fuller's earth were not different from rates determined under base-line conditions (Table 19). During this exposure a 50-gram fish consumed 5.6 mg 02 hol; a 150-gram fish consumed 12.4 mg Oo h-!, Sex influence on respiration rates was not apparent (Table 18). Oxygen consumption rates of toadfish during exposure to 1.58 g ad natural sediment were not different from base-line rates (Table 19). A 50-gram fish consumed 4.8 mg 07 h7!; a 150-gram fish consumed 9.7 mg Oo Ina in natural sediment suspensions. Male and female respiration rates during exposure to 1.58 g 1~! natural sediment differed in variance and elevation at the 5-percent level (Table 18). Toadfish were held in 10.37 g 17! natural sediment for 72 hours before oxygen consumption rates were determined in filtered river water. These rates were not different from base-line rates (Table 19). A 50-gram fish used 2.2 mg 0) h7!; a 150-gram fish used 7.3 mg Op h7!. A significant difference was observed between the variances of respiration rates for males and females during this experiment (Table 18). Respiration rates were determined for toadfish in 3.36 g 17! natural sediment after a 72-hour exposure to 11.09 g 17! of the same material. The variance associated with rates for fish in both the experimental and base-line groups were different (Table 19). Oxygen consumption rates of a 50- and 150-gram fish were 2.5 and 11.5 mg O09 h7!, respectively. Sex influence on respiration rates was not apparent (Table 18). Respiration rates of toadfish exposed to natural sediment suspensions for 72 hours were the same in filtered river water and in natural sediment suspensions (Table 19). 4. Discussion. Concentrations of suspended materials in an estuarine system are highly variable. Storms, floods, tidal scour, or engineering activities may increase concentrations of suspended particles. Naturally occurring suspended loads exceeding 1 g 17! are uncommon (Sherk, 1972). Masch and Espey (1967) reported concentrations exceeding 10 g 17! in dredge discharge plumes and 100 g 17! in dredge-generated density flows. Suspended solids concentrations used in this study typify those found near dredging opera- tions. 48 80 60 40 ) M S) Oxygen Consumption (mg O02 h'! re) Figure 15. A log Y= 0.548 log x + 0.457 n=l! r=0.678 p<0.05 / vA 4 / A 7 VA / 7 4 7 A 7 log Y=1.489 log x — 1.497 / n=9 r=0.728 p<0.05 20 40 60 80 |I00 200 400 Live Weight (gq) Oxygen consumption of white perch swimming at 1.02 ft/s following 72-hour exposure to 2.58 g 17! resuspended natural sediment (dashline) and under base-line conditions (solid line). 49 Table 17. Covariance analysis of oxygen consumption and live weight regressions of white perch}. Mean squared | Degrees of | Mean squared | Degrees of | Mean squared | Degrees of freedom- freedom- freedom- probability probability probability 0.033 0.164 lat swimming speeds of 1.58 ft/s in filtered water after 72-hour exposure to 2.58 g 1~* natural sediment and under base-line conditions. 2Not significant. Comparison Individuals Experimental Base line Table 18. Covariance analysis of See consumption and live weight regressions of male and female toadfish. [side viene | Comparison Individuals | Mean squared | Degrees of | Mean squared | Degrees of | Mean squared | Degrees of freedom- freedom- freedom- probability probability probability Fuller's earth} Males 0.043 Females 0.025 Natural sediment? Males Females Filtered water® Males Females Natural sediment® Males Females Base line Males Remales lrested in 2.20 g 17 = fuller's s pa {Not SrenLetcants 3In 1.58 g 1-} natural sediment. “In filtered pes water after 72-hour exposure to 10.37 g 17 macurad sediment. ein 3.36 g 1~* natural*’sediment after 72-hour exposure to 11.09 g i-! natural sediment. ®Under eee conditions. 50 *TeTieqeM sues 42 JO ;_T 3 GO°TT 03 esmsodxe anoy-z/ 1eqze JUeMTpes TeInjeU ;_[ 3 9¢*¢ 03 aunsodxa 3uring, “queTpes [evinzeu ,;_[ 3 L¢"OT 02 einsodxe Imoy-7, 1e1Ze 103M pateI{TF UL, 0z0°0 vs0°0 Ts0°O L8T°0 Lv0°0 £IT°O 9S0°0 aUuTT aseg g2USUTpes [eInjeN aut. eseg ,tUemTpes TeinzeN Z0000°0 SUT. oseg ££0°O gtUeltpas [einen £00°0 SuTT 9Stq Sz0°0 yuztee s,leting Aytteqeqosd | Aat 1 Fqeqoad -wopaery ~Wopsetz -wopadary uot Ie1AUaDU0D Azioysty sinsodxe Jo saai30q jo saaisaq | parenbs uray | Jo saei13eq | pestenbs uray [Te usmtsadxy aTotqyzed A10.eIOGeT uostieduo5 AatTtqeqord (,-T 3) QIUBTIBA [ENPTsoy “YSTJPBOI FO SuOTSSeIZe1 YZTOM ATT pue uotydumsuod use%Xkxo Jo stsk[Teue aoueTIeAD) “6 FTqeL S| The fish studied are from three ecologically distinct estuarine niches. Striped bass, a major sport and commercial fish, are anadromous and use the estuary aS a spawning and nursery area (Talbot, 1966). White perch make semianadromous migrations (Mansueti, 1964) and are usually restricted to a certain segment of the estuary throughout their lifespan. Toadfish are sedentary, demersal fish and inhabit the sediment-water interface. The swimming abilities of striped bass and white perch suggest that during periods of high turbidity these fish can move to more favorable areas. However, in laboratory experiments that prevented escape, suspen- sions of fuller's earth or Patuxent River sediments generally reduced oxygen consumption at controlled levels of swimming activity. Respiratory responses of striped bass and white perch to suspended solids were observed in the laboratory at concentrations exceeding those which occur naturally in estuaries. These concentrations may occur temporarily near dredge dis- charges. Toadfish exhibited no significant respiratory responses to suspensions of fuller's earth or natural sediment. The sediment-water interface is characterized by periods of low oxygen concentration, high turbidity, or both. Hall (1929, 1930) reported that oxygen consumption of toadfish is almost directly proportional to the oxygen tension of the water, and that the fish are able to remove all the oxygen from a limited volume of water before respiratory movement ceases. This may explain the absence of re- sponse. Suspension concentrations produced by dredging operations probably have a limited effect on striped bass and white perch respiration because of the mobility of these species. Toadfish are sedentary, but their high tolerance to suspended solids may minimize the effects of suspended solids on their respiration. VI. SUMMARY AND CONCLUSIONS Suspensions of particulate matter deposited in estuarine systems by nature or man can affect estuarine fish. Stress from suspended sediments may cause changes in growth, survival, and reproduction of fish. The effects of suspended particles on fish depend on the concentration and composition of the particles and the stress tolerance of the fish. Sus- pensions of commercial mineral solids were tested to determine the effects of suspended sediments of known composition, particle-size distribution, and organic matter content. Additional tests were run with resuspended Patuxent River estuary muds to test the effects of a natural sediment. Exposure to sublethal suspended solids concentrations increased micro- hematocrit, hemoglobin concentration, and red blood cell count in white perch, hogchoker, mummichog, and striped killifish. Increases in these hematological parameters raise the blood's oxygen exchange capacity. Hematological values probably change in response to suspended solids' interference with oxygen-carbon dioxide transport at the gill. No signifi- cant increases occurred in striped bass, spot, or toadfish. 