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NMFS is also charged with the development and implementation of policies for managing national fishing grounds, development and enforcement of domestic fisheries regulations, surveillance of foreign fishing off United States coastal waters, and the development and enforcement of international fishery agreements and policies. NMFS also assists the fishing industry through marketing service and economic analysis programs, and mortgage insurance and vessel construction subsidies. It collects, analyzes, and publishes statistics on various phases of the industry. The Special Scientific Report—Fisheries series was established in 1949. The series carries reports on scientific investigations that document long-term continuing programs of NMFS, or intensive scientific reports on studies of restricted scope. The reports may deal with applied fishery problems. The series is also used as a medium for the publica- tion of bibliographies of a specialized scientific nature. NOAA Technical Reports NMFS SSRF are available free in limited numbers to governmental agencies, both Federal and State. They are also available in exchange for other scientific and technical publications in the marine sciences. Individual copies may be obtained (unless otherwise noted) from D83, Technical Information Division, Environmental Science Information Center, NOAA, Washington, D.C. 20235. Recent SSRF’s are: 619. Macrozooplankton and small nekton in the coastal waters off Vancouver Island (Canada) and Washington, spring and fall of 1963. By Donald S. Day, January 1971, iii + 94 pp., 19 figs., 13 tables. 620. The Trade Wind Zone Oceanography Pilot Study. Part IX: The sea-level wind field and wind stress values, July 1963 to June 1965. By Gunter R. Seckel. June 1970, iii + 66 pp., 5 figs. 621. Predation by sculpins on fall chinook salmon, Oncorhynchus tshawytscha, fry of hatchery origin. By Benjamin G. Patten. February 1971, iii + 14 pp., 6 figs., 9 tables. 622. Number and lengths, by season, of fishes caught with an otter trawl near Woods Hole, Massachusetts, September 1961 to December 1962. By F. E. Lux and F. E. Nichy. February 1971, iii + 15 pp., 3 figs., 19 tables. 623. Apparent abundance, distribution, and migrations of albacore, Thunnus alalunga, on the North Pacific longline grounds. By Brian J. Rothschild and Marian Y. Y. Yong. September 1970, v + 37 pp., 19 figs., 5 tables. 624. Influence of mechanical processing on the quality and yield of bay scallop meats. By N. B. Webb and F. B. Thomas. April 1971, iii + 11 pp., 9 figs., 3 tables. 625. ‘Distribution of salmon and related oceanographic features in the North Pacific Ocean, spring 1968. By Robert R. French, Richard G. Bakkala, Masanao Osako, and Jun Ito. March 1971, iii + 22 pp., 19 figs., 3 tables. 626. Commercial fishery and biology of the freshwater shrimp, Macrobrachium, in the Lower St. Paul River, Liberia, 1952-53. By George C. Miller. February 1971, iii + 13 pp., 8 figs., 7 tables. 627. Calico scallops of the Southeastern United States, 1959-69. By Robert Cummins, Jr. June 1971, iii + 22 pp., 23 figs., 3 tables. 628. Fur Seal Investigations, 1969. By NMFS, Marine Mammal Biological Laboratory. August 1971, 82 pp., 20 figs., 44 tables, 23 appendix A tables, 10 appendix B tables. 629. Analysis of the operations of seven Hawaiian skipjack tuna fishing vessels, June- August 1967. By Richard N. Uchida and Ray F. Sumida. March 1971, v + 25 pp., 14 figs., 21 tables. For sale by the Superintendent of Documents, U.S. Government Printing Of- fice, Washington, D.C. 20402. 630. Blue crab meat. I. Preservation by freezing. July 1971, iii + 13 pp., 5 figs., 2 tables. Il. Effect of chemical treatments on acceptability. By Jurgen H. Strasser, Jean S. Lennon, and Frederick J. King. July 1971, iii + 12 pp., 1 fig., 9 tables: 631. Occurrence of thiaminase in some common aquatic animals of the United States and Canada. By R. A. Greig and R. H. Gnaedinger. July 1971, iii + 7 pp., 2 tables. 632. An annotated bibliography of attempts to rear the larvae of marine fishes in the laboratory. By Robert C. May. August 1971, iii + 24 pp., 1 appendix I table, 1 appendix I table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 633. Blueing of processed crab meat. II. Identification of some factors involved in the blue discoloration of canned crab meat Callinectes sapidus. By Melvin E. Waters. May 1971, iii + 7 pp., 1 fig., 3 tables. 634. Age composition, weight, length, and sex of herring, Clupea pallasii, used for reduc- tion in Alaska, 1929-66. By Gerald M. Reid. July 1971, iii + 25 pp., 4 figs., 18 tables. 635. A bibliography of the blackfin tuna, Thunnus atlanticus (Lesson). By Grant L. Beardsley and David C. Simmons. August 1971, 10 pp. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 636. Oil pollution on Wake Island from the tanker R. C. Stoner. By Reginald M. Gooding. May 1971, iii + 12 pp., 8 figs., 2 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 637. Occurrence of larval, juvenile, and mature crabs in the vicinity of Beaufort Inlet, North Carolina. By Donnie L. Dudley and Mayo H. Judy. August 1971, iii + 10 pp., 1 fig., 5 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 638. Length-weight relations of haddock from commercial landings in New England, 1931-55. By Bradford E. Brown and Richard C. Hennemuth. August 1971, v + 13 pp., 16 figs., 6 tables, 10 appendix A tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 639. A hydrographic survey of the Galveston Bay system, Texas 1963-66. By E. J. Pullen, W. L. Trent, and G. B. Adams. October 1971, v + 13 pp., 15 figs., 12 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 640. Annotated bibliography on the fishing industry and biology of the blue crab, Callinectes sapidus. By Marlin E. Tagatz and Ann Bowman Hall. August 1971, 94 pp. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 641. Use of threadfin shad, Dorosoma petenense, as live bait during experimental pole- and-line fishing for skipjack tuna, Katsuwonus pelamis, in Hawaii. By Robert T. B. Iversen. August 1971, iii + 10 pp., 3 figs., 7 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 642. Atlantic menhaden Brevoortia tyrannus resource and fishery—analysis of decline. By Kenneth A. Henry. August 1971, v + 32 pp., 40 figs., 5 appendix figs., 3 tables, 2 appendix tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 643. Surface winds of the southeastern tropical Atlantic Ocean. By John M. Steigner and Merton C. Ingham. October 1971, iii + 20 pp., 17 figs. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 644. Inhibition of flesh browning and skin color fading in frozen fillets of yelloweye snapper (Lutzanus vivanus). By Harold C. Thompson, Jr., and Mary H. Thompson. February 1972, iii + 6 pp., 3 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 645. Traveling screen for removal of debris from rivers. By Daniel W. Bates, Ernest W. Murphey, and Martin G. Beam. October 1971, iii + 6 pp., 6 figs., 1 table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 646. Dissolved nitrogen concentrations in the Columbia and Snake Rivers in 1970 and their effect on chinook salmon and steelhead trout. By Wesley J. Ebel. August 1971, iii + 7 pp., 2 figs., 6 tables: For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 647. Revised annotated list of parasites from sea mammals caught off the west coast of North America. By L. Margolis and M. D. Dailey. March 1972, iii + 23 pp. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. Continued on inside back cover. U.S. DEPARTMENT OF COMMERCE Frederick B. Dent, Secretary NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION Robert M. White, Administrator NATIONAL MARINE FISHERIES SERVICE NOAA Technical Report NMFS SSRF-677 Abundance of Benthic Macroinvertebrates in Natural and Altered Estuarine Areas GIL GILMORE and LEE TRENT yOUTOn, & % = Bs 3 Z Seattle, WA z BD April 1974 2 = vs ~~ 776 -491° WEE MaDe of Documents, U.S, Government Printing Office The National Marine Fisheries Service (NMFS) does not approve, rec- ommend or endorse any proprietary product or proprietary material mentioned in this publication. No reference shall be made to NMFS, or to this publication furnished by NMFS, in any advertising or sales pro- motion which would indicate or imply that NMFS approves, recommends or endorses any proprietary product or proprietary material mentioned herein, or which has as its purpose an intent to cause directly or indirectly the advertised product to be used or purchased because of this NMFS publication. Introduction Study area and methods Station description and environmental data Relative abundance Comparison between canal, marsh, and bay Discussion a i) = So 0> CONTENTS Figures Study area and sampling locations in the Jamaica Beach area of West Bay, Texas .......... Sand, silt, and clay fractions of the sediments by station and zone ...................-005- Average volumes of organic matter (cord grass roots, submerged marine grasses, and organic detritus combined) by station and zone, March-October 1969 ............. 0.0000 ee ee eee Average volume of organic matter by date, station, and zone ................. eee eeeeeeeee Average dissolved oxygen by date for all six stations combined and for the stations having theshishesiwandulowestgaveracenvalllc summer tars eaceistcic errs kicieiskec iin ocfeectere esters oii Average numbers and volumes (stations combined) of organisms caught per sample by taxonomic.eroupiandy zone) March-October O69 i lies yarcisverapst dei ree casieveheve) #1 ais. snersie eis ensauele Average number of polychaetes caught per sample by date, station, and zone .............. Average number of crustaceans caught per sample by date, station, and zone .............. Average number of pelecypods caught per sample by date, station, and zone ............... Average number of nemerteans caught per sample by date, station, and zone............... Average numbers and volumes of benthic macroinvertebrates caught per sample by area, taxonomic group, and for the groups combined, March-October 1969 ................---. Tables List of taxonomic groups and total numbers of specimens collected by station and zone ..... Comparisons between zones by taxon and station of the mean numbers of organisms col- lectedk(pained=comparisoni/-test) nner eer ORO EGC GORE CR ne toner Comparison between stations by taxon and zone of the mean numbers of organisms collected (woowavaeanaly sis) Ofsvallance) iy rer rene ceteris Sey alewe layin shah unshared), ac a ance ae Appendix Tables Numbers of animals caught by station, date, taxon, and volume of two samples in shore AYU SASH OO OODOPOD OD DONO DOOUDDOOOOU DU OO IUOOOUC OOOOH OOOO pOOD OO DOD ODO ODDO OOO OUDU Oto Numbers of animals caught by station, date, taxon, and volume of two samples in COTIAE GEARS BIVENS or Gro. DAREN CONC Ore Oe NEE ORCL Sr CORT ee DRE CE REL Cs CME OEIC Gea tetans Ory ata ill a) tet} gQ oO NINANN OD WwW won fpNNe [o-e) in Abundance of Benthic Macroinvertebrates in Natural and Altered Estuarine Areas! GIL GILMORE? and LEE TRENT? ABSTRACT The abundance of benthic macroinvertebrates during March-October 1969 in West Bay, Texas, was compared between 1) a natural marsh area, 2) an adjacent marsh area altered by channelization, bulk- heading, and filling, and 3) an open bay area. Animals representing four phyla were caught. Abundance indices (areas combined) of the four groups in terms of numbers were 66.4% polychaetes, 29.6% crusta- ceans, 2.5% pelecypods, and 1.5% nemerteans; volumes were 44.0% polychaetes, 40.8% pelecypods, 10.7% nemerteans, and 4.4% crustaceans. When all organisms were combined, they were slightly more abundant numerically and over twice as abundant volumetrically in the marsh than in the canals and were least abundant in the bay. Polychaetes were most abundant in the canals and least abundant in the bay; abundance was highest at stations with low to intermediate amounts of silt and clay or where vegetative matter was composed mostly of live sea grasses or detritus. Crustaceans were more abundant in the natural marsh than in the other two areas and showed a definite preference for sandy substrate in marsh areas. Pelecypods were numerically most abundant in the bay but volumetrically the marsh had the highest standing crop. Nemerteans were most abundant in the marsh and least abundant in the bay. In general, the seasonal abundance of polychaetes and nemerteans varied little during the study, whereas crustaceans and pelecypods were abundant only during the spring and early summer. An exception to this seasonal abundance pattern was the reduction in numbers of polychaetes at the upper- most canal station where the habitat was apparently unsuitable due to low oxygen levels during the summer and early fall. INTRODUCTION Development of bayshore property into housing sites by dredging, bulkheading, and filling is occurring in many estuaries. When this property is developed, shallow bay and tidal marsh areas are dredged out or filled with spoil, thus changing the environment for marine organisms. The effects of the resulting en- vironmental changes on the abundance of benthic or- ganisms are poorly understood. Some studies on the succession and abundance of benthic marine animals in canals following excavation indicate that succession may occur rapidly and that climax communities may be established within 2 yr (Brandt, 1897; Reish, 1956, 1957). For example, peaks in the number of species and specimens of benthic invertebrates were reached about 2 yr after seawater entered a newly constructed boat harbor in California, and the abundance and species diversity in the harbor * Contribution No. 335, Galveston Laboratory, Gulf Coastal Fisheries Center, National Marine Fisheries Service, Galveston, Texas. * Marine Laboratory, Texas A&M University, Galveston, TX 77550. ® Panama City Laboratory, Gulf Coastal Fisheries Center, Na- tional Marine Fisheries Service, NOAA, Panama City, FL 32401. 2 yr after opening were comparable to those in adja- cent natural areas (Reish, 1961). In other cases, however, climax communities may be altered or natural succession may not occur. Reish (1961) found that the benthic population in a boat har- bor in California decreased markedly during the third year, probably because of low dissolved oxygen. Taylor and Saloman (1968) concluded that soft de- posits in the canals of a Boca Ciega Bay housing de- velopment in Florida were in some way unsuitable for most benthic invertebrates that were found in the natural areas of the same bay. After 10 yr, recoloniza- tion of canal sediments by benthic organisms was neg- ligible, and it appeared doubtful that soft sediments of the canals would ever support a rich or diverse in- fauna. The habitat in Boca Ciega Bay was, however. changed drastically by channelization of the natural bay area which was sandy and shallow, and had con- tained numerous turtle grass (Thalassia testudinum) beds and oyster reefs. The objectives of our study were 1) to determine the relative abundance of benthic macroinvertebrates in three habitats: a natural marsh characterized by cord grass (Spartina alterniflora) and by sparse, submerged vegetation; a previously similar area altered by dredg- ing, bulkheading, and filling; and an open bay area; and 2) to relate invertebrate abundance within each area to sediment particle size, amount of plant matter, and dissolved oxygen. STUDY AREA AND METHODS The study area in West Bay, Texas, a part of the Galveston Bay System (Fig. 1), included a natural marsh area, an open bay area, and an area that was similar to the natural marsh prior to alteration by channelization, bulkheading, and filling for the Jamaica Beach housing development. The developed area, which included about 45 hectares of emergent marsh vegetation, intertidal mud flats, and subtidal water area prior to alterations was reduced to about 32 hectares of subtidal water area by dredging and filling between 1958 and 1960. The water volume (mean low tide level) was increased from about 184,000 to about 394,000 cubic meters. Six sampling stations—two in the canals of the al- tered area, three in the natural marsh, and one in the open bay—were established (Fig.1). The stations were numbered nonconsecutively and correspond to those reported by Trent, Pullen, and Moore (1972). Sam- pling was conducted in two zones, ‘‘shore’’ and “center,” at each station except at the open bay sta- tion. In the altered area, shore samples were taken on each side of the canal at each station (1 and 4) 1 m away from each bulkhead; center samples were taken near the center of the canal along a transect perpen- dicular to the bulkheads. Replicate samples were taken from the center at each station, so that two sam- ples were available from each zone for each sampling date; the average of the two samples was used as the observation in the statistical tests. Samples similar to those from the canal area were taken at stations 6, 7, and 8 in the intertidal zone adjacent to the cord grass and near the center of the bayou or lake along tran- sects perpendicular to the shoreline. Replicate sam- ples were taken at station 10 in the open bay. Bottom samples were taken at 14-day intervals from 25 March to 21 October 1969, with a metal cylinder 14 cm long and 9.6 cm in diameter. To obtain a sample the cylinder was pressed about 11 cm into the bottom sediments, capped on each end with plastic lids, and brought to the water’s surface. Each sample contained about 800 cm® of bottom materials and represented a surface area of 1/138 m?. The samples were refrigerated within 2 hr after col- lection. The following day, each sample was emptied into a sieve having a mesh size of 420 um, and the material was washed until the fine sediments passed through the sieve. The remaining material, including macroinvertebrates, was stored in a 10% Formalin solution. Macroinvertebrates and plant material in the pre- served samples were separated from the shell and sand. Animal volume for each pair of samples was determined to the nearest 0.1 ml by displacement in a graduated 10-ml centrifuge tube containing a previ- ously recorded volume of water. The volume of each phylum taken at each station and zone was determined at the end of the study by combining the individuals of a phylum from the 32 samples. The volume of plant material was determined to the nearest milliliter in a 30-ml graduated cylinder. The animals were separated and identified, usually to family, and the number of individuals in each group was recorded for each pair of samples. As suggested by Holme (1964), only whole animals or portions of animals containing the anterior end were counted to avoid recounting the same animal. The dissolved oxygen content of the water, taken 15 cm above the bottom at the center habitat of each station, was measured using a modified Winkler method. The water samples were taken about midday and midnight during the same 24-hr period that the bottom samples were collected. Samples for sediment analyses were taken at each station and zone on 12 August. Particle-size composi- tions (percents by weight) were determined using a series of sieves and soil hydrometers. Ranges in parti- cle size were: sand, 2.0-0.62 mm; silt, 0.061-0.004 mm; clay, 0.0039-0.001 mm. STATION DESCRIPTION AND ENVIRONMENTAL DATA Sampling stations were located at various distances from the West Bay shoreline (Fig. 1). Water depths (mean low tide level) in the shore zone ranged from 0.0 to 0.6 m and in the center zone from 0.2 to 1.6 m. The percentage compositions of bottom sediments varied considerably between stations and between zones (Fig. 2). In the canals, silt and clay components were more abundant in the center than in the shore zone. In the marsh, compositions of sediments were similar between zones at stations 7 and 8, but at station 6 a higher percentage of silt and clay occurred in the center than in the shore zone. The plant material taken in the samples was com- posed of live cord grass roots, attached marine grasses (mostly Diplanthera wrightii), algae (mostly Ectocarpus sp.), and detritus. In general, all organic matter collected at the canal stations (both zones) and in the open bay consisted of detritus and small amounts of attached algae. Cord grass roots were dominant in the samples from the shore of the marsh, whereas in the center, detritus was usually dominant, although attached grasses and algae were also present. The volume of plant material was many times great- er in the shore zone (stations 6, 7, and 8) of the marsh than in either zone within the canals (Fig. 3). Most of this vegetation, however, was not decomposed; thus, detrital and filter feeders could not utilize it. Differences in the volume of plant material in the center zones of all stations indicated that a major source of plant material in the canals originated from an outside source. The lowest volume of plant material GALVESTON BAY SYSTEM Figure 1.—Study “area and sampling locations in the Jamaica Beach area of West Bay, Texas. CANAL leak 60F _ it 40-+ MARSH BAY PERCENT SHORE 4of CENTER PERCENT 80F 100 A 6 . STATION Figure 2.—Sand, silt, and clay fractions of the sediments by station and zone. occurred at station 1, the farthest station from the open bay. Plant volume at station 4 (close to the bay) was similar to the volumes at stations 6 and 7 in the marsh. The highest volume of plant material occurred at station 8 where attached sea grasses were more abundant than at any other station. These grasses probably were effective in trapping detritus as it was flushed from the marsh by tidal action. Because almost no detritus was observed in the sediments from the open bay station, we think that most of the detritus in the altered area originated in the adjacent marsh. MILLILITERS Yy 7 | 7), _A_ m= lA | ZAE\Z. : 1 4 6 7 8 1 Figure 3.—Average volumes of organic matter (cord grass roots, sub- merged marine grasses, and organic detritus combined) by station and zone, March-October 1969. Seasonally, the volume of plant material changed little in the shore zone at stations 6, 7, and 8 where cord grass roots were abundant, but at the stations where detritus dominated the volume was much great- er during spring and early summer than in late summer and fall (Fig. 4). Dissolved oxygen values were consistently lower at station | in the deadend canal than at the other stations (Fig. 5); the observed values remained below 3 m1/liter from June through mid-August. During this period, zero oxygen values were observed at station 1 on three occasions (Corliss and Trent, 1971). Loa itr T T T T io Teen T Tea ol aa aaa T ~ | ah 77»SHORE STATION ae | ile O.5-F OF 1OF 5h (245}) 5.0F 40 20 nw- MILLILITERS 10 ob wr > —P | QE 4 1 1 4 1 4 4 = 1 4 1 JL 1 1 | 25 8 226 20 3 17 | 15 29 l2 26 9 23 7 2i MAR APR. MAY JUN JUL AUG SEP Oct. Figure 4.—Average volume of organic matter by date, station, and zone. LILITERS PER LITER Figure 5.—Average dissolved oxygen by date for all six stations com- bined and for the stations having the highest and lowest average val- ues. RELATIVE ABUNDANCE During the study, 8,397 specimens of macroinver- tebrates belonging to four phyla were collected (Table 1). The numbers of animals caught by station, family (phylum for nemerteans), date, and zone, and the vol- ume of the two samples by station and zone are shown in Appendix Tables 1 and 2. Polychaetes (Annelida) Table 1.—List of taxonomic groups and total numbers of specimens collected by station and zone. Station and zone Taxonomic group 1 4 6 7 8 10 Stations combined Sie S} Ce S} Ce Ss} G2 Si (ee S3 (CH S} (Cx Phylum Annelida: 480 292 1,631 234 399 174 281 309 S19 ees (*) 275 3,110 2,465 Class Polychaeta: Family: Nereidae 3 0 ul 3 28 20 68 40 36 247 ee) 15 142 102 Terebellidae 0 0 3 1 i 2 3} 6 8 IP ©) 2 21 23 Capitellidae 432) 2905 136015 9 9229 348 148 204 241 268 ~~ 1,100 + 1G) 188 2,853 2,196 Maldanidae 0 0 0 0 0 0 2 0 0 6) 1G): 0 2 6 Arenicolidae 0 0 0 0 7 0 0 2 3 0 (3) 0 10 D Unidentified 45 D: 20 1 9 4 4 20 4 39 (°) 70 82 136 Phylum Arthropoda: 197 16 11 34 967 123 240 235 322 333 (°) 4 1,737 745 Class Crustacea: Family: Ampeliscidae 0 0 9 32 318 98 92 228 64 313 (3) 4 483 675 Corophiidae 186 0 1 D) 257 17 18 5 62 qf (°) 0 524 31 Pinnotheridae 2 0 0 0 0 0 0 0 0 ): 1 0) 0 2 0 Unidentified 9 16 1 0 392 8 130 2 196 13 (°) 0 728 39 Phylum Mollusca: 4 0 33 24 13 2 4 4 9 82 (°) 38 63 150 Class Pelecypoda: Family: Tellinidae 0 0 8 18 3 0 0 2 0 Si) 6 11 34 Solecurtidae 3 0 4 0 7 0 4 0 6 64 (3) 0 24 64 Mactridae 1 0 0 0 0 0 0 0 0 Oe ©) 0 1 0 Mytilidae 0 0 0 0 2 0 0 0 0 OWN Fe) 0 2 0 Solenidae 0 0 0 0 0 0 0 0 0 Oi) 8 0 8 Semelidae 0 0 21 6 1 2 0 2 3 10 () 24 25 44 Phylum Nemertinea: 14 0 10 6 19 8 14 14 29 105° 1@) 3 86 4] Shore. 1 2 Center of waterway. 3 No sampling. were dominant comprising 66.4% of the number and 44.0% of the volume of organisms caught (Fig. 6). Crustaceans (Arthropoda) were second in number (29.6%) but lowest in volume (4.4%). Third in abun- dance (2.5%), but second in volume (40.8%), were cal- cified pelecypods (Mollusca). Nemerteans (Nemer- tinea) were lowest in number (1.5%) and third in vol- ume (10.7%). The average number of organisms of each phylum collected and the results of statistical comparisons of the data by zone and station are shown in Tables 2 and 3. Abundance values were compared between zones at each station with a paired t-test and between stations within each zone with a two-way analysis of variance. The average of two samples was used as the observa- tion. Average total catch (all phyla combined) was higher in the shore zone than in the center zone, but this difference was not consistent for all phyla or stations (Fig. 6, Table 2). In the canals (stations 1 and 4), the average catches for each phylum were greater along shore than in the center zone with the exception of crustaceans at station 4. Only 5 of the 16 differences, however, were statistically significant. Differences in average catch between zones in the marsh varied greatly among stations. At station 6 all phyla were caught in greater numbers along shore, significantly so for polychaetes and crustaceans. At station 7, average catches for each phyla were about the same in each zone. At station 8 the average numbers of polychaetes and pelecypods were significantly greater in the center, whereas nemerteans were significantly more abundant along shore. Staustically significant differences in abundance be- tween stations were found for each taxonomic group in each zone except nemerteans in the shore zone (Table 3). In the shore zone, average catches were highest at station 4 for polychaetes and pelecypods, at station 6 for crustaceans, and at station 8 for nemerteans. In the center zone, average catches were highest at station 8 for all groups except nemerteans, for which average catch was greatest at station 7. In general, catches of polychaetes and nemerteans exhibited only slight seasonal variations; crustaceans AVERAGE NUMBER AVERAGE VOLUME (ML) SHORE =] CENTER= POLYCHAETA CRUSTACEA PELECYPODA NEMERTEA Figure 6.—Average numbers and volumes (stations combined) of or- ganisms caught per sample by taxonomic group and zone, March- October 1969. Table 2.—Comparisons between zones by taxon and station of the mean numbers of organisms collected (paired-comparison /-test). Zone Taxon Station Shore Center d.f. t --— Mean number ---- Polychaeta 1 15.00 9.12 14 1.97 4 50.97 7.31 15 18.46 6 12.47 5.44 15 3} 117) q/ 8.78 9.66 15 —0.67 8 9.97 36.91 15 1—6.36 Crustacea 1 6.15 0.50 4 1.05 4 0.34 1.06 5 —1.38 6 30.22 3.84 11 22.53 7 7.50 7.34 10 0.05 8 10.06 10.41 9 —0.06 Pelecypoda 1 0.12 0.00 3 16.13 4 1.03 0.75 10 0.90 6 0.40 0.06 5 1.89 7 0.12 0.12 3 0.00 8 0.28 2.56 14 22.31 Nemertinea 1 0.44 0.00 8 16.42 4 0.31 0.19 8 0.77 6 0.59 0.25 9 1.36 a 0.44 0.44 9 0.00 8 0.91 0.32 12 9D) 1% significance level. 5% significance level. 1 2 Table 3.—Comparisons between stations by taxon and zone of the mean numbers of organisms collected (two-way analysis of variance). Station F-values Zone Taxon nn 1 4 6 7 8 10 Block Treatment wana nnn anne nnn nanan nena nee Mean number ----------------------------- Shore Polychaeta 15.00 50.97 12.47 8.78 9.97 (3) 1.19 197.75 Crustacea 6.15 0.34 30.22 7.50 10.06 (3) 13 225 1 4.62 Pelecypoda 0.12 1.03 0.40 0.12 0.28 (©) 22.02 1 4.75 Nemertinea 0.44 0.31 0.59 0.44 0.91 () 0.61 1.68 Center Polychaeta 9.12 7.31 5.44 9.66 36.91 8.59 1.06 18.11 Crustacea 0.50 1.06 3.84 7.34 10.41 0.12 12.40 2 2.30 Pelecypoda 0.00 0.75 0.06 0.12 2.56 1.19 13.24 1 9.93 Nemertinea 0.00 0.19 0.25 0.44 0.32 0.09 1.08 7) shi 1 1% significance level. ? 5% significance level. and pelecypods exhibited pronounced seasonal varia- tion (Fig. 7-10). Catches of crustaceans and pelecypods were highest during spring and early sum- mer. The significant F-values for blocks (seasons) for crustaceans and pelecypods and the nonsignificant F-values for polychaetes and nemerteans for the pooled station data (Table 3) substantiate these con- clusions. The exception to this general pattern was the decline in abundance, or absence, of all groups at sta- tion 1 in the center zone during June through Sep- aes STATION | AVERAGE NUMBER CAUGHT PER SAMPLE i ——— i 2 Se 1 ES DSMOMoo mon cOlS: olga lle cOMN2t26 89 25am, ell MAR. APR. MAY JUN JUL AUG SEP. OcT. Figure 7.—Average number of polychaetes caught per sample by date, station, and zone. CRUSTACEA STATION SO > SHORE i ° 4 50 CENTER 100 AVERAGE NUMBER CAUGHT PER SAMPLE Commo ecmomcO -sSmuilalomeconl2meGal Sikes mire MAR. APR. MAY JUN JUL AUG. SEP. « OCT. Figure 8.—Average number of crustaceans caught per sample by date, station, and zone. tember. This lowered abundance was probably caused by low dissolved oxygen during this period (Fig. 5). Benthic organisms in general were most abundant in areas with sediments composed of low to intermediate PELECYPODA nag Lemay OnouG Tre ean epee STATION | | SGuOXG DN0UMNG SVOUG DUW0OMD SuouGd AVERAGE NUMBER CAUGHT PER SAMPLE | E 10 4 Fe seas a 4 1 ee eT ht z 25 NeW oc} GeO ssruline lyn ee mMI2e26.9econaie vel MAR. APR. MAY JUN. JUL AUG. SEP. OCT. Figure 9.—Average number of pelecypods caught per sample by date, station, and zone. NEMERTINEA T rea. T a T T T T T an aaa T ler eat I | 2r STATION 4 IF | D> 4 OL Zire Mn. r 4 ae 4 4 2 SHORE 4 Qa Ni s s gq 0 wn Reale CENTER Ww Q 2+ = a5 {0} oO =) ee oO oar w | {ee} 2° Sie Bal g alr «xO Wye 4 2b J ab 10 1 (0) = il TE a 1 Ll 4 — 4 4 4 1 D5 Sooo eOL stale tuiSmeomileweGm Om Comic MAR APR MAY JUN. JUL. AUG. SEP. OcT. Figure 10.