52 White perch experienced gill tissue disruption and intensified mucus production on the gills during exposure to fuller's earth suspensions. Although fuller's earth concentrations below the 24-hour LCjg9 value were used, the gill tissue structure of white perch was adversely effected and the respiratory surface area was reduced in a 5-day period. High rates of liver glycogen depletion were recorded in hogchokers exposed to sublethal fuller's earth concentrations. This indicates carbo- hydrate utilization and drainage of metabolic reserves during periods of sediment stress. Hogchokers live at the sediment-water interface, but still demand extra energy for compensatory alterations of their physiology while exposed to suspended sediments. Oxygen consumption of striped bass and white perch increased with swimming speed in control tanks containing filtered river water. However, suspensions of fuller's earth or Patuxent River sediments generally reduced fish oxygen consumption rates at high levels of swimming activity. This indicates that sediment suspensions interfered with the fish's respiratory ability. Both striped bass and white perch are common to the open waters of the estuary. However, toadfish, which inhabit the turbid sediment-water interface, showed no significant respiratory responses to fuller's earth or natural sediment suspensions. It is customary and useful to establish suspended solids criteria by applying the lethal concentration levels causing 10- to 50-percent mor- tality over a defined period of exposure. however, this procedure ignores the biologically significant sublethal effects of suspended solids on estuarine fish. Concentrations of suspended sediments found in estuarine systems during storms, flooding, dredging, and dredged-material disposal are within the range of sublethal concentrations used in these experiments. Since the experimental suspensions induced stress responses in several fish species, preproject evaluations of the effects of dredging and related activities should include consideration of this effect. 53 LITERATURE CITED AMERICAN SOCIETY OF TESTING AND MATERIALS, 'Particle-size Distribution of Coating Clay,"' Pub. No. T649 su-68, Philadelphia, Pa., 1968. ANTHONY, E.H., ''The Oxygen Capacity of Goldfish (Carrasstus auratus) Blood in Relation to the Thermal Environment," Journal of Experimental Btology, WONG Ssh5 MOIS) oo, GSO BEAMISH, F.W.H., "Influence of Starvation on Standard and Routine Oxygen Consumption,'' Transactions of the American Fishertes Soctety, Vol. 93, Noe 96a pp lose BEAMISH, F.W.H., "Glycogen and Lactic Acid Concentrations in Atlantic Cod (Gadus morhua) in Relation to Exercise," Journal of the Fishertes Board, Canada, Vol. 25, 1968, pp. 837-851. 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EISLER, R., "Erythrocyte Counts and Hemoglobin Contents in Nine Species of Marine Teleosts,'' Chesapeake Setence, Vol. 6, No. 2, 1965, pp. 119-120. EUROPEAN INLAND FISHERIES ADVISORY COMMISSION, "Water Quality Criteria for European Freshwater Fish,'' Technical Paper No. 1, Report on Finely Divided Solids and Inland Fisheries, 1964. ELLIS, M., "Erosion Silt as a Factor in Aquatic Environments," Ecology, Vol. 17, 1936, pp. 29-42. ELLIS, M., "Detection and Measurement of Stream Pollution," Bulletin,U.S. Bureau of Fisheries, Vol. 48, 1937, pp. 365-473. 54 FARMER, G.J., and BEAMISH, F.W.H., "Oxygen Consumption of Ttlapta ntlotica in Relation to Swimming Speed and Salinity," Journal of the Fishertes Research Board, Canada, Vol. 26, 1969, pp. 2807-2821. FOLK, R.L., Petrology of Sedimentary Rocks, University of Texas, Austin, hex W968% FORSTER, R.P., and BERGLUND, F., "Osmotic Diuresis and its Effect on Total Electrolyte Distribution in Plasma and Urine of the Aglomerular Teleost Lophitus amertcanus ,"" Journal of General Phystology, Vol. 39, 1956, pp. 349-359. FRY, F.E.J., "The Effect of Environmental Factors on the Physiology of Fish," Fish Phystology, Vol. 6, Academic Press, New York, 1971, pp. 1-98. GLASS, N.R., "The Effect of Time and Food Deprivation on the Routine Oxygen Consumption of Largemouth Bass (Micropterus salmotdes), Ecology, Vol. 49, 1968, pp. 340-343. GOOD, C.A., KRAMER, H., and SOMOGYI, M., "The Determination of Glycogen," Journal of Brologtcal Chemtstry, Vol. 100, 1933, pp. 485-491. HALL, F.G., "The Influence of Varying Oxygen Tensions Upon the Rate of Oxygen Consumption in Marine Fishes," American Journal of Phystology, WO, 83.5 MIA)S os AIZSANs HALL, F.G., "The Ability of the Common Mackerel and Certain Other Marine Fishes to Remove Dissolved Oxygen from Sea Water," Amertecan Journal of Phystology, Vol. 93, 1930, pp. 417-421. HALL, F.G., GRAY, I.E., and LEPKOWSHY, S., "The Influence of Asphyxiation on the Blood Constituents of Marine Fishes," Journal of Biological Chemistry, Vol. 67, 1926, pp. 549-554. HEINLE, D.R., and MORGAN, R.P., Jr., ''Bioassay for Chronic Effects of Water from Baltimore Harbor,'' Reference No. 72-15, University of Maryland, National Research Institute, College Park, Md., 1972. HERBERT, D.W.M., et al., "The Effect of China Clay Waste on Trout Streams," Internattonal Journal of Atr and Water Pollution, Vol. 5, 1961, pp. 56-74. HERBERT, D., and MERKENS, J., "The Effect of Suspended Mineral Solids on the Survival of Trout," Internattonal Journal of Atr and Water Pollu- Ebony Volas5ss) LIGI. spp 46-55. HESSER, E.F., "Methods for Routine Fish Hematology," Progressive Fish Culture, Oct. 1960, pp. 164-171. 