—Average number of nemerteans caught per sample by date, station, and zone. amounts of silt and clay (Tables 2 and 3, Fig. 2). Abundance was higher for each taxon (except for crus- taceans at station 4) at stations 1, 4, and 6 in the shore zone where the percentages of silt and clay were lower than in the center zone. Abundance of each taxon and sediment compositions were similar between zones at station 7. The compositions of plant material, rather than the compositions of sediments which were similar, proba- bly caused the large differences in abundance between zones at station 8. With the exception of crustaceans, the abundance of organisms was much greater in the center than in the shore zone. Plant material in the center zone was mostly sea grasses and attached algae, whereas along shore the plant material was pre- dominantly live cord grass roots. COMPARISONS BETWEEN CANAL, MARSH, AND BAY Based on a comparison of mean values for all groups combined (Fig. 11), benthic organisms were slightly more abundant numerically and over twice as abun- dant volumetrically in the marsh than in the canals; they were least abundant in the bay. When each group was considered separately, however, numeric and volumetric abundance by area varied. Polychaetes were most abundant numerically in the canals, most abundant volumetrically in the marsh; they were least abundant in the bay. Crustaceans were over three times as abundant in the marsh as in the other two areas. Pelecypods were numerically most abundant in the bay; volumetrically, they were most abundant in the marsh. Nemerteans were most abundant in the marsh and least abundant in the bay. DISCUSSION This and other studies (Reish, 1961; Taylor and Saloman, 1968) imply that production of benthic or- ganisms will decrease as a result of the type of altera- tion of the environment studied here. The magnitude of the reduction, however, is dependent on many fac- tors. The type of vegetative productivity, the segment of the area that is developed, and the configuration of the canals are of paramount importance in determining changes in benthic productivity. In many of the es- tuarine areas in Florida, vegetative production occurs primarily on the sand flats. Usually, these flats are the segments which are developed (extrusion of the shoreline). In the estuaries along the northern Gulf coast, including Texas, most of the vegetative produc- tion occurs in the intertidal zone; this and adjacent inland areas are usually the areas developed (intrusion of the shoreline). We think, from an ecological stand- point, that the types of developments (extruded and intruded) should be reversed in respect to the types of estuarine area described above. If reversed, the de- POLYCHAETA MEAN NUMBER PER SAMPLE MEAN NUMBER PER M2 MEAN VOLUME (ML) PER SAMPLE MEAN VOLUME (ML) PER M2 CANAL MARSH BAY Figure 11.—Average numbers and volumes of benthic macroinverte- brates caught per sample by area, taxonomic group, and for the groups combined, March-October 1969. crease in benthic productivity might be less in each type of area. The configuration of the canals deter- mines the rate and extent of eutrophication. Low dis- solved oxygen resulting primarily from poor water circulation has been identified as a major problem in relation to maintaining biological productivity in de- velopment canals (Reish, 1961; Taylor and Saloman, 1968; Moore and Trent, 1971; Corliss and Trent, 1971; Lindall, Hall, and Saloman, 1973). LITERATURE CITED BRANDT, K. 1897. Das vordringen mariner Thiere in den Kaiser Wilhelm- Canal. Zool. Jahrb. Ab. Syst., Geol. Biol. Tiere 9:387-408. CORLISS, J., and L. TRENT. 1971. Comparison of phytoplankton production between natural and altered areas in West Bay, Texas. Fish. Bull., U.S. 69:829-832. HOLME, N. A. 1964. Methods of sampling the benthos. Jn F. S. Russell (editor), Advances in marine biology, Vol. 2, p. 171-260. Academic Press, N.Y. LINDALL, W.N.,JR.,J. R. HALL, and C. H. SALOMAN. 1973. Fishes, macroinvertebrates, and hydrological conditions of upland canals in Tampa Bay, Florida. Fish. Bull., U.S. 71:155-163. MOORE, D., and L. TRENT. 1971. Setting, growth and mortality of Crassostrea virginica in a natural marsh and a marsh altered by a housing develop- ment. Proc. Natl. Shellfish. Assoc. 61:51-58. REISH, D. J. 1956. An ecological study of lower San Gabriel River, Califor- nia, with special reference to pollution. Calif. Fish Game 42:51-61. 1957. Effect of poilution on marine life. Ind. Wastes 2:114-118. 1961. A study of benthic fauna in a recently constructed boat harbor in Southern California. Ecology 42:84-91. TAYLOR, J. L., and C. H. SALOMAN. 1968. Some effects of hydraulic dredging and coastal develop- ment in Boca Ciega Bay, Florida. U.S. Fish Wildl. Serv., Fish. Bull. 67:213-241. TRENT, W. L., E. J. PULLEN, and D. MOORE. 1972. Waterfront housing developments: their effect on the ecology of a Texas estuarine area. Jn Mario Ruivo (editor), Marine pollution and sea life, p. 411-417. Fishing News (Books) Ltd., Lond. Appendix Table 1.—Numbers of animals caught by station, date, taxon, and volume of two samples in shore zone. Annelida Arthropoda Mollusca Nemertinea Ba lay Seg g 4a 2 5 o) o = 3 3s r= = 3 z 2 9 s EU Go By oe 3 2D 3 € § 5 = < 3 & g 5 = 3 $2} jude Identified Totals Gop rey = ° € o os 5 5 o rah ic eal Bok Ete ye ONinS Ss ae ame only to. (> So SiationsaDateez, = OS < GOura S So Aes aS) Ane phylum Number Volume 1 25 Mar. 46 as) Ss 2 2230.6 8 Apr. 34 18 3 1 1 Sif tail 22 Apr. 39 1 40 0.1 6 May 76 76 (0.5 20 May 53 2 55 0.4 6June 2 65 3 70 0.3 17 June 31 1 320.4 lJuly 1 20 1 1 1 24 (18 15 July 17 1 18 0.1 28 July 10 1 3 14 0.2 11 Aug. 5 5S 0.1 25 Aug. 3 31 Qi 22 Sept. 1 1 Dd Oil 6 Oct. 9 37 2 48 0.2 20 Oct es 23 4 rte Yaa eG OPEB 04 Total 3 432 45 186" 29 29 Saal 14 695 6.4 4 25 Mar. 115 1 2 1 119 0.6 8 Apr. 119 2. 3 2 126 0.9 22-Apre)) | 125 2 2 I 1 2 1 135 1.6 GiMay a2 Pi iT 1 4 4 123 0.8 20 May 128 1 2 131 1.6 6 June 97 2 1 100 l57/ 17 June 2. 1 73 1.5 1 July 145 5 2 1 153 2.5 15 July 1 42 2 45 0.4 28 July 1 84 2 87 0.6 11 Aug. 3 80 2 2 87 «0.7 25 Aug. 118 1 119 0.5 8 Sept 127 I 2 130 = 0.7 22 Sept. 70 3 73 0.5 6Oct. 1 101 11 2 115 0.9 20Och eee. 67 1 poodle zt Ss) Wa Total 73) 10601 20 9 1 1 Sane 21 10 1,685 16.2 6 25Mar. 6 27 ce 5) 90 lie al 1733. 0.7 8 Apr. 1 32 l 11 120 118 3 286 1.4 22 Apr. 1 13 1538 175 ili 2 2285 04) 6 May 10 185 57 8 260 0.2 20 May 2 ll 49 1 63 © 0.2 6June 7 2 23 1 1 340.4 17 June 1 13 31 2 AT aanO2 lJuly 1 46 1 6 61 0.8 July 2 33 35.0.3 28July 2 27 3 3 350.4 MeAUIe. ly 62 6 9 0.3 25vAug. 1 26 3 1 3 35 2.1 8 Sept. 1 27 1 1 1 31 0.5 22 Sept. 2 23 22 29 «0.3 6 Oct. 1 39 7 1 1 49 1.1 20 Oct. 3 13 4 a 3 23 0.6 Motall 28. 7) 348 Tic SIS) 257, 392 shi 7 2 1 19 1398 enna 10 Appendix Table 1.—Continued. Annelida Arthropoda Mollusca Nemertinea Of vou Reekoee ees og P qs s See Bee gs ce aCe gee ee Se Jame gs Bo eo. a5 Sake O56 ge eee eo Se Se Identified Totals ho Tat ese FES ise! EP Wey dies ge ee Oa See ee Ome ac nly t a : Opes he Gh [she=stape Behe t=] OO oO 6 only to Station Date Z & Of > <> > TO) ERE GS) Sho MA re VP ann 0 phylum Number Volume hie 25.Mar: 1 17 104 2 124 0.2 8 Apr. 3 5 1 11 1 21 0.2 22 Apr. 1 7 1 14 4 2 29 0.2 6 May 2 12 2 44 6 3 69 0.6 2OMayei Ul 9 11-28 1 22 1 2 66 1.6 6 June 13 2 3 1 19 0.3 17 June Pd 92 4 1 1 10 0.3 1 July 5 11 2 18 0.2 15 July 4 11 15 0.2 28 July 9 6 1 16 0.7 11 Aug. 10 20 3 1 1 35 Te5 25 Aug. fi 21 1 29 0.6 8 Sept. 3 15 1 19 0.5 22 Sept. 6 36 2 2 46 0.5 6 Oct. 2 14 2 18 0.4 QOOCtN eas a 22 Eo 5 0.1 Total 6833) 204) 2 4 92 «+18 130 4 14 539s 14.1 8 25 Mar. 1 9 1 9 76 96 0.2 8 Apr. 2. 3 1 33 49 2 2 92 0.3 22 Apr. 2 20 1 10 20 68 2 3 128 0.4 6May 4 15 18 3 1 41 0.3 20 May 18 5 23 0.2 6 June 3 9 1 18 2 33 153 17 June 2 22 7 3 34 0.3 1 July 1 26 1 1 29 0.3 15 July 9 9 0.1 28 July 3 3 6 0.4 11 Aug. I -4- 14 5 4 28 0.2 25 Aug. - 6 18 3 27 0.3 Sisepts. 3) 22> 126 4 35 1e2 a2Sept. 4 1 3 2 1 4 42 4.5 6 Oct. 5 31 1 2 39 0.7 1D) Oi oa a oe 2 puliTey 04 Total 36 8 268 324 64 62 196 6 3 29 679 11.1 11 Appendix Table 2.—Numbers of animals caught by station, date, taxon, and volume of two samples in center of waterway. Annelida Arthropoda Mollusca Nemertinea o vo ho) 2 o mo} o Meg ed ea 32 & en & S| pie Ge ar aos § 2 5 2 Identified Totals 7) o = Sie ss) ae Ones el 9 5 _ ae Sse Pe Bes aie i eso only to Station Date 7A (= Oy ee ae >) Cope OS) HE A A DH phylum Number Volume 1 25 Mar. 37 2 39 0.3 8 Apr. 36 14 50 1.4 22 Apr. 91 91 0.2 6 May 2 72 0.8 20 May 21 21 0.4 6 June 15 15 0.4 17 June 6 6 0.1 1 July 8 8 0.1 6 Oct. 2 2 0.1 20 Oct. Pe! ae = 24 Oth Total 290 2 16 308 3.9 4 25 Mar. 4 6 2. 6 18 1.1 8 Apr. 16 12 2 30 0.4 22 Apr. 5 18 2 2 27 0.2 6 May 31 2 2 35 0.2 20 May 12 12 0.2 6 June 1 13 2 16 0.2 17 June 118 2 120 0.8 1 July 18 2 20 0.2 15 July 1 1 0.2 8 Sept. 6 6 0.1 6 Oct. 4 4 0.2 20Oct) 2 2 1 4 Sak Te a 9 02 Total 3 12229 l 3242. 18 6 6 298 4.0 6 25 Mar. 2 21 2 28 «6 59 0.8 8 Apr. 11 11 3 25 0.4 22 Apr. 12 34 46 0.2 6 May 24 6 2 32 0.2 20 May 11 2 13 0.1 6 June 4 15 2 3 24 0.8 17 June 4 10 25 39 0.6 1 July 2 2 0.1 15 July 2 2 0.1 28 July 12 2D 2 16 0.6 11 Aug. 7 2 9 0.1 25 Aug. 10 10 0.1 8 Sept. 12 2 14 0.1 22 Sept. 6 2 8 0.1 Oct sits. 2 3 8 0.1 Total 20 2 148 4 98 17 8 2 8 307 4.4 12 Appendix Table 2.—Continued. Annelida Arthropoda Mollusca Nemertinea By o ws x Os Gel o A ep er ee eee gee wens ro oeae (son ae 2 6 6 gf y 8 Identified Totals Ce Om ao emSe acc Fe 2 Ss 2 2 ¢€ only to ares = o a) a Ss 6 < ° & o ° io) o Station Date Z & O = < D Ol =) FAB D phylum Number Volume 7 25 Mar. 2 12 40 3 2 2 2 63 0.4 8 Apr. 18 2 4 P32 98 1.5 22 Apr. 8 42 2 52 0.4 6 May 22 2 62 4 90 0.6 20 May 34 3 10 2 49 0.6 6 June 4 2 2 8 0.2 17 June 26 2 28 0.2 1July 10 10 20 0.8 15 July 14 14 0.4 28 July 4 4 8 0.2 11 Aug. 12 12 24 0.6 25 Aug. 14 14 0.2 8 Sept. 2 15 2 19 0.2 22 Sept. 5 34 if 46 0.4 GOcty 11 4 2 17 0.4 20 Oct. 10 praca Vegas = Phen mal 2 016) Total 40 6 241 2 20 2283) 50 2 2 2 14 562 Toll 8 25 Mar. 68 6 4 16 4 98 3.0 8 Apr. aD 30 8 2 14 56 1.2 22 Apr. 2 68 12 IGE gf Dil 2 295 5.2 6 May 48 53 2 2 105 2.8 20 May 10 2 94 8 1 Dy 2) 2 121 4.0 6 June 98 2 4 104 7.0 17 June 7) 2 94 22 2 122 1.0 1 July 6 54 6 4 24 2 2 98 2.6 15 July 86 2 2 2 94 4.0 28 July 70 2 72 0.4 11 Aug. 4 132 6 142 16.4 25 Aug. 2 84 2 88 0.4 8 Sept. 40 2 2 44 0.2 22 Sept. 2 2 2 6 0.2 6 Oct. 74 21 2 2 2 101 0.8 AV OSs, es Set Bante A eae) 2260] 1056: Total 24 12 1,100 6 39 sie 7 il 8 64 10 10 1,606 49.8 10 25 Mar. 5 2 2 5 14 0.4 8 Apr. 2 1 4 7 0.2 22 Apr. 3 3 1 2 2 11 0.8 6 May 16 2 3 7 28 0.6 20 May 1 17 2 20 0.6 6 June 19 19 0.1 17 June 3 1 13 1 18 0.2 1 July 3 8 6 1 18 0.2 15 July 7 2 9 0.3 28 July 36 1 37 0.3 11 Aug. 5 19 3 1 28 0.2 25 Aug. 13 10 23 0.2 8 Sept. 6 7 1 14 0.2 22 Sept. 4 14 1 19 0.3 6 Oct. 8 28 36 0.2 AW OlAi re slits ae Pion SEE at plist 19 0.3 Total 15 2 188 70 4 6 8924! 3 320 Sil 13 % U.S. GOVERNMENT PRINTING OFFICE: 1974—796-121 /4 REGION 10 hiacitinnd of axe sky Rah oh nema han“ ae 22 <= - eos xe ¢ ~ s rey ee ve Me Cc > i ya’ @ 3 = 1 i i atin.) ii { { T } . hie i % i { ro : at i f P it ; i b nN) i Aa 648. Weight loss of pond-raised channel catfish (Ictalurus punctatus) during holding in processing plant vats. By Donald C. Greenland and Robert L. Gill. December 1971, iii + 7 pp., 3 figs., 2 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 49. Distribution of forage of skipjack tuna (Euthynnus pelamis) in the eastern tropical Pacific. By Maurice Blackburn and Michael Laurs. January 1972, iii + 16 pp., 7 figs., 3 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 650. Effects of some antioxidants and EDTA on the development of rancidity in Spanish ‘mackerel (Scomberomorus maculatus) during frozen storage. By Robert N. Farragut. February 1972, iv + 12 pp., 6 figs., 12 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 651. 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For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 656. The calico scallop, Argopecten gibbus. By Donald M. Allen and T-. J. Costello. May 1972, iii + 19 pp., 9 figs., 1 table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 657. Making fish protein concentrates by enzymatic hydrolysis. A status report on Tesearch and some processes and products studied by NMFS. By Malcolm B. Hale. November 1972, v + 32 pp., 15 figs., 17 tables, 1 appendix table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. : 658. List of fishes of Alaska and adjacent waters with a guide to some of their literature. _ By Jay C. Quast and Elizabeth L. Hall. July 1972, iv + 47 pp. For sale by the Superinten- dent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 659. The Southeast Fisheries Center bionumeric code. Part I: Fishes. By Harvey R. - Bullis, Jr., Richard B. Roe, and Judith C. Gatlin. July 1972, x] + 95 pp., 2 figs. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 660. A freshwater fish electro-motivator (FFEM)-its characteristics and operation. By James E. Ellis and Charles C. Hoopes. November 1972, iii + 11 pp., 9 figs. 661. A review of the literature on the development of skipjack tuna fisheries in the cen- tral and western Pacific Ocean. By Frank J. Hester and Tamio Otsu. January 1973, iii + 13 pp., 1 fig. For sale by the Superintendent of Documents, U.S. Government Printing Of- fice, Washington, D.C. 20402. 662. Seasonal distribution of tunas and billfishes in the Atlantic. By John P. Wise and Charles W. Davis. January 1973, iv + 24 pp., 13 figs., 4 tables. For sale by the Superinten- dent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 663. Fish larvae collected from the northeastern Pacific Ocean and Puget Sound during April and May 1967. By Kenneth D. Waldron. December 1972, iii + 16 pp., 2 figs., 1 table, 4 appendix tables. For sale by the Superintendent of Documents, U.S. Government Print- ing Office, Washington, D.C. 20402. 664. Tagging and tag-recovery experiments with Atlantic menhaden, Brevoortia tyran- nus. By Richard L. Kroger and Robert L. Dryfoos. December 1972, iv + 11 pp., 4 figs., 12 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 665. Larval fish survey of Humbolt Bay, California. By Maxwell B. Eldridge and Charles F. Bryan. December 1972, iii + 8 pp., 8 figs., 1 table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 666. Distribution and relative abundance of fishes in Newport River, North Carolina. By William R. Turner and George N. Johnson. September 1973, iv + 23 pp., 1 fig., 13 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 667. An analysis of the commercial lobster (Homarus ariericanus) fishery along the coast of Maine, August 1966 through December 1970. By James C. Thomas. June 1973, v + 57 pp., 18 figs., 11 tables. Fowsale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 668. An annotated bibliography of the cunner, Tautogolabrus adspersus (Walbaum). By Fredric M. Serchuk and David W. Frame. May 1973, ii + 43 pp. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 669. Subpoint prediction for direct readout meteorological satellites. By L. E. Eber. August 1973, iii + 7 pp., 2 figs., 1 table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 670. Unharvested fishes in the U.S. commercial fishery of western Lake Erie in 1969. By Harry D. Van Meter. July 1973, iii + 11 pp., 6 figs., 6 tables. For sale by the Superinten- dent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 671. Coastal upwelling indices, west coast of North America, 1946-71. By Andrew Bakun. June 1973, iv + 103 pp., 6 figs., 3 tables, 45 appendix figs. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 672. Seasonal occurrence of young Gulf menhaden and other fishes in a northwestern Florida estuary. By Marlin E. Tagatz and E. Peter H. Wilkins. August 1973, iii + 14 pp., 1 fig., 4 tables. For sale by the Superintendent of Documents, U.S. Government Printing Of- fice, Washington, D.C. 20402. 673. Abundance and distribution of inshore benthic fauna off southwestern Long Island, N.Y. By Frank W. Steimle, Jr. and Richard B. Stone. December 1973, iii + 50 pp., 2 figs., 5 appendix tables. 674. Lake Erie bottom trawl explorations, 1962-66. By Edgar W. Bowman. January 1974, iv + 21 pp., 9 figs., 1 table, 7 appendix tables. UNITED STATES DEPARTMENT OF COMMERCE NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION POSTAGE AND FEES PAID NATIONAL MARINE FISHERIES SERVICE U.S. DEPARTMENT OF COMMERCE SCIENTIFIC PUBLICATIONS STAFF COM-210 ROOM 450 1107 N.E. 45TH ST. SEATTLE, WA 98105 OFFICIAL BUSINESS Library he Division of Fishes U. S. National Museum Washington, D.C. 20560 NITED STATES SEATTLE, WA ctober 1974 NOAA TR NMFS SSRF-681 DIVISION OF FISHES U.S. NATIONAL MUSEUM NOAA Technical Report NMFS SSRF-681 U.S. DEPARTMENT OF COMMERCE FEB 5 1973 National Oceanic and Atmospheric Administration National Marine Fisheries Service Physiological Response of the Cunner, Jautogolabrus adspersus, to Cadmium NOAA TECHNICAL REPORTS . ' National Marine Fisheries Service, Special Scientific Report—Fisheries Series The major responsibilities of the National Marine Fisheries Service (NMFS) are to monitor and assess the abundance and geographic distribution of fishery understand and predict fluctuations in the quantity and distribution of these resources, and to establish levels for optimum use of the resources. NMFS is also the development and implementation of policies for managing national fishing grounds, development and enforcement of domestic fisheries regulations, surveillai fishing off United States coastal waters, and the development and enforcement of international fishery agreements and policies. NMFS also assists the fishing ini marketing service and economic analysis programs, and mortgage insurance and vessel construction subsidies. It collects, analyzes, and publishes statistics on v the industry. The Special Scientific Report—Fisheries series was established in 1949. The series carries reports on scientific investigations that document long-term continuing pr 2 of NMFS, or intensive scientific reports on studies of restricted scope. The reports may deal with applied fishery problems. The series is also used as a medium for tion of bibliographies of a specialized scientific nature. NOAA Technical Reports NMFS SSRF are available free in limited numbers to governmental agencies, both Federal and State. They are also available ii other scientific and technical publications in the marine sciences. Individual copies may be obtained (unless otherwise noted) from D83, Technical Information Environmental Science Information Center, NOAA, Washington, D.C. 20235. Recent SSRF’s are: 619. Macrozooplankton and small nekton in the coastal waters off Vancouver Island (Canada) and Washington, spring and fall of 1963. By Donald S. Day, January 1971, iii + 94 pp., 19 figs., 13 tables. 620. The Trade Wind Zone Oceanography Pilot Study. Part IX: The sea-level wind field and wind stress values, July 1963 to June 1965. By Gunter R. Seckel. June 1970, iii + 66 pp., 5 figs. 621. Predation by sculpins on fall chinook salmon, Oncorhynchus tshawytscha, fry of hatchery origin. By Benjamin G. Patten. February 1971, iii + 14 pp., 6 figs., 9 tables. 622. Number and lengths, by season, of fishes caught with an otter trawl near Woods Hole, Massachusetts, September 1961 to December 1962. By F. E. Lux and F. E. Nichy. February 1971, iii + 15 pp., 3 figs., 19 tables. 623. Apparent abundance, distribution, and migrations of albacore, Thunnus alalunga, on the North Pacific longline grounds. By Brian J. Rothschild and Marian Y. Y. Yong. September 1970, v + 37 pp., 19 figs., 5 tables. 624. Influence of mechanical processing on the quality and yield of bay scallop meats. By N. B. Webb and F. B. Thomas. April 1971, iii + 11 pp., 9 figs., 3 tables. 625. Distribution of salmon and related oceanographic features in the North Pacific Ocean, spring 1968. By Robert R. French, Richard G. Bakkala, Masanao Osako, and Jun Ito. March 1971, iii + 22 pp., 19 figs., 3 tables. 626. Commercial fishery and biology of the freshwater shrimp, Macrobrachium, in the Lower St. Paul River, Liberia, 1952-53. By George C. Miller. February 1971, iii + 13 pp., 8 figs., 7 tables. 627. Calico scallops of the Southeastern United States, 1959-69. By Robert Cummins, Jr. dune 1971, iii + 22 pp., 23 figs., 3 tables. 628. Fur Seal Investigations, 1969. By NMFS, Marine Mammal Biological Laboratory. August 1971, 82 pp., 20 figs., 44 tables, 23 appendix A tables, 10 appendix B tables. 629. Analysis of the operations of seven Hawaiian skipjack tuna fishing vessels, June- August 1967. By Richard N. Uchida and Ray F. Sumida. March 1971, v + 25 pp., 14 figs., 21 tables. For sale by the Superintendent of Documents, U.S. Government Printing Of- fice, Washington, D.C, 20402. 630. Blue crab meat. I. Preservation by freezing. July 1971, iii + 13 pp., 5 figs., 2 tables. II. Effect of chemical treatments on acceptability. By Jurgen H. Strasser, Jean S. Lennon, and Frederick J. King. July 1971, iii + 12 pp., 1 fig., 9 tables. 631. Occurrence of thiaminase in some common aquatic animals of the United States and Canada. By R. A. Greig and R. H. Gnaedinger. July 1971, iii + 7 pp., 2 tables. 632. An annotated bibliography of attempts to rear the larvae of marine fishes in the laboratory. By Robert C. May. August 1971, iii + 24 pp., 1 appendix I table, 1 appendix 0 table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 633. Blueing of processed crab meat. II. Identification of some factors involved in the blue discoloration of canned crab meat Callinectes sapidus. By Melvin E. Waters. May 1971, ili + 7 pp.. 1 fig.. 3 tables. 634. Age composition. weight, length, and sex of herring, Clupea pallasii, used for reduc- tion in Alaska, 1929-66. By Gerald M. Reid. July 1971, iii + 25 pp., 4 figs., 18 tables. Continued on inside back cover. 635. A bibliography of the blackfin tuna, Thunnus atlanticus (Lesson) Beardsley and David C. Simmons. August 1971, 10 pp. For sale by the Su Documents, U.S. Government Printing Office, Washington, D.C. 20402. 636. il pollution on Wake Island from the tanker R. C. Stoner. By Gooding. May 1971, iii + 12 pp., 8 figs., 2 tables. For sale by the Superinten Documents, U.S. Government Printing Office, Washington, D.C. 20402. 637. Occurrence of larval, juvenile, and mature crabs in the vicinity of Beaufo North Carolina. By Donnie L. Dudley and Mayo H. Judy. August 1971, iii + 10 pp., 5 tables. For sale by the Superintendent of Documents, U.S. Government Printi Washington, D.C. 20402. : 638. Length-weight relations of haddock from commercial landings in 1931-55. By Bradford E. Brown and Richard C. Hennemuth. August 1971, v figs., 6 tables, 10 appendix A tables. For sale by the Superintendent of Doi Government Printing Office, Washington, D.C. 20402. 639. A hydrographic survey of the Galveston Bay system, Texas 1963-66. By W. L. Trent, and G, B, Adams. October 1971, v + 13 pp., 15 figs., 12 tables. Foi Superintendent of Documents, U.S. Government Printing Office, Washi 20402. 7 640. Annotated bibliography on the fishifg industry and biology of th Callinectes sapidus. By Marlin E. Tagatz and Ann Bowman Hall. August 1971, sale by the Superintendent of Documents, U.S. Government Printing Office, D.C. 20402. 641. Use of threadfin shad, Dorosoma petenense, as live bait during experi and-line fishing for skipjack tuna, Katsuwonus pelamis, in Hawaii. By rt Iversen. August 1971, iii + 10 pp., 3 figs., 7 tables. For sale by the Superinte Documents, U.S. Government Printing Office, Washington, D.C. 20402. : 642. Atlantic menhaden Brevoortia tyrannus resource and fishery—analysis 0} By Kenneth A. Henry. August 1971, v + 32 pp., 40 figs., 5 appendix figs., ta appendix tables. For sale by the Superintendent of Documents, U.S. Government Prir Office, Washington, D.C. 20402. yi 643. Surface winds of the southeastern tropical Atlantic Ocean. By John M. S! Merton C. Ingham. October 1971, iii + 20 pp., 17 figs. For sale by the Supe! Documents, U.S. Government Printing Office, Washington, D.C. 20402. — i 644. Inhibition of flesh browning and skin color fading in frozen fillets f yel snapper (Lutzanus vivanus). By Harold C. Thompson, Jr., and Mary F hi Government Printing Office, Washington, D.C. 20402. 645. Traveling screen for removal of debris from rivers. By Daniel W. Bat Murphey, and Martin G. Beam. October 1971, iii + 6 pp., 6 figs., 1 tab 01 Superintendent of Documents, U.S. Government Printing Office, Was! 20402. 646. Dissolved nitrogen concentrations in the Columbia and Snake River their effect on chinook salmon and steelhead trout. By Wesley J. Ebel. Aug pp., 2 figs., 6 tables. For sale by the Superintendent of Documents, Printing Office, Washington, D.C. 20402. 647. Revised annotated list of parasites from sea mammals caught off North America. By L. Margolis and M. D. Dailey. March 1972, iii + 23 py Superintendent of Documents, U.S. Government Printing Office, V 20402. U.S. DEPARTMENT OF COMMERCE Frederick B. Dent, Secretary NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION Robert M. White, Administrator — NATIONAL MARINE FISHERIES SERVICE reas Robert W. Schoning, Director ATMOSp NO i, oP Ac UP OC, WATIONA, e NOAA Technical Report NMFS SSRF-681 Physiological Response of the Cunner, Tautogolabrus adspersus, to Cadmium YOUTOn, e Ye > 3 < Zi 2 SEATTLE, WA we = October 1974 e7a ~~ /?> g1° For sale by the Superintendent of Documents, U.S. Government Printing Office 6-1 Washington, D.C. 20402 The National Marine Fisheries Service (NMFS) does not approve, rec- ommend or endorse any proprietary product or proprietary material mentioned in this publication. No reference shall be made to NMFS, or to this publication furnished by NMFS, in any advertising or sales pro- motion which would indicate or imply that NMFS approves, recommends or endorses any proprietary product or proprietary material mentioned herein, or which has as its purpose an intent to cause directly or indirectly the advertised product to be used or purchased because of this NMFS publication. CONTENTS Page Introduction and experimental design, by Anthony Calabrese, Ries SuColliervandJamesi wMillerie ss. ee eee eens e 1 Uptake of cadmium by organs and tissues, by Richard A. Greig, Al- bert: EyAdams-sandiBetty7AmNelsonm a) .75 =. sche aaa e ee ee 5 Changes in osmoregulation and oxygen consumption, by Frederick P. Thurberg and Margaret A.Dawson ................ 11 Effects on the immune system, by Richard A. Robohm and Maureen HYeIN ACK OWS Kittens fo eit cc nih eee eae ineatemnen mrire, tt Rober ai eaR ees 15 Observations on the biochemistry, by Edith Gould and John J. Karo- NO i. SERNtet ee ERE ETL. Learn | PRES neers ne ch eae ae Rams Be 21 Histopathology, by Martin W. Newman and Sharon A. MacLean .. . 27 iil SUMMARY ABSTRACT The cunner, Tautogolabrus adspersus, was exposed to six concentrations of cadmium, as cadmium chloride (CdCl, -2'2 H, O), for 96 hr. At the end of this exposure period, tests of blood serum osmolality and gill tissue oxygen consumption were performed. High levels (48 ppm) of this metal resulted in abnormally high serum osmolality, and an exposure as low as 3 ppm reduced the normal rate of oxygen consumption. Both of these parameters may be related to observed tissue damage. The histopathological effects of acute exposure of the cunner to cadmium were manifested in the kidney, intestine, hemopoietic tissue, epidermis, and gill. Few significant changes were noted in fish exposed to concentrations less than 48 ppm. The results implicate renal failure as the probable cause of death subsequent to acute exposure to cadmium. Clearance of intracardially injected bacteria from the blood of cunners exposed to 12 ppm cad- mium was examined. The rate of bacterial uptake in the cells of the liver and spleen was increased, but the bacterial death rate within these cells was decreased. Exposure of fish at 3 to 24 ppm failed to influence antibody production against sheep red blood cells. The activity of two liver enzymes changed significantly with exposure to cadmium. Aspartate aminotransferase was lower in the exposed fish, and a magnesium-linked oxidoreductase in exposed fish required 10 times as much added magnesium to reach the same level of activity as in the control fish. Chemical analyses were made for uptake and clearance of cadmium from exposed cunners. In the uptake study, cadmium residues averaged 8.5 times higher in liver than in gills. In the clearance study, substantial reductions in cadmium residues were found in the gills and blood of fish held in clean seawater for 6 wk after exposure to cadmium, as compared to fish sacrificed immediately after exposure. Muscle and carcass samples from the “‘cleared’’ fish showed little reductions in cadmium levels. ACKNOWLEDGMENT The authors of each section of this study thank Rita S. Riccio for her critical reading and typing of this manuscript. iv Physiological Response of the Cunner, Tautogolabrus adspersus, To Cadmium. I. Introduction and Experimental Design ANTHONY CALABRESE, RIES S. COLLIER, and JAMES E. MILLER’ INTRODUCTION Like most metals, cadmium is stable and does not degrade in the environment. Thus, as increasing amounts of cadmium are refined, more and more of it is circulated in the environment, and increasing amounts may reach man. Cadmium becomes an air and water pollutant, through a variety of industrial processes, and is being used in increasing amounts by the storage battery, plastics, plating, and petroleum industries (U.S. Council on Environmental Quality, 1971). There is no evidence that cadmium is biologically essential or beneficial but, on the other hand, has caused severe human health problems (McKee and Wolf, 1971). Since cadmium salts are most likely to be found in estuarine areas that are im- portant nursery areas for marine fish and shellfish lar- vae and juveniles, adult marine fish and shellfish are a potential source of cadmium in the human diet. Relatively little is known about the effect of cad- mium on aquatic animals, particularly those in the marine environment. Most studies on the effect of cadmium to aquatic species have been performed with freshwater forms. More recently, however, research emphasis has been directed toward the effect of cadmium salts on various marine organisms (Shuster and Pringle, 1968, 1969; Gardner and Yevich, 1969, 1970; Jackim, Hamlin, and Sonis, 1970; Hisler, 1971; Eisler, Zaroogian, and Hennekey, 1972; Calabrese et al., 1973). These studies have progressed from the more classical bioassay tests for simply determining TL, (that concentration of toxicant causing 50% mortality) to those of physiological stress caused by sublethal levels of the pollutant being tested. Studies conducted at sublethal concentrations of a contaminant material so as to determine physiological damage to the organism concerned may be more important than mortality itself. The gradual elimination of valued marine species by low concen- trations of toxicants is no less serious than instan- _ taneous death of those species. In a sense, it is more _ serious because it is less likely to be obvious and to be _ traced to its source in time to permit recovery of the | 4 Milford Laboratory, Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, Milford, CT 06460. environment. Studies of physiological stress caused by sublethal levels of a toxicant generally require chronic or long-term exposure, but some physiological parameters can be examined in short-term tests. Parameters of physiological damage that can be ex- amined