55 HOLTEN, G.F., and RANDALL, D.J., ''The Effects of Hypoxia Upon the Partial Pressure of Gases in the Blood and Water of Afferent and Efferent Gills of Rainbow Trout," Journal of Experimental Biology, Vol. 64, 1967, pp. 317-327. HOUSTON, A.H., and DeWILDE, M.A., "Hematological Correlations in the Rainbow Trout, Salmo gairdnert,"" Journal of Fisheries Research Board, Canada, Vol. 25, No. 1, 1968, pp. 173-176. INGRAM, P., "Uridine Diphosphate Glucose-Glycogen Transferase from Trout Liver,'' International Journal of Biochemistry, Vol. 1, 1970, pp. 263-273. KAMRA, S.K., "Effects of Starvation and Refeeding on Some Liver and Blood Constituents on Atlantic Cod (Gadus morhua L.),"' Journal of Ftshertes Research Board, Canada, Vol. 23, 1966, pp. 975-982. LARSEN, H.N., “Comparison of Various Methods of Hemoglobin Determination on Catfish Blood," Progressive Fish Culturist, Vol. 26, No. 1, 1964, pp. 11-15. LARSEN, H.N., and SNIEZKO, S.F., "Comparison of Various Methods of Deter- mination of Hemoglobin in Trout Blood," Progressive Fish Culture, Voll 12355, Noe ES SGI pp Siac MANSUETI, R.J., "Eggs, Larvae, and Young of the White Perch, Roccus americanus, with Comments on its Ecology in the Estuary," Chesapeake Setence, Vol. 5, No. 1-2, 1964, pp. 3-45. MASCH, F.D., and ESPEY, W.H., "Shell-Dredging - A Factor in Sedimentation in Galveston Bay,"' Technical Report No. 7, University of Texas, Center for Research in Water Resources, Austin, Tex., 1967, p. 168. MAY, E.B., "Environmental Effects of Hydraulic Dredging in Estuaries," Alabama Marine Resources Bulletin Number 9, 1973, pp. 1-85. McERLEAN, A.J., and BRINKLEY, H.J., "Temperature Tolerance and Thyroid Activity of the White Perch Roccus (=Morone) amertcanus ,"" Journal of Fish Biology, Vol. 3, 1971, pp. 97-114. MONTGOMERY, R., ''Determination of Glycogen," Arch-Btochemical Btophystcs, Vol. 67, 1957, pp. 378-386. O'CONNOR, J.M., "Tidal Activity Rhythm in the Hogchoker, Tritnectes maculatus (Bloch and Schneider) ," Journal of Experimental Marine Biology and Ecology, Vol. 9, 1972, pp. 173-177. O'CONNOR, J.M., NEUMANN, D.A., and SHERK, J.A.,'''Lethal Effects of Sus- pended Sediments on Estuarine Fish," TP 76-20, U.S. Army, Corps of Engineers, Coastal Engineering Research Center, Fort Belvoir, Va., Dec. 1976. 56 OSTROUMOVA, I.N., ''Properties of the Blood of Trout During Adaptation to Different Oxygen and Salt Contents of the Water," In: Privol 'hev, T. I. (Ed.), Fish Phystology tn Acclimattzation and Breeding, Bulletin of St. Science Research Institute of Lake and River Fish, Vol. 58, 1964, pp. 24-34. PERKIN-ELMER CORPORATION, "Analytical Methods for Atomic Absorption Spec- trophotometry," 1971. PHYLLIPS, A.M., Jr., "The Effects of Asphyxia Upon the Red Blood Cell Content of Trout Blood," Copeta, 1947, pp. 183-186. PROSSER, C.L., and BROWN, F.A., Comparative Animal Phystology, 2d ed., Saunders, Philadelphia, Pa., 1961. PROSSER, C.L., et al., "Acclimation to Reduced Oxygen in Goldfish," Phystology of Zoology, Vol. 30, 1957, pp. 137-141. RANDALL, D.J., "Gas Exchange in Fish," Fish Phystology, Vol. IV, The Nervous System, Circulation and Respiration, Academic Press, New York, 1970. RITCHIE, D.W., "Project F: Fish," Gross Physical and Biological Effects of Overboard Spoil Disposal in Upper Chesapeake Bay, Special Report No. 3, Natural Resources Institute, University of Maryland, College Park, Md., 1970. ROGERS, B.A., ''The Tolerance of Fishes to Suspended Solids," M.S. Thesis, University of Rhode Island, Kingston, R.I., 1969. SHERK, J.A., Jr., ''Current Status of the Knowledge of the Biological Effects of Suspended and Deposited Sediments in Chesapeake Bay,"' Chesapeake Sctence, Vol. 13, (Suppl.), 1972, pp. S137-S144. SHERK, J.A., and O'CONNOR, J.M., "Effects of Suspended and Deposited Sediments on Estuarine Organisms, PHASE II,'' Reference No. 71-4D, Annual Report Project Year II, N.R.I., University of Maryland, College Park, Md., 1971. SHERK, J.A., O'CONNOR, J.M., and NEUMANN, D.A., “Effects of Suspended and Deposited Sediments on Estuarine Organisms, PHASE II,'' Reference No. 72-9E, Project Year II, N.R.I., University of Maryland, College Park, Md., 1972. SHERK, J.A., O'CONNOR, J.M., and NEUMANN, D.A., "Effects of Suspended Sediments on Selected Estuarine Plankton,'' MR 76-1, U.S. Army, Corps of Engineers, Coastal Engineering Research Center, Fort Belvoir, Va., Jan. 1976. 57 SIMPSON, G.G., ROE, A., and LEWONTIN, R.C., Quantitative Zoology, Harcourt, Brace and World, Inc., New York, 1960. SLANINA, D., ''Beitrag sur Wirkung Mineralischer Suspensionen auf Fische," EIFAC Technical Paper No. 1, European Inland Fisheries Advisory Com- mission Working Party on Water Quality Criteria for European Freshwater Fish, 1964. SNEDECOR, G.W., and COCHRAN, W.G., Stattstteal Methods, 6th ed., Iowa State University Press, Ames, Iowa, 1967. SOIL TESTING AND PLANT ANALYSIS LABORATORY, "Laboratory Procedures," Cooperative Extension Service, Athens, Ga., 1970. SOUTHGATE , B.A., "Water Pollution Research, 1959," River Pollutton II Causes and Effects, Butterworth, London, 1962. SUMMERFELT, R.; LEWIS, C., and ULRICH, M., ''Measurement of Some Hematol- ogical Characteristics of the Goldfish," Progressive Fish Culturtst, Wools A795) NOs. A ISO 5 Tes: LS—A0)s SWALLOW, R.L., and FLEMING, W.R., "The Effect of Starvation, Feeding, Glucose, and ACTH on the Liver Glycogen Levels of Ttlapta mossambtca,"' Comparative Biochemistry and Phystology, Vol. 28, 1969, pp. 95-106. TALBOT, G.B., "Estuarine Environmental Requirements and Limiting Factors for Striped Bass," A Sympostum on Estuarine Fishes, American Fisheries Society, 1966, pp. 37-49. TRASK, P.D., Recent Marine Sediments, 2d ed., Dover Publications, New York, 1968. WALLEN, I.E., ''The Direct Effect of Turbidity on Fishes," Bulletin, Oklahoma Agriculture and Mechantcal College, Stillwater, Okla., Vol. 435ed 951s hppa 276 WILSON, D.S., "Food Size Selection Among Copepods," Ecology, Vol. 54, No. 4, 1973, pp. 909-914. WILSON, J.N., "Effects of Turbidity and Silt on Aquatic Life," Brologtcal Problems tn Water Pollution, First Seminar, U.S. Public Health Service, Washington, D.C., 1956. WINTROBE, M.M., Clinteal Hematology, Lea and Febiger, Philadelphia, Pa., 1956. 58 APPENDIX A LIVER GLYCOGEN CONCENTRATIONS IN FOUR ESTUARINE FISH AND LIVER GLYCOGEN DEPLETION RATES IN WHITE PERCH iP ielintsoductaon To derive useful data from studies of glycogen utilization in response to suspended solids, several species of estuarine fish were screened and "natural" liver glycogen values were established under field conditions. Glycogen depletion studies were conducted to establish the expected rates of glycogen mobilization in the selected species. Liver glycogen determinations were performed on four species: White perch, striped bass, hogchokers, and spot. A glycogen depletion study was conducted at two temperatures with white perch. 2. Materials and Methods. Liver glycogen was determined for each of the four species within 8 hours of capture. Glycogen was extracted according to the method of Good, Kramer, and Somogyi (1933) and quantified by the phenolsulfuric acid method (Montgomery, 1957). Glycogen mobilization of white perch is dependent on the breakdown of glycogen (a long-chain polymer of glucose) to glucose 6-phosphate (Black, Robertson, and Parker, 1961; Ingram, 1970). This enzymatic reaction is highly dependent on temperature. Glycogen depletion in white perch was determined at 10 + 2° and 20 + 2° Celsius. On the day of capture, white perch were divided into two groups of 60 fish and placed in separate holding tanks maintained at either 10° or 20° Celsius by immersion in a water bath. Ten fish were removed at random from each group at the initiation of the experiment and after 2, 3, 7, and 8 days in the holding tanks. Liver glycogen was deter- mined according to the methods previously described. 