by long-term exposure include growth, life span, reproductive success, adaptation to en- vironmental stress, feeding and mating behavior, changes in respiration and osmoregulation, pathological effects, biochemical anomalies, and genetic alterations. It is apparent that increases in human population and technological development are producing serious stresses on the marine environment, with a resulting decrease in its effective use. These events, plus natural events, are fostering conditions that diminish the harvest of marine resources. The National Marine Fisheries Service (NMFS), as part of the National Oceanic and Atmospheric Administration (NOAA), is concerned about the threat to marine life and is providing a national focus for marine research to generate the basic knowledge and understanding of marine environmental processes required for effective management of the marine environment and its resources. The New York Bight, which is receiving inter- national attention because of the large amount of waste material being dumped into it, borders the most heavily populated and industrialized complex in the country. Because the Middle Atlantic Coastal Fisheries Center of the NMFS is located within this geographical area, it is important that this Center un- dertake studies to determine the impact of man upon the living marine resources of this area. This Center, comprising laboratories in Sandy Hook, N.J.; Milford, Conn.; and Oxford, Md., has the facilities and scientists to undertake studies of this type. The present study was designed to determine the short- term (96 hr) physiological response of a local fish, Tautogolabrus adspersus, commonly known as the cunner, to cadmium. A multidisciplinary approach was used to determine the following: 1) uptake of cad- mium into various tissues and organ systems; 2) changes in osmoregulation and oxygen consumption rates; 3) changes in enzymological patterns; 4) im- mune response to various antigens; and 5) induction of histopathological abnormalities. Results of these studies are reported in the following sections of this technical report. METHODS AND MATERIALS Collection and Conditioning Cunners were collected in modified eel pots, a cylinder of 2-inch-mesh hardware cloth with a funnel in one end and a hinged door at the other end. Pots were baited with cracked hard clams, Mercenaria mercenaria, and fished in 10-25 feet of water in the Stratford to New Haven, Conn., area of Long Island Sound. Fish were transported to the laboratory in 15- liter polyethylene buckets and placed in tanks of flow- ing seawater at ambient temperature. Before being exposed to cadmium, the fish were transferred to tanks of recirculating artificial seawater (Zaroogian, Pesch, and Morrison, 1969), adjusted to 25 ppt salini- ty, and maintained at room temperature for at least 1 wk for acclimation. The fish were fed Purina Trout Chow? during this time, but were unfed for 2 days prior to and during the experiment. Exposure For exposure to cadmium, the fish were placed in glass aquaria filled to 60 liters, with artificial seawater (Instant Ocean), which was aerated throughout the entire exposure period. Cadmium, as cadmium chlo- ride (CdCl,*2’2 H,O), was added to test aquaria at concentrations of 0, 3, 6, 12, 24, and 48 ppm of Cd**. Stock solution for all tests was made up with reagent grade cadmium chloride dissolved in water at 50 g Cd?+ per liter and acidified to a pH of 2.5 to maintain stability. Proper aliquots of the cadmium stock solu- tion were added immediately prior to the addition of the artificial seawater to obtain desired cadmium con- centrations, and aeration was begun a few minutes prior to the introduction of test fish. Temperatures ranged from 21° to 25°C and pH levels remained between 7.3 and 7.6 during the entire study. The above five concentrations of cadmium were tested in duplicate, with two aquaria serving as controls, in each of a series of seven tests. Four cunners were plac- ed in each aquarium and were observed daily throughout the 96-hr exposure period, and dead fish were removed each day. At the termination of each test the fish were made available to resident scientists at the Milford laboratory; in addition, specimen samples were prepared for histopathological examina- tion by scientists at the Oxford laboratory, Oxford, Md. Supplementary tests of the same design were subsequently performed for those research projects needing further samples. > Reference to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA. bho Of a total of 500 fish tested, 126 were sampled for weight and length, averaging 45.2 g in weight and 146.3 mm in length, with a range in length from 115 to 170 mm. RESULTS AND DISCUSSION Although the intent of this study was to determine the physiological response of cunners exposed to sub- lethal levels of cadmium, rather than to determine TL,,, some mortality data were nevertheless obtained. Concentrations of cadmium (in ppm water) tested and percent mortality (in parentheses) follow: con- trols 0 (1.8%), 3 (3.5%), 6 (5.4%), 12 (1.8%), 24 (10.7%), and 48 (26.8%). It is obvious from these data that cunners can tolerate high concentrations of cad- mium for at least 96 hr. It was also noted in other phases of this study, however, that those fish exposed to 48 ppm of cadmium for 96 hr and then placed in clean seawater all died within a few days. Hisler (1971) reported that the tautog, Tauwtoga onitis, a fish in the same family as the cunner, can also tolerate high levels of cadmium for a short duration. He also reported that the sheepshead minnow, Cyprinodon variegatus, and the mummichog, Fundulus heteroclitus, had TLso values of 50 and 55 ppm, respectively, for a 96-hr exposure to cadmium. Although it appears that marine teleosts are relatively resistant to cadmium, it will be apparent from the sections that follow that cadmium does, in fact, cause physiological stress at sublethal concentrations. LITERATURE CITED CALABRESE, A., R.S. COLLIER, D. A. NELSON, and J. R. MacINNES. 1973. The toxicity of heavy metals to embryos of the American oyster Crassostrea virginica. Mar. Biol. (Berl.) 18:162-166. EISLER, R. 1971. Cadmium poisoning in Fundulus heteroclitus (Pisces: Cyprinodontidae) and other marine organisms. J. Fish. Res. Board Can. 28:1225-1234. EISLER, R., G. E. ZAROOGIAN, and R. J. HENNEKEY. 1972. Cadmium uptake by marine organisms. J. Fish. Res. Board Can. 29:1367-1369. GARDNER, G.R., and P. P. YEVICH. 1969. Toxicological effects of cadmium on Fundulus hetero- clitus under various oxygen, pH, salinity and temperature regimes. Am. Zool. 9:1096. 1970. Histological and hematological responses of an estuarine teleost tocadmium. J. Fish. Res. Board Can. 27:2185-2196. JACKIM, E., J. M. HAMLIN, and S. SONIS. 1970. Effects of metal poisoning on five liver enzymes in the killifish (Fundulus heteroclitus). J. Fish. Res. Board Can. 27:383-390. McKEE, J. E., and H. W. WOLF. 1971. Water quality criteria. 2nd ed. Calif. State Water Qual. Control Board, Publ. 3-A, 548 p. SHUSTER, C.N., JR., and B. H. PRINGLE. 1968. Effects of trace metals on estuarine mollusks. Proc. 1st Mid-Atl. Ind. Waste Conf., Univ. Del. (CE-5):285-304. 1969. Trace metal accumulation by the American eastern oyster, Crassostrea virginica. Proc. Natl. Shellfish. Assoc. 59:91-103. U.S. COUNCIL ON ENVIRONMENTAL QUALITY. 1971. Toxic substances. U.S. Gov. Print. Off., Wash., D.C., 25 p. ZAROOGIAN, G. E., G. PESCH, and G. MORRISON. 1969. Formulation of an artificial sea water media suitable for oyster larvae development. Am. Zool. 9:1144. ir Physiological Response of the Cunner, Tautogolabrus adspersus, to Cadmium. II. Uptake of Cadmium by Organs and Tissues RICHARD A. GREIG, ALBERT E. ADAMS, and BETTY A. NELSON ' ABSTRACT Cadmium uptake and clearance data were obtained on cunners, Tautogolabrus adspersus, exposed to various concentrations of this metal in artificial seawater. In the uptake study, cunners were exposed to 0, 3, 6, 12, 24, and 48 ppm cadmium in seawater for 4 days. Cadmium residues averaged 8.2 times higher in livers than in gills. At the 48 ppm cadmium exposure level, the livers averaged 195 ppm, as compared to 33.5 ppm for gills (wet weight values). In the clearance study, cunners were exposed to 24 ppm cadmium in seawater for 4 days, after which time half of the fish were placed in clean flowing seawater for 1 mo and half were sacrificed immediately to determine initial cadmium residue concentrations. Gill, liver, blood, muscle, and carcass samples were analyzed. Substantial reductions in cadmium residues were found in the gills and blood of fish held in clean seawater, as compared to samples from fish sacrificed immediately after exposure to cadmium. Liver samples produced variable results: livers of fish held in clean seawater for 1 mo contained 62-155 ppm cadmium for four fish and 5- 11 ppm for three fish, as compared to 30-117 ppm for livers from eight fish sacrificed immediate- ly after exposure to cadmium. Muscle and carcass samples from the ‘‘cleared”’ fish showed very little reduction in cadmium levels. INTRODUCTION Freshwater and marine organisms have the ability to concentrate metals far in excess of the levels found in the waters they inhabit. Mollusks and other shellfish, in particular, selectively concentrate chemical materials (including metals) up to many hundreds of times the levels in their environment (Pringle et al., 1968). There are three major sources for uptake of metals by water-inhabiting organisms: 1. The water column (the metals are dissolved in the water); 2. Particulate matter (the metals are adsorbed to particles suspended in the water column); and 3. Food (the metals are incorporated in the material the organism consumes). There are relatively few reports in the literature dealing with the uptake of cadmium by marine organisms. Of those few marine animals studied for cadmium uptake, mollusks have received the most attention and finfish the least (Pringle et al., 1968; Shuster and Pringle, 1969; Eisler, Zaroogian, and Hennekey, 1972). Several investigators have studied 5 Milford Laboratory, Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, Milford, CT 06460. nN the toxic effects of cadmium in both freshwater and marine finfish, but these workers did not include in- formation on the uptake of cadmium (Ball, 1967; Gardner and Yevich, 1969, 1970; Roberts, 1963; Eisler, 1971). Our objective was to obtain data on the uptake of cadmium by liver and gill tissues of cunners exposed to solutions of CdCl,°2'% H,O in artificial seawater. In addition, information was obtained on the extent of clearance of cadmium from various tissues and organs of cunners that were returned to clean seawater after exposure to cadmium. METHODS AND MATERIALS Fish Holding Uptake study.—The methods of exposure of the cunner to various concentrations of cadmium chloride in artificial seawater for the uptake study are describ- ed in Part I of this collaborative report. Clearance study.—An independent study was con- ducted to determine the extent of clearance of cad- mium from various tissues of the cunner, after the ex- posed fish were held in clean seawater. Sixteen fish were exposed to 24 ppm cadmium (as CdCl.*2'2 H,O) in artificial seawater for 96 hr, and eight fish were maintained as controls. The fish weighed from 28-89 g (X = 62.3 g). After the 96-hr exposure, eight of the ex- posed fish and four control fish were sacrificed to determine cadmium residues in various parts of the fish. The remaining eight cadmium-exposed fish and four control fish were placed in flowing seawater for 1 mo, after which time they were sacrificed for cad- mium analysis. Sampling Procedures Uptake study.—For the uptake study, data were collected from three separate experiments, in each of which five different concentrations of cadmium were used, plus a control. A single pooled sample was made of the livers from four to five fish per exposure level. The same sampling procedure was followed with the gills, which were rinsed in clean seawater immediately after dissection to avoid possible adherence of cad- mium chloride to the gill surfaces. Clearance study.—Muscle, gills, liver, red blood cells, serum, and carcass were analyzed for cadmium residues. The sampling was as follows: Muscle.—Paired fillets were taken from each fish, skinned, and ground and combined into a single sam- ple per fish. The skin was added to the carcass sample (described below). Gill and liver.—Samples were taken as described above for the uptake study, except that the samples were analyzed individually. Red blood cells and serum.—Blood samples were pooled from four fish per treatment, except in the case of the cadmium-exposed fish held 1 mo in clean run- ning seawater; only seven fish survived, and their blood pools represented four and three fish, respec- tively. Whole blood was taken from the cunner by heart puncture, placed in a test tube, and allowed to clot at room temperature for 45-60 min. The serum was removed from the clot and centrifuged at 350-500 XG for 10 min. The clarified serum was frozen-stored until analysis. For the red blood cells samples, the cellular residue remaining after centrifugation was combined with the clot. Carcass.—The remainder of the fish after removal of the samples described above was called the carcass, and included the skin removed from the fillets. Chemical Analyses For analysis of cadmium in cunner tissues, samples were placed in 50-ml glass beakers, dried at 110°C for 18 hr, and heated over a Bunsen burner to char the tissue. The samples were brought to 400°C in a muffle furnace, removed after 1 hr at that temperature, and cooled; a small amount of concentrated HNO, was added to wet the ash, and the samples were returned to the muffle furnace at room temperature and brought to 400°C again. This process was repeated until only a white residue remained in the beakers, usually after 3-5 additions of HNO;. The residue was rinsed from each beaker with 10% HNO, and filtered through Whatman” No. 2 paper. The filtrate was brought to a final 10-ml volume and subsequently analyzed with an atomic absorption spectrometer, employing a deuterium background corrector (Perkin Elmer? Model 403). RESULTS Uptake Study Cadmium accumulation was far greater in the liver than in the gills (Table 1). Cadmium concentrations averaged 8.2 (range 3-15) times higher in liver than in gill tissue for all concentrations tested. Although there was substantial variation in results for the three up- take experiments, the averaged data show a nearly linear relation for cadmium concentrations in liver versus cadmium exposure levels (Fig. 1). Variation in liver-cadmium concentration was greatest at 24 and 48 ppm exposure levels, and least at 3, 6, and 12 ppm levels. Uptake of cadmium into gill tissues was curvilinear in form, as shown in a plot of cadmium concentrations in gills versus cadmium exposure levels (Fig. 2). Cad- mium concentrations in gill tissue for 3 and 6 ppm * Reference to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA. 0267 200 180 160 140 120 100 O Experiment 1 & Experiment 2 O Experiment 3 @ Average of 1-3 80 60 40 20 Cadmium Concentration In Livers(ppm,wet wt.) 036 12 24 48 Cadmium Concentration In Seawater (ppm) Figure 1.—Uptake of cadmium by the livers of cunner held 96 hr in various concentrations of cadmium (as cadmium chloride) in — artificial seawater. Table 1.