3. Results and Interpretation. Mean liver glycogen values for white perch, striped bass, and spot resembled one another on the day of capture (Table A-1). Liver glycogen values for hogchokers were significantly greater than those for the other three species. The precision of liver glycogen determinations was satis- factory in perch, bass, and hogchokers (Table A-1). In spot the variation was greater; however, the standard error of the mean was only 15.5 percent of the mean. These data show that levels of glycogen reserves were rela- tively constant in a population. The rate of glycogen mobilization in starved white perch increased with temperature. At 10° Celsius, glycogen stores decreased by 59.7 Se) Table A-1. Liver glycogen determined on day of.capture for four species of estuarine fish. Species Individuals Liver glycogen SEs Lee Geen | ) (mg 100 mg! + S.E. xz!) | pet of mean 10.5 White ipeeeramenaner ar) Striped bass Hogchoker Spot lstandard error of the mean. percent, from a mean of 5.41 + 0.57 mg 100 mg7! liver at the time of cap- ture to 2.22 + 0.39 mg 100 mg~! (Table A-2). At 20° Celsius, glycogen decreased from 5.41 + 0.57 mg 100 mg~! to 0.098 + 0.015 mg 100 meu eal 98.2-percent reduction over the 8-day holding period (Table A-2). The glycogen mobilization curve for white perch at both temperatures was changed to linear form by straight-line regression. Based on this regression line, mobilization at 10° Celsius occurred at the rate of 0.39 mg 100 mg7! per day. At 20° Celsius, mobilization almost doubled to 0.67 mg 100 mg~! per day. 60 Table A-2. Liver glycogen depletion and regression analysis of white perch at 10° and 20° 1 Liver glycogen value Celsius~. Days after Individuals start (No. ) I+ I+ I+ I+ I+ I+ So 0. So 0. 5. 0. Sr 0. Bo O. I+ Oe Oo COW OM OoOW I+ I+ Regression analysis Temperature Slope Intercept Correlation coefficient (°C) 1Values of mean liver glycogen are given for each sampling day plus or minus standard error of the mean. 6| APPENDIX B HEMATOLOGICAL CORRELATIONS IN ESTUARINE FISH 1. Introduction. Red blood cell count, microhematocrit (packed blood cell volume), and hemoglobin concentration in fish blood have been estimated by several ana- lytical techniques (Hesser, 1960; Anthony, 1961; Larsen and Snieszko, 1961; Larsen, 1964; Summerfelt, Lewis, and Ulrich, 1967; Berinati and Crowley, 1972). From these parameters other useful hematological indexes may be calculated, such as mean microhematocrit and hemoglobin content of individ- ual red blood cells (Wintrobe, 1956; Holton and Randall, 1967). Several attempts have been made to establish predictive correlations between microhematocrit and red blood cell count or. hemoglobin concentra- tion in both marine and freshwater teleosts (Eisler, 1965; Summerfelt, Lewis, and Ulrich, 1967; Houston and DeWilde, 1968). A highly predictive regression of microhematocrit with red blood cell count and hemoglobin con- centration may enable workers to derive useful hematological data from a single microhematocrit measurement without red blood cell enumeration and hemoglobin determination. Houston and DeWilde (1968) showed that an estimate of microhematocrit for the rainbow trout, Salmo gatrdnert, may be used to predict red blood cell counts and hemoglobin concentration in routine assessments of hema- tological status. However, the prediction was not sufficiently exact for research purposes. 2. Materials and Methods. Hematological data were taken from five species common to the Patuxent River estuary: White perch, Morone americana; striped bass, M. saxatilts; spot, Letostomus xanthurus; hogchoker, Trinectes maculatus; and menhaden, Brevoortta tyrannus. Male and female fish from each species were used to study hematological response to suspensions of mineral solids (Sherk and O'Connor, 1971). Fish were captured by otter trawl from the Patuxent River estuary. Blood samples were taken from both the control fish and the fish exposed to sublethal concentrationsjof fuller's earth. The data represent hematology of fish under normal laboratory conditions (18 + 1° Celsius, salinity 5.5 parts per thousand), and fish under stress from suspended sediment. Blood was collected in heparinized pipets and mixed before analysis. Microhematocrit was determined according to methods outlined by Hesser (1960), and was read on an International Equipment Company microcapillary reader. Hemoglobin was determined by the cyanmethemoglobin method. Sam- ples were centrifuged at 11,500 revolutions per minute for 20 minutes to remove red cell nuclei from suspension before taking a reading (Larsen, 1964). Optical density of the hemoglobin samples was determined at 540 62 nanometers, using a Coleman Junior II spectrophotometer. Concentration was plotted against a Hycel (mammalian) standard curve. Red blood cell counts were made at X 100, using an improved Newbauer hemacytometer. A modified Hayme's solution (Heinle and Morgan, 1972) was the diluting medium for red blood cell enumeration. Regression and correlation analyses were done according to Snedecor and Cochran (1967). Simple and partial correlation coefficients were calculated for each combination of parameters. Microhematocrit data were first trans- formed to log to the base 10 (log;g) to permit the use of parametric sta- tistical procedures throughout the analyses. 3. Results and Interpretation. Regression analyses between independent pairs of hematological param- eters in the five fish species showed significant correlation between microhematocrit and hemoglobin concentration in white perch, spot, and striped bass (p < 0.01, Table B-1). Correlation between microhematocrit values and red blood cell counts were also found to be significant in white perch, spot, and hogchokers. Correlation of hemoglobin concentration and red cell counts was significant in white perch and spot (p < 0.01). The significance of correlation is important in estimating a parameter from a statistical relationship between it and another parameter. The predictive capacity of the mathematical model is also important; e.g., the correlation data for white perch. All three paired comparisons are sig- nificant at the 0.01 level; i.e., chances are 1 in 100 (or less) that the relationship established between any two of the blood parameters could be due to chance alone. However, the correlation coefficients (r) differ by as much as 0.21 between the microhematocrit with hemoglobin concentration correlation (0.885) and the red blood cell count with hemoglobin concen- tration correlation (0.676). These correlations may be significant at the same probability levels, but the predictive capacity of the relationship differs. This is shown by the coefficient of determination (r2), a meas- ure of correlation which estimates the proportion of variance accounted for by the correlation. Given a microhematocrit value with the hemoglobin concentration correlation r* = 0.784 from a white perch, the hemoglobin content (y) is estimated by regression, knowing that the microhematocrit value accounts for 78.4 percent of the variance in the hemoglobin concen- tration estimate. In the estimation of red blood cell count from microhematocrit, r2 = 0.464; only 46.4 percent of the variance of the predicted red blood cell count can be accounted for by the microhematocrit. The correlation coefficients were highly significant when ‘estimating two blood parameters from the same microhematocrit value, but the predictive capacity of the former relationship was almost 80 percent; the latter was below 50 percent. The coefficients of determination must exceed 0.75 for a paired rela- tionship before estimated parameters may be used for research purposes. 63 Table B-1. en = eS Striped bass Menhaden (ered paired hematological parameters from estuarine fish. Comparison Microhematocrit versus hemoglobin concentration Microhematocrit versus red blood cell count Hemoglobin concentration versus red blood cell count Microhematocrit versus hemoglobin concentration Microhematocirt versus red blood cell count Hemoglobin concentration versus red blood cell count Microhematocrit versus hemoglobin concentration Microhematocrit : versus red blood cell count Hemoglobin concentration versus red blood cell count Microhematocrit versus hemoglobin concentration Microhematocrit versus red blood cell count Hemoglobin concentration versus red blood cell count Microhematocrit versus red blood cell count Individuals (No. ) 64 4.1040 1.0206 7.2995 9.9183 Correlation coefficient 0.8854 0.6813 0.6760 0.8750 0.5869 0.8373 0.2828 0.5547 Probability Regression, significance of correlation, and coefficients of determination of Coefficient of determination 0.7839 0.4642 0.4570 0.3629 0.3445 0.7011 0.0800 0.1708 0.3077 0.4295 0.2904 0.7451 Coefficients of determination between 0.60 and 0.74 were sufficient in routine estimates of hematological status. Coefficients of determination sufficient for research purposes were found in the correlation of microhematocrit with hemoglobin concentration for white perch and spot (Table B-1). The correlation of microhematocrit with red blood cell count in hogchokers was of no predictive value in parametric estimates for research purposes (x2 = 0.7451). The microhema- tocrit with hemoglobin concentration correlation in striped bass was suf- ficient for routine hematological work (r2 = 0.7011). The correlation of microhematocrit with red blood cell count and the correlation of hemoglobin concentration with red blood cell count, did not account for sufficient variance to use regression methods in predictive estimates of hematological parameters in perch, spot, striped bass, and menhaden (Table B-1). The three hematological parameters are closely related in a physical and biological sense. Microhematocrit measures the percent volume of red blood cells in a sample. Red blood cells in most vertebrates transport the respiratory pigment, hemoglobin. Therefore, the predictive correlations between microhematocrit and hemoglobin concentration in white perch and spot are largely dependent on the quantity of red blood cells present. To establish whether the correlation of microhematocrit and hemoglobin con- centration was significant and independent of red blood cell count, partial correlation coefficients were determined for microhematocrit-hemoglobin concentration-red blood cell count interrelationships. This statistic estimated the correlation of two variables, microhematocrit and hemoglobin concentration; the red blood cell count variable was held constant. Partial correlation coefficients were determined for all species in which the three variables were studied (Table B-2). Partial correlation coefficients showed the relationship of microhematocrit and hemoglobin concentration in white perch and spot statistically independent of red blood cell count (Table B-2). The relationships established for these species have significant value. The ability to estimate blood parameters in estuarine fish from a simply determined value, such as microhematocrit, may facilitate physiological studies of estuarine fish in the field and in the laboratory. These hema- tological studies can estimate stress responses in fish (Hesser, 1960; Summerfelt, Lewis, and Ulrich 1967). Physiological and hematological field studies could increase the value of onsite environmental disturbance studies. Estimates of sublethal effects of various pollutants on fish populations should prove useful to estuarine biologists. 65 Table B-2. Analysis of partial correlation of microhematocrit with hemoglobin concentration eliminating the effect of red blood cell count. Species : Partial Degrees of Probability correlation freedom coefficient! White perch Spot Striped bass Menhaden 66 APPENDIX C PRELIMINARY OBSERVATION ON THROUGH-GUT TRANSPORT OF SUSPENDED SOLIDS BY ESTUARINE FISH 1. Introduction. The objective of this study was to determine the accumulation of par- ticulate matter on the gills and in the alimentary canal of fish exposed to sublethal concentrations of suspended solids. Observations were made on white perch, striped bass, and hogchokers. 2. Materials and Methods. The following suspended solids were used: (a) Kaolinite clays: (1) Hydrite MP, median particle size 9 micrometers. (2) Hydrite Flat-D, median particle size 4.5 micrometers. (3) Hydrite-10, median particle size 0.55 micrometer. (b) Fuller's earth, median particle size 0.50 micrometer. (c) Natural bottom muds taken from Long Point, Patuxent River, Maryland. The particle-size distribution of the natural Patuxent River mud is shown in Figure C-1. Fish were captured by otter trawl in the Patuxent River estuary and transported to the laboratory in a flow-through system of river water. All specimens were starved 72 to 96 hours before exposure to suspended solids, and were not fed during an exposure. Groups of 6 to 10 fish, dependent upon size and species, were exposed to graded concentrations of suspended solids for 24 hours. The gills, stomach, and intestine of each individual were examined to determine the accumulation of suspended solid following an exposure. Accumulation of solids on the gills, in the stomach, and in the intes- tine was scored on a scale from 0 (no accumulation) to 4 (continuous coat- ing of particulate matter). Scoring was based on visual observation by a Single, trained observer. Mean accumulation for each group of fish exposed to each concentration was plotted as a histogram. Replicate exposures of each species to graded concentrations of each solid were not done. Data are from the preliminary observations. 67 100 90 @ (e) “ (e) Pct Finer By Weight o {o) 50 lOO 7 OS 4 3 2 10 O8 O06 Stokes Diameter (/2m) Figure C-l. Particle-size distribution of natural mud collected at Long Point, Patuxent River, Maryland. 