—Uptake of cadmium by livers and gills of cunners, Tautogolabrus adspersus, exposed for 96 hr at various concentrations of cadmium, as CdCl,*H,0, in artificial seawater. Concentrations of cadmium in tissues Concentration of cadmium in artificial Individual experiments Individual experiments seawater (ppm) Ay. 1 2 3 Av. 1 2 3 o--------- ppm wet weight basis’--------- ----------ppm dry weight basis'---------- LIVER TISSUE 0 1.2 0.95 1.65 0.85 5.5 3.6 6.7 ao 3 16.0 13.5 21.5 13.0 54.5 41.0 75.0 47.5 6 34.5 39.0 36.5 27.5 119.5 125.0 131.0 102.0 12 55.0 54.5 65.0 45.0 198.7 182.5 236.0 177.5 24 110.7 143.0 109.0 80.0 390.0 454.0 386.5 329.5 48 195.0 267.0 160.5 157.0 761.3 928.0 744.0 611.5 GILL TISSUE 0 ile Heil 0.9 3 5.4 5.4 5.0 eS, 3 3.0 4.3 2.3 2.5 16.5 21.5 11.5 16.5 6 3.4 5.1 2.4 Pil 17.5 28.0 12.5 13.0 12 6.3 7.5 5.8 5.6 31.8 38.5 28.0 29.0 24 11.9 16.0 12.0 7.8 66.5 88.5 60.5 44.0 48 33.5 43.0 27.5 30.0 171.3 226.5 135.0 152.5 ‘ Liver and gill tissues from four to five fish per exposure level were composited and analyzed in duplicate for each experi- ment. The values shown for individual experiments are averages of the duplicate analyses. D Experiment 1 Experiment 2 O Experiment 3 @ Average of 1-3 Cadmium Concentration In Gills(ppm,wet wt.) 036 12 24 48 Cadmium Concentration In Seawater (ppm) Figure 2.—Uptake of cadmium by the gills of cunner held 96 hr in various concentrations of cadmium (as cadmium chloride) in artificial seawater. cadmium exposure levels were virtually the same, and moderate increases occurred at 12 and 24 ppm levels. There was a very sharp increase, however, at the 48 ppm level, which may be related to physiological gill damage. Thurberg and Dawson (this report, Part III) found a marked depression in oxygen consumption rates of gill tissues, as well as a breakdown of os- moregulation in cunners exposed to 48 ppm cadmium. Clearance Study Substantial reductions in cadmium residues were found in the gills, red blood cells, and serum of cadmium-exposed fish held in clean running seawater for 1 mo after exposure. In contrast, muscle and car- cass samples of these fish showed very little reduction in cadmium residues, as compared to fish examined immediately after exposure to cadmium (Table 2). Liver samples produced variable results (Table 2). Cadmium concentrations in livers of fish examined immediately after exposure varied from 30 to 117 ppm (x = 64.2), a nearly 4-fold difference. Cadmium con- centrations in livers of fish held 1 mo in clean running seawater after exposure, varied only 5-11 ppm (x = 10) in three of the fish, but varied from 62 to 155 ppm (x = 92) in the other four fish. In spite of the pooling of blood samples (which reduced the number of possible observations), cad- mium concentrations in both serum and red blood cells were as variable as those observed in the in- dividual liver samples (Table 2). All blood pools from “cleared”? fish, however, had substantially lower levels of cadmium than those from fish sacrificed im- mediately after exposure to cadmium. Gill tissues showed a greater clearance of cadmium than did the other tissues examined. Gills of fish sacrificed immediately after exposure contained 6.2- 10.6 ppm cadmium (x = 8.1), and gills of fish held in Table 2.—Clearance of cadmium from organs and tissues of cunners, Tautogolabrus adspersus, held in flowing natural seawater for 1 mo after a 96-hr exposure to 24 ppm cadmium, as CdCl,-2!2 H,O. Cadmium Concentration Organ or Immediately after exposure tissue Average (range) Flesh 0.17 (0.11 - 0.22) Liver 64.2 (30.5 - 11722) Gills 8.1 (Ge 10.6 ) Red blood cells 6.6 (5:2) ‘and £8:0)) Serum 5.9 (5.9 and 6.0 ) Carcass 4.8 (0:9) “and 1622") Flesh 0.06 - Liver 0.7 (0.6 and 0.8 ) Gills 0.4 - Red blood cells 0.4 - Serum 0.4 = Carcass 0.09 (0.08 and _ 0.10) After 1 mo in clean seawater Average (range) TEST FISH 0.12 (0.08 - 0.22) 92.0 (62.0 - 155.0 )! 10.0 ( 5.0 5.) allt) )} 3.5 ( 2.8 - 4.7 ) 1.8 (0.8 and 2.8 ) 1.5 ( 0.7 - 2.3 ) 3.5 ( 2.8 - 4.2 ) CONTROL FISH 0.05 - 1.0 0.3 0.4 0.5 0.12 ' The livers of 4 fish had cadmium concentrations in the range of 62-155 ppm, whereas the livers of 3 other fish had cadmium concentrations in the range of 5-11 ppm. clean running seawater for 1 mo after exposure con- tained 2.8-4.6 ppm cadmium. Concentrations of cadmium found in tissues of con- trol fish (Table 2) were all less than 1 ppm. DISCUSSION In the literature, studies of the uptake of cadmium and other metals by marine animals deal predominantly with shellfish. Pringle et al. (1968), who studied the uptake of five metals by several species of shellfish, examined only the soft-shell clam, Mya arenaria, for cadmium uptake. Clams exposed to 0.05 ppm cadmium (nitrate) in flowing seawater for 70 days accumulated 8 ppm (whole-body wet weight), and clams exposed to 0.1 ppm for 56 days ac- cumulated 9 ppm. Shuster and Pringle (1969) exposed Kastern oysters, Crassostrea virginica, to cadmium (nitrate) in flowing seawater for 20 wk. At a 0.1 ppm exposure level, oysters accumulated 90-100 ppm cad- mium (whole-body wet weight) within 13 wk, whereas at a 0.2 ppm exposure level, the animals accumulated the same concentrations of cadmium within 8-10 wk. After only 1 wk at 0.1 and 0.2 ppm exposure levels, the oysters accumulated 7-24 ppm cadmium, with no ap- parent difference due to exposure levels. Eisler et al. (1972) studied cadmium uptake by Eastern oysters; American lobsters, Homarus americanus; bay scallops, Aquipecten irradians; and mummichog, Fundulus heteroclitus. The animals were held for 21 days in flowing seawater containing 10 ppb cadmium (CdCl, «22 H, O). Accumulation of cadmium was highest in the oysters, with 1.49 ppm (whole-body wet weight) in the exposed animals and 0.33 ppm in control animals. The mummichog had whole-body residues of 0.48 ppm cadmium for ex- posed fish and 0.33 ppm in control fish. For comparison with these studies, a combined es- timate of all data was made for whole-body residues in cunners exposed to 24 ppm cadmium for 96 hr (see clearance study, Table 2). The whole-body residues were calculated to be 2-4 ppm (wet weight) for ex- posed fish and 0.1-0.2 ppm for control fish. Although a direct comparison of these data with those cited above for oysters is not possible, it would appear that cunner accumulates cadmium to a much lesser extent than the oyster. LITERATURE CITED BALL, I. R. 1967. The toxicity of cadmium to rainbow trout (Salmo gairdnert Richardson). Water Res. 1:805-806. EISLER, R. 1971. Cadmium poisoning in Fundulus heteroclitus (Pisces: Cyprinodontidae) and other marine organisms. J. Fish. Res. Board Can. 28:1225-1234. EISLER, R., G. E. ZAROOGIAN, and R. J. HENNEKEY. 1972. Cadmium uptake by marine organisms. J. Fish. Res. Board Can. 29:1367-1369. GARDNER, G. R., and P. P. YEVICH. 1969. Toxicological effects of cadmium on Fundulus hetero- clitus under various oxygen, pH, salinity and temperature regimes. Am. Zool. 9:1096. 1970. Histological and hematological responses of an estuarine teleost tocadmium. J. Fish. Res. Board Can. 27:2185-2196. PRINGLE, B. H., D. E. HISSONG, E. L. KATZ, and S.T. MULAWKA. 1968. Trace metal accumulation by estuarine mollusks. J. Sanit. Eng. Div., Proc. Am. Soc. Civ. Eng. 94:455-475. ROBERTS, H. 1963. Cadmium toxic to rainbow trout. Prog. Fish-Cult. 25:216. SHUSTER, C. H., and B. H. PRINGLE. 1969. Trace metal accumulation by the American eastern oyster, Crassostrea virginica. Proc. Natl. Shellfish. Assoc. 59:91-103. THURBERG, F. P., and M. A. DAWSON. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. II]. Changes in osmoregulation and oxygen consumption. Jn Physiological response of the cunner, Tautogolabrus adspersus, to cadmium, p. 11-13. NOAA Tech. Rep. NMFS SSRF 681. Physiological Response of the Cunner, Tautogolabrus adspersus, to Cadmium. III. Changes in Osmoregulation and Oxygen Consumption FREDERICK P. THURBERG and MARGARET A. DAWSON!’ ABSTRACT The cunner, Tautogolabrus adspersus, was exposed to various concentrations of cadmium, as cadmium chloride (CdCl, -2'’2 H,O), for 96 hr. At the end of this exposure period tests of blood serum osmolality and gill tissue oxygen consumption were performed. High levels (48 ppm) of this metal resulted in an abnormally high serum osmolality and an exposure as low as 3 ppm reduced the normal rate of oxygen consumption. Both of these parameters may be related to observed tissue damage. INTRODUCTION Cadmium, which is neither essential nor beneficial to aquatic organisms (McKee and Wolf, 1971), has been detected in increasing amounts in the tissues of a number of such animals (Mullin and Riley, 1956; Peden et al., 1973). The use of cadmium in a variety of industrial processes has increased in recent years, making this metal an immediate concern as an en- vironmental pollutant (U.S. Council on Environmen- tal Quality, 1971; Dean, Bosqui, and Lanouette, 1972). A number of investigators have demonstrated the toxicity of this metal to aquatic animals (Eisler, 1971; Calabrese et al., 1973; Collier et al., in press); however, little is known of the sublethal effects of cad- mium on finfish. Other metals have been shown to alter serum osmolality and respiration of freshwater fish (McKim, Christensen, and Hunt, 1970; Lewis and Lewis, 1971). Few experiments in this area have been conducted with metals and marine fish and fewer still with cadmium as the test pollutant. The present study was undertaken to determine the effect of cadmium on osmotic regulation and oxygen con- sumption in the cunner, Tautogolabrus adspersus. METHODS AND MATERIALS Cunners were exposed to 0, 3, 6, 12, 24, and 48 ppm cadmium for 96 hr by the method of Calabrese, Collier, and Miller (this report, Part I). At the end of this exposure period, a blood sample was drawn by heart puncture using a scalpel and a disposable Pasteur pipette. Pooled blood samples from three to 4 Milford Laboratory, Middle Atlantic Coastal Fisheries Center, " National Marine Fisheries Service, NOAA, Milford, CT 06460. four fish per cadmium concentration were collected in chilled 15-ml centrifuge tubes and spun at 1,720 X g for 20 min at 4°C. The osmolality in milliosmoles per Kg H,O (mOsm) of 0.2-ml serum samples was read on an Advanced 3L Osmometer.’ Gill tissues from these same fish were dissected out and placed in 15-ml Warburg-type flasks chilled on ice; each flask con- tained 5 ml of cadmium-treated seawater from the tank from which the fish were removed. Oxygen con- sumption was monitored over a 4-hr period in a Gilson Differential Respirometer at 20°C. Oxygen consump- tion. rates were calculated as microliters of oxygen consumed per hour per milligram dry weight of gill tissue (yl/hr/mg) corrected to microliters of dry gas at standard temperature and pressure. RESULTS Cunners exposed to 3-24 ppm cadmium for 96 hr showed no change in serum osmolality from the nor- mal value of approximately 340 mOsm determined in control fish. This value is lower than that of the sur- rounding seawater (630 mOsm). Osmoregulatory dif- ficulties were noted in fish exposed to 48 ppm. Serum osmolality in these fish rose to an average value of 390 mOsm. These data are presented in Figure 1; each point on the curve represents the mean of six pooled samples, one from each of six exposures at a given cadmium concentration. Cadmium reduced the gill tissue oxygen consump- tion rates at all concentrations tested. A normal rate of 0.750 « l/hr/mg was reduced to approximately 0.510 ul/hr/mg after exposure to cadmium at concentrations ? Reference to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA. Oxygen Consumption(p2/hr/mg) (@) 3 6 12 24 48 Cadmium Concentration(ppm) _ 400 T 5 dl z 5 375 = 2 T fo) eos ag Ih E = o YO 325 Figure 1.—Cunner gill tissue oxygen consumption and serum osmolality after a 96-hr exposure to cadmium. The upper por- tion of the graph represents gill tissue oxygen consumption rate (ul/hr/mg) vs. cadmium concentration; the lower portion represents serum osmolality (mOsm) vs cadmium. Both curves show the mean value and standard error. of 3, 6, 12, and 24 ppm cadmium. Oxygen consump- tion was slightly higher (0.580 ul/hr/mg) after ex- posure to 48 ppm, the same concentration at which os- moregulatory stress was observed. These results are presented in Figure 1; each point on the curve represents the mean value of gill tissue oxygen con- sumption of 18 fish. DISCUSSION Marine teleosts maintain a normal blood serum osmolality considerably below that of the surrounding medium (Krogh, 1965; Parry, 1966). In the present study cunner serum osmolality rose considerably above its normal level after a 96-hr exposure to 48 ppm cadmium. Exposures below this level did not alter serum osmolality. Teleost kidneys excrete salts and thus maintain a normal osmotic concentration. Newman and MacLean (this report, Part VI) detected gross pathology in the kidneys of certain cunners used in this study. They reported that kidneys of cunners exposed to 48 ppm cadmium for 96 hr were nearly nonfunctional, while those exposed to cadmium con- centrations below 48 ppm appeared normal. Gill and gut tissues are also involved in osmoregulatory func- tion (Krogh, 1965; Prosser and Brown, 1961). Other investigators have reported gill and kidney tissue damage in marine teleosts after exposure to cadmium (Gardner and Yevich, 1970; Eisler, 1971), and New- man and MacLean (this report, Part VI) noted some gill and gut damage in cadmium-exposed cunners. Osmoregulatory difficulty at 48 ppm is, therefore, ap- parently due to kidney failure, although gill and gut damage may be contributory. Gill tissue oxygen consumption was depressed after exposure to 3-48 ppm cadmium. The slight rise (although still well below the normal level) in oxygen consumption at 48 ppm was attributed to increased osmoregulatory stress at that concentration. The depression of oxygen consumption may have been due to gill damage. Newman and MacLean (this report, Part VI) noted gill tissue abnormalities in fish ex- posed to cadmium, and Ledgerwood and Brown (1973) reported cadmium-induced aneurysms in the gill lamellae of threespine sticklebacks, Gasterosteus aculeatus. Greig, Adams, and Nelson (this report, Part II) found elevated levels of cadmium present in gill tissues of all exposed cunners examined. In summary, the results of this study demonstrated two physiological effects of cadmium on the cunner. High levels (48 ppm) of this metal resulted in an ab- normally high serum osmolality, and an exposure as low as 3 ppm reduced the normal rate of oxygen con- sumption. Both of these parameters may be related to observed tissue damage. LITERATURE CITED CALABRESE, A., R. S. COLLIER, and J. E. MILLER. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. I. Introduction and experimental design. Jn Physiological response of the cunner, Tautogola- brus adspersus, to cadmium, p. 1-3. NOAA Tech. Rep. NMFS SSRF 681. CALABRESE, A., R.S. COLLIER, D. A. NELSON, and J. R MacINNES. 1973. The toxicity of heavy metals to embroys of the American oyster Crassostrea virginica. Mar. Biol. (Berl.) 18:162-166. COLLIER, R.S., J. E. MILLER, M.A. DAWSON, and F. P. THURBERG. In press. Physiological response of the mud crab, Eury- Panopeus depressus, to cadmium. Bull. Environ. Contam. Toxicol. DEAN, J. G., F. L. BOSQUI, and K. H. LANOUETTE. 1972. Removing heavy metals from waste water. Environ. Sci. Technol. 6:518-522. EISLER, R. 1971. Cadmium poisoning in Fundulus heteroclitus (Pisces: Cyprinodontidae) and other marine organisms. J. Fish. Res. Board Can. 28:1225-1234. GARDNER, G.R., and P. P. YEVICH. 