68 3. Results. White perch accumulated little Hydrite MP clay (mean particle size 9 micrometers) in a 24-hour period. Accumulation from concentrations of 6 to 13 g 17! was greatest in the intestine and least on the gills. Accu- mulation in the intestine at 9 and 13 g 1-! Hydrite MP clay was approxi- mately double the accumulation at 6 g 17! (Fig. C-2). More Hydrite Flat-D (mean particle size 4.5 micrometers) than Hydrite MP clay particles were accumulated by white perch (Fig. C-3). At 6.7 g 1~!, Flat-D accumulation in the stomach and the intestine was greater than MP accumulation by a factor of three. Less accumulation occurred at 16.3 g 17! than at 6.7 g 17!. Particle accumulation in the intestine exceeded 2 at concentrations of 7, 24, and 37 g 17}. Fuller's earth accumulation on the gills of white perch was greater than either of the kaolinite clays, regardless of concentration (Fig. C-4). Accumulation of fuller's earth on the gills of fish exposed to 6.7, 8.3, and 10.7 g 17! ranged between 1.5 and 2;little or no fuller's earth was detected in the stomachs or the intestines. White perch exposed to suspensions of 6.7 to 36.2 g 17! natural muds for 24 hours, showed greatest accumulation on the gill, in the stomach, and in the intestine at 6.7 g ae (Fig. C-5). Least accumulation was noted aml ie Gill accummlationtwasthagh lath 2549) ¢ 1-1. Accumulation in stomachs and intestines was approximately the same (between 1.5 and 2) at 23.9 amd) S6,2 @ Pe, White perch exposed to lower concentrations of natural muds for 72 hours had approximately the same accumulation scores, from 5.6 to 17.8 g 123 (Fig. C-6). Intestinal accumulation scores remained between 2 and 2.5 over the range of concentrations. Particle accumulation in stomachs and on gills was slightly more variable. Gill accumulation was greatest for esi exposed to 5.6 g 17! and least accumulation occurred at 17.8 @ Wo, Striped bass accumulation scores for Hydrite MP particles ranged from 0.75 to 1.5 which was relatively low (Fig. C-7). Accumulation on the gills, in the stomach, and in the intestines was similar over concentrations rang- ing from 6 to 13 g 17!. Accumulation of Hydrite MP by striped bass was greater than accumulation by white perch by a factor of three or more (Eales (C=2))2 Hydrite Flat-D accumulation by striped bass was greatest on the gill (Fig. C-8). Particle accumulation in stomachs and intestines ranged from ONtonl.2) in) concentrations of 3.3 to I7el ¢ sl, except fora value of 2 in the stomachs of bass exposed to 5 g 17! Flat-D. Single exposure of striped bass and hogchokers to natural muds suspen- sions resulted in large accumulation on the gills, moderate accumulation 69 6 9 13 Suspended Solids Concentration (g £7') Figure C-2. Relative accumulation of solids in white perch exposed to graded concentrations of Hydrite MP (G = gill, S = stomach, I = intestine). rae) 3.0 25 ! I I 2.0 S S 15 I 5 WN 1.0 GS S G 05+ ¢ G ; | | 6.7 16.3 24.| 37.4 Suspended Solids Concentration (g £7!) Figure C-3. Relative accumulation of solids in white perch exposed to graded concentrations of Hydrite Flat-D (G = gill, S = stomach, I = intestine). 7 6.7 8.3 10.7 Suspended Solids Concentration (g £7!) Figure C-4. Relative accumulation of solids in white perch exposed to graded concentrations of fuller's earth (G = gill, S = stomach, I = intestine). 72 4.0 GS G 3.5 ! 3.0 25 2.0 1 I GS G 1.5 1.0 0.5 0 Ba 14.1 23.9 36.2 Suspended Solids Concentration (g £7') Score Figure C-5. Relative accumulation of solids in white perch exposed to graded concentrations of natural Patuxent River mud for 24 hours (G = gill, S = stomach, I = intestine). 73 3.0 G S I 2.5 2.0 ! G 1.5 ss S So | a 1.0 0.5 ) | | 5.6 11.6 17.8 Suspended Solids Concentration (g £7') Figure C-6. Relative accumulation of solids in white perch exposed to graded concentrations of natural Patuxent River mud for 72 hours (G = gill, S = stomach, I = intestine). 74 Score 6 9 13 Suspended Solids Concentration (g £7') Figure C-7. Relative accumulation of solids in striped bass exposed to graded concentrations of Hydrite MP (G = gill, S = stomach, I = intestine). igo 3.0 2.5 G G 2.0 S G I. S ; I I I S 0. S 3.3 3.6 5.0 17.1 Score ro) on on (e) Suspended Solids Concentration (Gg 24) Figure C-8. Relative accumulation of solids in striped bass exposed to graded concentrations of Hydrite Flat-D (G = gill, S = stomach, I = intestine). 76 in the stomachs, and little or no accumulation in the intestines (Figs. C-9 and C-10Q). 4. Conclusions. A mechanism for gill cleansing in white perch, striped bass, and hog- chokers in highly turbid water was entrapment of particulate matter on the gills and transport of entrapped particles through the alimentary canal. Extremely fine particles, such as resuspended bottom muds, fuller's earth, and Hydrite Flat-D, accumulate more than larger particles (Hydrite MP). 5. Additional Observations and Discussion. Microscopic examination of gills revealed a possible mechanism for through-gut transport of suspended particles. Gills examined at X 30 showed that fine particles become entrapped between gill filaments and between the secondary lamellae. Fish exposed to the suspended solids had streams of particle-laden mucus on the gill and attached to the pharyngeal teeth on the inner margin of the gill arch. A function of the pharyngeal teeth is to assist in passing food from the mouth to the esophagus, and it is likely that the mucus streams on the gill and on the pharyngeal teeth were being ingested. However, the hogchoker, a demersal fish, had a reduced accumulation of particulate matter in the gut when exposed to Similar concentrations of the solids used with white perch and bass. Few of these data may be directly compared because of the wide range of suspended solids concentrations. However, it is evident that accumu- lation of particles was much the same in several instances, regardless of concentration. Finer solids accumulated the most; i.e., fuller's earth (75 percent <2 micrometers, median size 0.5 micrometer), Hydrite Flat-D (40 percent <2 micrometers, median size 4.5 micrometers), and resuspended natural muds (70 percent <2 micrometers, median size 0.87 micrometer). Mucus stream transport of particles exposes the entrapped material to normal digestive processes and, thus, to a wide range of chemical environ- ments. Particles are exposed for varying periods of time to acid conditions in the stomach. The pH ranges from 2 to 3 and material undergoes a strong acid hydrolysis. The pH environment changes from acid to moderately basic as material passes from the stomach to the intestine (pH 7 to 8). Hydrol- ysis of food material in the intestine is carried out by enzymatic processes. Particles in the stomach are exposed to approximately the same condi- tions as absorbed materials stripped from particulate matter for chemical analysis. Potentially toxic materials such as heavy metal ions, pesticide residues, petrochemical residues, and various biocides of organic origin, may become available to the organism. Through-gut transport of particles removed from suspension provides an avenue for accumulation of noxious material in the tissue of fish. CU 229 G 2.0 15 S 2 1.0 (ep) 05 I 0 7.8 Suspended Solids Concentration (gq £7') Figure C-9. Relative accumulation of solids in striped bass exposed to graded concentrations of natural Patuxent River mud (G = gill, S = stomach, I = intestine). 