1970. Histological and hematological responses of an estuarine teleost tocadmium. J. Fish. Res. Board Can. 27:2185-2196. GREIG, R. A., A. E. ADAMS, and B. A. NELSON. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. II. Uptake of cadmium by organs and tissues. In Physiological response of the cunner, Tautogolabrus adspersus, to cadmium, p.5-9. NOAA Tech. Rep. NMFS SSRF 681. KROGH, A. 1965. Osmotic regulation in aquatic animals. IncsaNeYes 242sp: LEDGERWOOD, R. D., and G. W. BROWN, SR. 1973. Cadmium induced aneurysms in gill lamellae. Fish. Univ. Wash., Seattle, Contrib. 375. LEWIS, S. D., and W. M. LEWIS. 1971. The effect of zinc and copper on the osmolality of blood serum of the channel catfish, Jctalurus punctatus Rafinesque, and golden shiner, Notemigonus crysoleucas Mitchill. Trans. Am. Fish. Soc. 100:639-643. McKEE, J. E., and H. W. WOLF. 1971. Water quality criteria. 2nd ed. Calif. State Water Qual. Control Board. Publ. 3-A, 548 p. McKIM, J. M., G. M. CHRISTENSEN, and E. P. HUNT. 1970. Changes in the blood of brook trout (Salvelinus fontinalis) after short-term and long-term exposure to copper. J. Fish. Res. Board Can. 27:1883-1889. Dover Publ. Res. 13 MULLIN, J. B., and J. P. RILEY. 1956. The occurrence of cadmium in seawater and in marine organisms and sediments. J. Mar. Res. 15:103-122. NEWMAN, M. W., and S. A. MacLEAN. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. VI. Histopathology. Jn Physio- logical response of the cunner, Tautogolabrus adspersus, to cadmium, p. 27-33. NOAA Tech. Rep. NMFS SSRF 681. PARRY, G. 1966. Osmotic adaptation in fishes. 41:392-444. PEDEN, J.D., J.H. CROTHERS, C.E. WATERFALL, and J. BEASLEY. 1973. Heavy metals in Somerset marine organisms. Pollut. Bull. 4:7-9. PROSSER, C. L., and F. A. BROWN. 1961. Comparative animal physiology. W. B. Saunders, Phila., 688 p. U.S. COUNCIL ON ENVIRONMENTAL QUALITY. 1971. Toxic substances. U.S. Gov. Print. Off., DC: 25 p: Biol. Rev. (Camb.) Mar. Wash., Petite” opel bh Paks rl ibe eho Physiological Response of the Cunner, Tautogolabrus adspersus, to Cadmium. IV. Effects on the Immune System RICHARD A. ROBOHM and MAUREEN F. NITKOWSKI'! ABSTRACT Two elements of the immune system in cunners, Tautogolabrus adspersus, were examined after 96-hr exposure to cadmium: 1) clearance of intracardially injected bacteria from the bloodstream and 2) ability to produce antibody against intraperitoneally injected sheep red blood cells (SRBC). Exposure to 12 ppm cadmium increased the rates of bacterial uptake in phagocytes of the liver and spleen but significantly decreased the rates of bacterial killing within these cells. Exposure of fish at 3 to 24 ppm cadmium failed to influence antibody produc- tion against SRBC. These results indicate that cadmium affects one aspect of cellular immunity but not humoral immunity in cunners. This effect may increase susceptibility to infection. INTRODUCTION There is evidence that cadmium poisoning in teleosts disrupts respiratory processes (Schweiger, 1957; Mount and Stephan, 1967) and damages kidneys (Gardner and Yevich, 1970) and gills (Mount and Stephan, 1967; Gardner and Yevich, 1970). The exact mechanisms of these effects are unknown, although some evidence in liver, kidney, and other tissues of mammals (Simon, Potts, and Gerard, 1947) and fish (Jackim, Hamlin, and Sonis, 1970) indicates that enzyme systems are inhibited. If there is inhibi- tion in rapidly growing or metabolizing cells, then cells of the immune system in fish may also be affected. Identification of such effects, in addition to being useful as indicators of toxicity, may, in part, ex- plain environmental mortalities of fish. For example, if a pollutant can inhibit production of antibody by the lymphocytes or in some way reduce the effec- tiveness of reticuloendothelial system phagocytes, fish may become susceptible to infection. A report by Pip- py and Hare (1969) linking bacterial infection of salmon with sudden spikes of copper and zinc levels in a Canadian river is indicative that metal pollution may indeed lower fish immunity. The following investigation was undertaken to determine whether short-term exposure of the cunner to sublethal cadmium levels would limit antibody production or reduce clearance of bacteria from the blood, spleen, and liver. Reprinted from NOAA Technical Report NMFS SSRF-681 METHODS AND MATERIALS Fish Holding and Cadmium Exposure Cunners, Tautogolabrus adspersus, were captured from the wild, acclimated to artificial seawater, and exposed to 3 to 48 ppm Cd*+ (as CdCl,°2'2 H,O), as described by Calabrese, Collier, and Miller (this report, Part I). Some fish were immunized (as described below) with sheep red blood cells (SRBC), removed from Cd?+ treated water, and held an ad- ditional 10 days at ambient temperature (23 + 2°C). In two experiments these immunized fish were held in 20-gal fiber glass aquaria containing recirculating seawater under constant charcoal filtration. The fish (with dorsal spine, pectoral fin, or pelvic fin clipped to denote levels of cadmium treatment) were held nine or less per tank, and the water changed every second day. In two later experiments with immunized fish, fins were no longer clipped, and fish were held in run- ning seawater (20°-23°C) in 500-gal fiber glass tanks partitioned off with polyethylene mesh. Some fish were exposed to 12 ppm Cd** treated water then removed and held for 1 or 2 days in the running seawater system. These fish were challenged by intracardial injection of bacteria, as described below. Immunization and Collection of Antisera Sheep red blood cells in Alsever’s solution were washed in phosphate buffered physiological saline (PBS), pH 7.2, until the supernatant was free of hemoglobin and then diluted in PBS to 0.5% suspen- sion (packed cell volume/volume PBS). Fish were weighed and injected intraperitoneally with 0.2 m! of the suspension per 40-g fish (i.e., 2.6 X 10° ml packed SRBC/g fish weight). SRBC injections were made at 0 hr, at 0 minus 24 hr or at 0 plus 24 hr relative to the start of the 96-hr cadmium-exposure period. Second “booster’’ injections of SRBC were given 7 days after each initial injection. Serum was drawn from each fish 6 or 7 days after the second SRBC injection. Blood was removed from the ventral aorta using a Pasteur pipette equipped with a small, rubber finger bulb after nicking the artery with a scalpel blade. Blood was allowed to clot at room temperature and the clots to retract at 4°C. After removing red cells from the serum by centrifugation, sera were stored at —25°C until assayed. Hemagglutination Assay The microtiter system of the Cook Engineering Co., Alexandria, Va., was used in making dilutions and setting up hemagglutination assays. Serial twofold dilutions of fish serum were made in PBS, pH 7.2, containing normal rabbit serum (NRS) at 1/100 con- centration (the rabbit serum was preabsorbed with SRBC). Washed SRBC were suspended to 1% concen- tration (v/v) in the PBS-NRS diluent and added to each serum dilution (0.025-ml serum plus 0.025-ml SRBC suspension). Controls included normal nonim- mune fish serum, known positive serum from im- munized fish and PBS alone. After 2-hr incubation at room temperature and overnight at 4°C, the titers of each serum were read by examining the degree of SRBC agglutination based on a 0 to 4+ rating scale. The last dilution causing a 2+ agglutination of SRBC was taken as the titer. A 2+ rather than a 1+ end- point was used because it gave more reproducible results. Heat inactivation was not done because it created a gel in the serum. Hemolysis of red cells was not a problem because it occurred only after 48-hr in- cubation—a time long after final readings had been made. Growth and Injection of Bacteria Cells of Bacillus sp (biochemical tests consistent with Bacillus cereus) were grown well into stationary phase culture (72 hr) in Trypticase soy broth. The medium in the culture vessel was constantly agitated by an air-driven magnetic stirrer while the vessel was held in a water bath at 37°C. After incubation the culture consisted of about 70% single cells and 30% cells attached in pairs (with less than 0.1% spores) by phase-contrast microscopy. Cells were diluted in physiological saline, containing 0.1% peptone, and counted by the pour plate method in Trypticase soy agar. The following day the bacteria were washed 2X at 3°C with 0.15 M PBS, pH 7.2, and resuspended to about 5 X 10° cells/0.1 ml in PBS based on the counts of the previous day. Fish which had been held for 24 or 48 hr in running seawater after termination of 96-hr exposure to 0 ppm or 12 ppm Cd’* were injected intracardially on a vol/wt basis with the bacterial suspension. For exam- ple, a 40-g fish received 0.1 ml, a 60-g fish received 0.15 ml, etc. Actual numbers of viable bacteria in- jected per 40-g fish varied from 5 X 10° to 5 X 108 because of some cell loss and death during washing in the PBS diluent. Actual numbers were determined by bacterial counts on the suspension after injecting all the fish on a particular day. Measurement of Bacterial Clearance Intracardially injected fish were placed in 4-gal polyethylene pails containing seawater at ambient temperature (23°C). After 30 or 90 min, bacterial counts were made of the blood, liver, and spleen using the following procedures: Blood was removed from the ventral aorta with a premarked, heparinized Natelson blood collecting pipette; blood was drawn to the mark (0.125 ml) using mouth suction on the end of a short rubber tube with mouthpiece (after first wetting the heparin with a small amount of blood). Blood was diluted immediately into a tube of physiological saline containing 0.1% peptone. In order to minimize the amount of standing blood (containing bacteria) in the organs, aspiration of blood was continued until the fish was bled dry. The liver and spleen were removed, weighed to three places, and each diluted in an aqueous solution of 0.5% peptone. In many in- stances, blood from the pericardial space spilled onto the liver during removal of that organ. When this happened, the liver was washed with sterile, distilled water and blotted on sterile paper toweling prior to weighing. Each liver and spleen was ground in a sterile, motor driven tissue grinder with teflon pestle and further diluted serially in 0.1% peptone-saline diluent. Plate counts of the blood, liver, and spleen were made in Trypticase soy agar within 10 min of organ removal. Values were recorded as percent of the initial bacterial dose present in each organ. Calculations for total bacteria in the blood stream were based on a blood volume of 3% of the fish body weight (this was approximated from values given by Thorson, 1961). RESULTS Antibody Response to SRBC Injections In order to examine the possibility that cadmium could affect protein formation or cell division in newly produced, immunocompetent cells, fish were given priming doses of SRBC followed by a second antigen dose 7 days later. Production of antibody was mea- sured by ability of fish serum to agglutinate washed SRBC. Table 1 compares the reciprocal hemagglutina- tion titers of cadmium-treated fish and fish receiving no cadmium during the 96-hr holding period. Al- though antigen was injected into fish on the day be- fore, the day of, or the day after the start of cadmium Table 1.—Serum hemagglutination titers of fish immunized by intraperitoneal injection of sheep red blood cells (SRBC) at the time of 96-hr cadmium exposure. Sera were drawn 2 wk later (1 wk after a second SRBC injection). Cadmium concentration (ppm) 0 3-6 12-24 No. No. No. tested Titer? tested Titer tested Titer Immunized fish 12 338411 95} 29+6 17 30+5 Nonimmunized fish 9 6+1 12 3841 12 6+1 ' Titer is shown as the reciprocal of the mean serum dilution+standard error. treatment, no differences were noted in eventual amount of antibody produced; therefore, the data in the table are grouped as though all antigen injec- tions were given on the same day. It may be seen from the table that treatment of fish with low doses (3-6 ppm) or high doses (12-24 ppm) of Cd?* did not cause any significant differences in an- tibody response over those produced by fish not ex- posed to Cd**. The table also shows that fish had a natural, low-level agglutinin to SRBC which was dis- tinguishable from immune agglutination by its lower titer. About half of the fish tested had no natural SRBC agglutinins. All fish treated with 48 ppm cad- mium died within the 2-wk holding period even though held in fresh, Cd*+free water. Effects of Cadmium on Bacterial Clearance Experiments were run to determine whether cadmium exposure would affect another aspect of im- munity in cunners, namely, clearance of bacteria by phagocytic cells of the reticuloendothelial system. The two primary elements of this system, the liver and spleen, were examined. Bacteria injected into the bloodstream via intracardial route were counted after 30 and 90 min for their remaining levels in the blood, for quantities picked up in the liver and spleen, and (by subtraction) for quantities killed within the 30- and 90-min time intervals. Five experiments were run in which a total of 10 or 11 fish were used for each of the following four variables: 0 ppm Cd’+ at 30 min, 12 ppm Cd’* at 30 min, 0 ppm Cd?+ at 90 min, and 12 ppm Cd**+ at 90 min. Fish caught and used in the warm summer months had significantly greater clearance rates than those caught and used in the autumn; however, the relationships of the 30- and 90- min effects and the 0 ppm and 12 ppm Cd?**+ effects were approximately the same. Therefore, to get a representative mean (one in which the low count ex- periments would carry as much weight as high count experiments), each value within an experiment was multiplied by a factor for that experiment according to the following example: mean of all values Factor, = mean for experiment #1 This allowed each experiment to be representative in calculating the mean while preserving all the treat- ment effect relationships within that experiment. Figure 1 depicts the effects of Cd** on clearance of bacteria. Wilcoxon’s signed rank test for paired obser- vations was used to determine significance of effects O ppm CADMIUM 12 ppm CADMIUM Significant Differences: z B (.02 treated fish were matched by weight +30% with non- treated fish within each experiment). Wilcoxon’s two- sample test for unpaired observations was used to test significance between 30- and 90-min time intervals. Figure 1 shows that within the first 30 min, Cd** ex- posed fish clear bacteria from the blood stream more rapidly and take up greater numbers of bacteria in the liver and spleen than fish not exposed to Cd** Differences in counts in the blood and liver between Cd?+ treated and nontreated fish are significant. However, counts in the cadmium-treated fish change very little during the next 60 min, i.e., only 6.3% ad- ditional cells are eliminated. By contrast, the counts in fish not exposed to cadmium continue to decrease in the blood, liver, and spleen between 30 and 90 min. Values for the blood and the total remaining cells in the blood + liver + spleen are significantly different; 28.7% of the initial bacterial load is eliminated (or killed) within the 60-min time interval. Thus, at 90 min the total remaining bacteria in the non-Cd?