78 2.0 Score w 0.5 11.6 Suspended Solids Concentration (g Lely Figure C-10. Relative accumulation of solids in hogchokers exposed to graded concentrations of natural Patuxent River mud (G = gill, S = stomach, I = intestine). 79 APPENDIX D ANALYSIS OF SEDIMENTS 1. Introduction. Experimental work during 1971 and 1972 used artificial (commercially available) mineral solids to provide base-line data for biological effects of (a) different concentrations of solids, (b) different particle-size distributions, and (c) different mineral types of solids. Work during 1973 concentrated on the biological effects of naturally occurring sedimentary material. The material was collected by anchor dredge at Long Point (38°29'30"' N., 76°39'45'' W.) in the Patuxent River and stored in large polyethylene tanks before use in experiments. The sediment surface was covered with a layer of water (salinity range 4 to 6 parts per thousand) to maintain the natural ionic equilibria between sedi- ment and water occurring in the Patuxent River. A microoxidized sediment layer developed at the sediment-water interface in these tanks after a few days of storage. Analyses were performed on both the commercially available mineral solids and the naturally occurring sediments. The sediment characteristics measured were organic matter content (weight loss on ignition), inorgan- ically bound heavy metals (atomic absorption), and particle-size distribu- tions (settling diameter). The particle-size distributions were determined in distilled water, and may represent the basic particles which can be bound into aggregates by atomic and molecular forces. The composite units are stable under dis- persion methods. The basic particles also may form aggregates in saline water; however, these units are relatively weakly bonded by electrostatic forces, surface tension, and ''sticky" organic matter. 2. Materials and Methods. a. Size Distribution. Artificial sediments (mineral solids) were as follows: (a) Kaolinite (1) Hydrite-10 (Georgia Kaolin Company) (2) Hydrite Flat-D (Georgia Kaolin Company) (3) Hydrite MP (Georgia Kaolin Company) (b) Fuller's earth (Fisher No. F-90) Particle-size distributions were determined by the sedimentation method (American Society for Testing and Materials, 1968) for paper-coating clays. 80 The natural sediments collected from the Patuxent River were analyzed as follows: Preliminary work showed this material was approximately 75- to 80-percent salt and water by weight. Appropriate triplicate yolumes. of natural sediments were removed from the holding tanks. The volumes were calculated to contain between 5- and 10-gram inorganic dry solids, and were corrected upward for the weight of organic matter present (see method of analysis below). Measured quantities of solids were placed in 1-liter- capacity Pyrex beakers and an appropriate amount of 30-percent hydrogen peroxide (H,0.) was added. The volume of H20 needed to oxidize the organic matter present in the sediment produced a final 5-percent concentration of H,0 in the sediment volume. The oxidation reaction was initially violent. It proceeded overnight in a hood with air bubbling slowly through the sedi- ment-H20, mixture to remove the excess H209. When gas evolution had ceased, 750 milliliters of deionized, glass- distilled water were added to each beaker. The sediment was resuspended by stirring with a glass rod and allowed to settle. The supernatant was carefully decanted, and another 750 milliliters rinse of deionized, glass- distilled water was added to each beaker. A 0.2-milliliter sample of supernatant water was taken from each beaker and the dissolved ion concentration of each solution was determined with the freezing-point depression osmometer. Salt concentration was read from a standard curve relating freezing-point depression and osmolal concentra- tion to sodium chloride (NaCl) concentration in milligram per kilogram (mg kg-!) water. If the salt concentration was greater than 300 mg NaCl kg water, the suspension was allowed to settle, the clear supernatant was decanted, and an additional rinse of 750-milliliter deionized distilled water was added to each beaker. The sediment was resuspended and allowed to settle. The clear supernatant was decanted and the beaker containing the washed sediment was filled to 500 milliliters with fresh, deionized, glass-distilled water and placed into an ultrasonic bath (45 kilohertz) for 30 minutes. The suspension was placed in a glass cylinder, made up to volume with deionized distilled water, and analyzed as described in American Society for Testing and Materials (1968), but the dispersing agent sodium pyrophosphate (NaiP 207), was not added. il Values are reported as percent by weight remaining in suspension (per- cent finer than) plotted against equivalent spherical diameters according to Stokes' law. b. Organic Matter Content. Samples of the natural sediment collected from the Patuxent River at Long Point were ovendried for 24 hours at 100° Celsius, ground fine with a porcelain mortar and pestle, and ashed for 3 hours at 500° Celsius. Organic matter values are reported as percent of dry weight lost on ignition. No appreciable loss of inorganic carbonate occurred during the ashing procedure, as evidenced by nonsignificant weight losses of calcium carbonate (CaC0O3) samples ashed along with the ovendried natural sediments. BI c. Heavy Metals. Amounts of extractable cations in the mineral solids and in the natural sediment samples were determined through mild acid extraction and atomic absorption analysis hy Mr. Dayid Boon, Seafood Processing Laboratory, Crisfield, Maryland. Tests for inorganically bound cations as described by Soil Testing and Plant Analysis Laboratory (1970) and Perkin-Elmer Corporation (1971) were conducted for zinc, copper, iron, manganese, lead, cobalt, nickel, chromium, and cadmium. Total mercury values are reported from sediments digested for 1 minute in boiling aqua regia (Dow Method, CAS-AM-70.13, 22 June 1970 revised, Chlorine Institute, Madison Avenue, New York, New York). Metal values are mg kg! dry weight of solids. 3. Results and Discussion. a. Size Distributions. Particle-size distributions of the extremely fine mineral solids and the natural sediment are listed in Figure D-1 and Table D-1. Materials are ranked coarsest to finest by median size as follows: Hydrite MP, kaolinite (Georgia Kaolin Company), median size = 9.5 micrometers, <2 micrometers = 12 percent; Hydrite Flat-D, kaolinite (Georgia Kaolin Company), medium size = 4.5 micrometers, <2 micrometers = 34 percent; Patuxent River silt (composite less organic matter fraction, 11.5 percent of dry weight), median size = <0.8 micrometer, <2 micrometers = 72 percent; fuller's earth, montmorillonite, and attapulgite (Fisher No. F-90), median size = <0.5 micrometer, <2 micrometers = 82 percent; Hydrite-10, kaolinite (Georgia Kaolin Company), median size = <0.5 micro- meter, <2 micrometers = 92 percent. Graphic solutions (Folk, 1968) and mathematical calculations (Trask, 1968) can be used to determine the second, third, and fourth moments of these distributions. Additional size-distribution analyses for the natural sediments (by date of collection) are presented in Figure D-2 and Table D-2. Median sizes ranged from a high of approximately 1.1 to a low of <0.5 micrometer (August collection). Fraction by weight finer than 2 micrometers ranged from a high of approximately 82 percent to a low of 65 percent (August collection). These particle-size distributions of solids (Tables D-1 and D-2, Figs. D-1 and D-2) are comparable with those reported by May (1973) in the mudflow from a shell dredge (Table D-3). b. Organic Matter Content. Organic matter content of natural sediment samples tended to increase throughout the summer of 1973 from 8.9 percent in June to over 11 percent in August and September (Table D-4). A compar- ison of mean organic matter values (Table D-5) showed the differences between early and late samples were significant. Organic matter, which has settled out at Long Point, may come from marshes which line the shores of the Patuxent watershed. Organic matter analyses were also conducted on the mineral solids. Ashing caused no significant weight loss in fuller's earth solids. Sub- stantial weight losses in the kaolinites (about 11 percent of dry weight) were attributed to the bound water lost (at temperatures of 500° Celsius) 82 | = Hydrite MP 2=Hydrite Flat-D 3 = Patuxent River Silt 4= Fuller's earth 2 | 10 5 = Hydrite-10 oo Pct Finer By Weight S (oe) SS 90 95 re ae / vA ae Stokes. é. Pe Se NEE: 99 phi 4 5 6 7 8 9 10 i pm 62.5 31.3 15.6 7.8 3.9 1.95 0.98 0.49 Figure D-1. Particle-size distribution of sediments used in this project. 83 "[-@ ean3ty UT SOUTT 03 Jezoad sasoyjuerted ut sLoqunn, * 19} OWOLTO TU UT JojJoWeTp ,SeyxoIs ueYyI WYySTEM Aq TOUTF (JuUddI1ed se pesseidxe) UOoTJOeIF = LOUTF JUSdIEd, (=). Gl) 2 ArTNONMNAO e Amn~oOnANAOr mM OnAatON rE ise) MrmRONNMNMme-A nD Or 6°0 Loa vee br) 8°72 ies T £°6 eonn~nrtToOouoOo ee Oa a cay BBN E TD Ae) (1) dW 237T2pAH (Z) d-381d 99TIpAH (Ss) 0T-93T2p4H z(p) yqtee s,JeTINg *,z9eford Sty UT pesn SjUOWTpes TRTOTJTILe FO SUOTINGTIISTP 9ZTS-aTITIWeq “T-d STIL 84 Collection Date Sample No. tS) 14 Mar. 1973 4 12 June 1973 5 10 27 Aug. 1973 1,6 25 Sept. 1973 23 Pct Finer By Weight 98 99 phi 7 8 9 10 1 um 7.8 3.9 1.95 0.98 0.49 Stokes Diameter Figure D-2. Particle-size distributions of natural Patuxent River silt samples (two replicate determinations) collected by anchor dredge at Long Point. 85 *Z-@ ean3ty ut siaqumu atdues 03 teje1 sesoyquered ut szequmn, *IaZWOIOT UT IoJOWeTp ,Sayxo0IS ueYy YBtTEem Aq LouTF (qued1ed se passaidxa) uot IVF = IOUTF aussie ° ° ° ° Sononanam ° no @ oO 0O ° ° ° ° ° nnoonns NOomMnNnsTnmMNOM eA ° e 8 orTMANAAA BOO orTDUMNMNOAM ° ee TMOANAN AAA HOO omonoonoma ° 8°0 6°0 OT Cm eT s‘T 6°2 6°v (s) 7. prow ies 82 asn3ny £2 aunt ZT yore vT * £261 SuLInp 2UTOg BuoT 1e pazIeT{[OD satdues juouTpes ZaATYyY QUSXNZeg [eINqeU sATJeJUSSeIdeL JO SUOTINGTIISTP 9ZTS-9TITIWeg *Z-d PIGEL 86 E91 LE lEG Heel ebalag un 6T 02 gg | wn 6¢ 03 79 (2y8tem Aq 39d) a8uet 9ZTS as1eyostp WOLF 9dULIST "(fL6T ‘AeW) e8perp TTeys B WoLZ MOT FpNU ay} UT SPT[TOs pepuedsns Fo AYystem Aq sodejuedted ezts-9TIT eq “¢-d STqPL 87 Table D-4. Organic matter content of natural mud collected by anchor dredge from the Patuxent River (Long Point)!. Collection date | Sample No. Organic content Standard error of the mean (1973) + standard deviation) 9.412 - 0038 10.358 .0272 10.940 - 3270 - 3808 -4127 -6079 0.3397 0.3205 0.2740 0.1762 11.4217 11.8750 11.2200 and pestle, then ashed for 3 hours at 500° Celsius. Organic matter values reported are percent loss of dry weight on ignition. Table D-5. Comparison of means of organic matter determinations by collection date. Sample collection dates (1973) 17 June p < 0.001 28 June ° p < 0.001 14 July p < 0.001 27 Aug. 18 Sept. 1Not significant. 88 from these clays (Michael Taranto, Georgia Kaolin Company, personal com- munication, 1973). c. Heavy Metals. The mineral solids contained biologically insignif- icant amounts of metal (Table D-6). The values reported for Patuxent silt (Long Point) are in the "natural" range of metal found in similar estuarine salinity ranges by Huggett (Virginia Institute of Marine Science, personal communication, 1973) in the York, the James, and the Elizabeth Rivers, which drain into the Virginia part of the Chesapeake Bay system. 89 *pe3sed ION, - ‘ueou 94} JO TOILE pLepueys F ULdW, *,- 34 Su ore sonte, *Adoosorq9eds uot ydazosqe atuojye Aq stskTeue pue poe ITINF[Ns-dTLoTYyoorpAy [ewxLou ¢/0°Q Aq WOTIDeIIXY, ($1 ziTts Juexnzed yqiee s,JeT [ny dW 93TIpAH 93TIpAH 01-93 TIpAH qUSsWe TY *,JUAUTpes [TeInzeU puke TeTOTFTIIe UT SUOTIed puNog AyyTeotuesi1ouy *9-q eTqeL 90 £79 €-LL *ou daigcn* €0ZOL “€000-9-1L2-ZLMOV 39P1RU0D = *1aRUeD YoIReSEeY SuTiseuTsug TeIseoD *S*n : SeTIes “AI ‘E-// ‘ou azaded Teotuyoay ‘equej yoieasey BuTiseuTsuq [eq3seoD *S*f : SeTIesS *[I] ‘Aoyjne qutof **y°q ‘uuewneN “IIT "eTIFL “I ‘puepArey ‘iteaTy Juexnqeg ‘4 “ABoTODe suTIeNnjs_ *€ “*SeYysT_ *Z ‘*SjJueWTpes papuedsns *1 *yqIee S,AeTTNJ eTqgeTteae AT[TetToteummod Jo ‘pueTAieW ‘Azenjsa AeATY Juexnjeg ay} wojz peutejgo ‘jueutpes [einjeu jo 319M suofsuedsns ay, ‘*swezsks ouTienqse ut ySTjJ 94} UO sTeTiejzeW papuedsns Jo suoTjerqusoU0D TeYyJeTqns jo ‘Aue JF *s}0esFe By aUTWIejzep 02 sem Apnqs sty Jo aAFIoelqo sayy ‘yg *d : AydersotTqrg *pueTArey jo AjRTSireafun faqgnqzTAsuy seoinosey TeinqenN Aq peszedeig (€000-0-12-Z2MOVd * 1e3U99 yoieesey BufiseutTzug [e}seog *s*n — seded Teotuysel,) “TIT : *d 6g “/L6, ‘19qUeD YoIessey BuTiseuT3ug TeyseoD *S*n : “eA fATOATeg FA0q — *[*Te Jo] ***uueUNeN *y'q ‘10uU0D,0 ‘W'r 4q / YSTJ sUTIeNIsa uo sjUueUTpes pepuedsns jo sjoezze TeyIeTQnsS “Wp ‘zouu0) .O £29 €-2£ ‘ou daigcn* €0ZOL “€000-0-1L-7LMOVA 39P1}U0D §=*A9}USD YIPEeSeYy SuTiseutsug TeIseoD *S*n : Setzeg “AL ‘“€-// “ou azvded Teoruyay ‘leqUeD YOieesey BSuTAeeuTSuq Te IseoD *S*p : SeTIeg “III ‘Aoyqne jqutof ‘*y*q ‘uuewneN “II ‘“aTATL “I ‘puepArey] ‘teaTY Juexnqeg *y *ASoTOD9 sutTaenisg *€ ‘“‘SoUustq *Z ‘“*SjueuTpes pepuedsns *1 *yqaee S,laT [NJ eTqgeTteae ATTepforeumos 10 ‘pueTArzey ‘AieNjse AeATY Juexnjeg asyq worFZ peuteqjgo ‘juewtpes [Teinjeu jo 3v1em suotsuedsns ayy *suaqshs suTIienqsa ur 4ST} 9y} UO STeTiezeU pepuedsns Jo suoT}eIqUaDUOD TeYJeTqns jo “Aue JT ‘sqoeyye ay} euTMIeqap 079 sem Apnjs sty? 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