*fish are significantly lower (P.01) than in the Cd** treated fish. Statistical analyses were done on all possible relationships between elements of the 0 ppm and 12 ppm Cd**treated fish and between the 30- and 90-min time intervals. Any probabilities not shown were found to be nonsignificant. DISCUSSION From the experimental data presented in this paper, one can conclude that short-term exposure of the cunner to CdCl, at toxic or near toxic levels does not affect the production of antibody against SRBC. Although this conclusion is based upon early antibody production which, undoubtedly, had not reached a peak, it seems safe to assume that significant differences, which were to appear, would show up as a delay or lag in these early responses. Fish could not be held for long periods of time because of limitations in holding space. Although the second SRBC injection (to hasten the rise in antibody titer) was given after fish had been in Cd?-free water for 3 days, it is certain that fish still had high Cd?* levels at this point. The data of Greig, Adams, and Nelson (this report, Part Il) show continued high levels of Cd?*+in cunners after 4 wk of holding in Cd?+-free water. Others have shown that the half-life of Cd** after a single exposure does is in excess of 200 days in rats, mice, dogs, and mon- keys (Friberg, Piscator, and Norberg, 1971, p. 66). The low-level agglutination titers observed in about half the nonimmunized fish (see titers in Table 1) are not unusual. Natural or nonspecific agglutinins are common among fish, as well as other animals. This does not interfere with immunization experiments as long as these agglutinins are low enough in titer that they are not confused with the results of specific im- mune stimulation. In contrast to the results on antibody production, Cd** did have significant effects on uptake and destruction of bacteria by phagocytes in the liver and spleen. Cadmium at 12 ppm was used in these studies because it was the highest level at which there was consistent survival of fish during post-Cd?*+ holding. Fish exposed at this level exhibited a more rapid in- itial uptake of bacteria by cells of the liver and spleen, but a slower bacterial destruction rate than fish not exposed to Cd** These results are consistent with con- clusions drawn by Holmes, Page, and Good (1967) that the metabolic events accompanying phagocytosis can be separated into two categories: 1) events associated with particle uptake and 2) events associated with degranulation within the phagocyte. In the present study it appears that Cd?* stimulates the metabolic events responsible for bacterial uptake but inhibits degranulation or those events responsible for delivering bactericidal substances to the inter- nalized bacteria. The initial, relatively rapid clearance of bacteria from the blood cannot be entirely credited to phagocytosis in the liver and spleen since phagocytic cells in the kidney and gill tissues could also con- tribute to bacterial uptake and destruction. However, cells of the liver and spleen probably take up the ma- jor portion of the injected antigen. This is assumed for two reasons: 1) in some instances, the liver and spleen contained as much as 80% of the tota! bacteria initial- ly injected and 2) studies in other animals indicate that cells of the liver and spleen are responsible for removing the majority of intravenously injected par- ticulate antigens. Benacerraf, et al. (1957) found that the liver and spleen of rats removed 85 to 98% of in- jected carbon or saccharated iron oxide. McCloskey (1972) showed that the liver and spleen of mice retain- ed higher proportions of injected bacteria than other organs. However, data in the latter two references show the liver as the organ of major uptake; whereas in cunners, the spleen usually contains more bacteria than the liver. Since the anterior kidney has been shown to be a site of antibody production in rainbow trout (Chiller et al., 1969), it is assumed that this organ may also take up significant numbers of bacteria; however, for the reasons already given, it is unlikely that this uptake in the cunner of is the same magnitude as that of the liver and spleen. Lack of significant differences between levels of bacteria in the liver and spleen at 30 and 90 min (as seen in Fig. 1) does not indicate lack of activity within their phagocytic cells. As bacteria are destroyed within loaded phagocytes, additional bacteria can be taken from the blood stream to reload the phagocytes. Hence, the bacterial levels in these organs may appear to be static when, in fact, there is a rapid turnover. The destruction rate of the bacteria (28.7%/hr in the normal fish) can be greatly increased when bacteria from 18-hr growth cultures are used (rather than 72-hr cultures); in one experiment (unpublished data) viable bacteria were reduced so quickly that they dropped below the counting range in 30 min. Presumably, the older (72-hr) bacteria are in a more dormant state, which is less susceptible to an- tibacterial metabolites. One clue regarding the mechanism of cadmium ac- tion is given by this work. It appears that within phagocytic cells cadmium may prevent the delivery of lysosomal substances to the phagocytic vacuole or in- hibit the action of these substances on bacteria. On the other hand, cadmium does not appear to inhibit events leading to protein formation. This is indicated because lymphocytic cells exposed to cadmium in vivo actively proliferate and produce antibody protein to the same extent as cells not exposed to cadmium. This work also suggests one way in which cadmium could reduce fish populations. Since bacteria are more slowly killed within phagocytes of cadmium-exposed fish, it follows that certain marginally pathogenic bacteria may multiply within phagocytes and even- tually overwhelm the fish with infection. Studies of the effects of chemical agents in other animals suggest a common mechanism for phagocytic dysfunction. Laurenzi et al. (1963) found reduced clearance of aerosolized bacteria from lungs of mice exposed to ethanol, cortisone, or cigarette smoke. Green and Carolin (1967) found that cigarette smoke inhibited the capacity of rabbit pulmonary macrophages to in- activate bacteria. Kass and Finland (1953) reviewed a large body of literature showing that treatment of animals with cortisone and other adrenocortical hor- mones increases severity of bacterial, viral, fungal, protozoan, and helminth infections. Sidransky, Verney, and Beede (1965) showed that mice treated with cortisone or cytotoxic cancer therapy drugs became highly susceptible to pneumonia from aerosols of Aspergillus flavus spores. Merkow et al. (1968) then demonstrated that lysosomes in phagocytes of cortisone-treated mice failed to fuse with vacuoles containing the spores. Consequently, the substances in these lysosomes were not delivered to the vacuole. Jones and Hirsch (1972) have also demonstrated absence of lysosomal fusion with phagocytic vacuoles containing living toxoplasma parasites. These studies indicate a possible universal mechanism for shutting off microbicidal activities within phagocytes. If so, it is likely that a number of environmental pollutants may be found to cause similar phagocytic dysfunction in fish. LITERATURE CITED BENACERRAF, B., G. BIOZZI, B. N. HALPERN, and C. STIFFEL. 1957. Physiology of phagocytosis of particles by the R.E.S. In B. N. Halpern, B. Benacerraf, and J. F. Delafresnaye (editors), Physiopathology of the reticulo-endothelial system, p. 52-77. Blackwell Sci. Publ., Oxford. CALABRESE, A., R. S. COLLIER, and J. E. MILLER. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. I. Introduction and experimental design. In Physiological response of the cunner, Tauto- golabrus adspersus, to cadmium, p. 1-3. NOAA Tech. Rep. NMFS SSRF 681. CHILLER, J.M., H.O. HODGINS, V.C. CHAMBERS, and R. S. WEISER. 1969. Antibody response in rainbow trout (Salmo gairdneri). J. Immunol. 102:1193-1201. FRIBERG, L., M. PISCATOR, and G. NORDBERG. 1971. Cadmium in the environment. CRC Press, Cleveland, 166 p. GARDNER, G. R., and P. P. YEVICH. 1970. Histological and hematological responses of an estuarine teleost to cadmium. J. Fish. Res. Board Can. 27:2185- 2196. GREEN, G. M., and D. CAROLIN. 1967. The depressant effect of cigarette smoke on the in vitro antibacterial activity of alveolar macrophages. N. Engl. J. Med. 276:421-427. GREIG, R. A., A. E. ADAMS, and B. A. NELSON. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. I. Uptake of cadmium by organs and tissues. In Physiological response of the cunner, Tautogolabrus adspersus, to cadmium, p. 5-9. NOAA Tech. Rep. NMFS SSRF 681. HOLMES, B., A. R. PAGE, and R. A. GOOD. 1967. Studies of the metabolic activity of leukocytes from patients with genetic abnormality of phagocytic function. J. Clin. Invest. 46:1422-1482. JACKIM, E., J. M. HAMLIN, and S. SONIS. 1970. Effects of metal poisoning on five liver enzymes in the killifish (Fundulus heteroclitus). J. Fish. Res. Board Can. 27:383-390. JONES, T.C., and J. G. HIRSCH. 1972. The interaction between Toxoplasma gondii and mam- malian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J. Exp. Med. 136:1173-1194. KASS, E. H., and M. FINLAND. 1953. Adrenocortical hormones in infection and immunity. Annu. Rev. Microbiol. 7:361-388. LAURENZI, G. A., J. J. GUARNERI, R. B. ENDRIGA, and J. P. CAREY. 1963. Clearance of bacteria by the lower respiratory tract. Science (Wash., D.C.) 142:1572-1573. McCLOSKEY, R. V. 1972. Uptake and killing of Mima polymorpha and Herellea vaginicola by the reticuloendothelial system of neonatally thymectomized nonwasted mice. Infect. Immun. 6:21- 26. MERKOW, L., M. PARDO, S. M. EPSTEIN, E. VERNEY, and H. SIDRANSKY. 1968. Lysosomal stability during phagocytosis of Aspergillus flavus spores by alveolar macrophages of cortisone- treated mice. Science (Wash., D.C.) 160:79-81. MOUNT, D.I., and C. E. STEPHAN. 1967. A method for detecting cadmium poisoning in fish. J. Wildl. Manage. 31:168-172. PIPPY, J. H. C., and G. M. HARE. 1969. Relationship of river pollution to bacterial infection in salmon (Salmo salar) and suckers (Catostomus commersoni). Trans. Am. Fish. Soc. 98:685-690. SCHWEIGER, G. 1957. Die toxikologische einwirkung von schwermetallsalzen auf fische und fischnahrtiere. /Engl.summ.j/ Arch. Fisch- ereiwiss. 8:54-78. SIDRANSKY, H., E. VERNEY, and H. BEEDE. 1965. Experimental pulmonary aspergillosis. 79:299-309. Arch. Pathol. 20 SIMON, F. P., A. M. POTTS, and R. W. GERARD. 1947. Action of cadmium and thiols on tissues and enzymes. Arch. Biochem. 12:283-291. THORSON, T. B. 1961. The partitioning of body water in Osteichthyes: Phylogenetic and ecological implications in aquatic verte- brates. Biol. Bull. (Woods Hole) 120:238-254. Physiological Response of the Cunner, Tautogolabrus adspersus, to Cadmium. V. Observations on the Biochemistry EDITH GOULD and JOHN J. KAROLUS' ABSTRACT In the liver of cunner, Tautogolabrus adspersus, exposed to 3 ppm and to 24 ppm Cd for 96 hr, aspartate aminotransferase activity was 71% and 59%, respectively, of the activity in livers of control fish. In the livers of cunners exposed to 24 ppm Cd, nicotinamide-adenine dinucleotide reductase activity required 20 mM Mg for activation of the same order that 2 mM Mg produced in control livers. Although individual variation precludes generalization here, what may be a metal- complexing group of proteins in the serum of cadmium-exposed cunner warrants further elec- trophoretic study. INTRODUCTION In the collective effort to determine the effects of heavy metals on the marine ecology, relatively little attention has been directed toward possible biochemical malfunctions in marine animal tissue. Yet apart from acute physical trauma, such as gross occlusion of gill tissue, the earliest response of a marine animal to physiological challenge by sublethal concentrations of heavy metals is at the molecular level. Normal metabolic response to the ingestion or ab- sorption of heavy metals is their temporary inactiva- tion by serum proteins, which sequester and transport the metals to the liver for further processing for removal from the body. Furst, Flessel, and Kelly (1972) observed that noncancer-causing metals, such as zinc and iron, are carried by a- and _ 6-globulins, whereas those heavy metals definitely known to be able to cause cancer, such as nickel and cadmium, are carried by albumins, the major metal-transport pro- tein of blood. Serum electrophoretic patterns may conceivably be a means of detecting an abnormal proportion of metal-protein complexes. If the normal biochemical mechanisms are unable wholly to inactivate the heavy metals, toxic effects follow. The ionic character of the blood serum becomes seriously deranged (Lewis and Lewis, 1971), with consequent osmoregulatory distress (Thurberg and Dawson, this report, Part III). Key ligand af- finities of some enzymes, particularly those for which divalent cations act as positive or negative effectors, can be distorted by a changing ionic environment, ‘Milford Laboratory, Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, Milford, CT 06460. 21 with consequent changes in their capacity to react (Gould, 1969, 1971). Such effects on biochemical systems are most readi- ly assayed by measuring changes in the activity of their constituent enzymes. Because it is not always clear what systems are involved, one must look either to enzymes that are known to sequester metals or to those that require metals for their proper catalytic functioning. Jackim, Hamlin, and Sonis (1970), for example, in their acute-static study of heavy-metal poisoning in the liver of mummichog, Fundulus heteroclitus, selected three metal-requiring enzymes (alkaline phosphatase, xanthine oxidase, and catalase), the metal-sensitive ribonuclease, and acid phosphatase, an enzyme involved in mineral metabolism. In the multidisciplinary study reported here, the cunner, Tautogolabrus adspersus, was exposed for 96 hr to varying concentrations of cadmium, a soft Lewis acid with the capacity to bind strongly and irrevers- ibly to sulfur groups. It may be well to note here that the nature of cadmium-protein bonding has been observed to differ with the duration of actual metal challenge (Nordberg, Piscator, and Lind, 1971): in mouse liver shortly after a single injection of cad- mium, the cadmium-protein complex was of high molecular weight, whereas in livers of mice surviving for more than 24 hr after injection, the cadmium was bound to a protein of low molecular weight—probably the sulfur protein metallothionein, whose synthesis by the liver is stimulated by prolonged exposure to cad- mium (Anonymous, 1972). More importantly, in vitro binding of cadmium by liver homogenates—which would be rapid—is nonselective, and will inhibit sulfhydryl-dependent enzymes, whereas a metallothionein-cadmium complex has no such in- hibitory effect. In long-term exposure to sublethal concentrations of heavy metals, therefore, an animal’s biochemistry may be adapted in ways that will mask effects observed in acute-static studies. In the initial seven experiments of this study, results of which are published throughout this collaborative report, skeletal muscle was the only tissue available in sufficient quantity for biochemical testing. Although the relatively slow metabolism of the muscle would not be expected to reflect the biochemical response of rapidly metabolizing liver, for instance, the muscle sarcoplasm was, nevertheless, examined for possible changes in malic enzyme (ME)? and a-glycerophosphate dehydrogenase activities, which in vertebrates require manganese and magnesium, respectively, for optimal activity. Subsequent experiments provided liver and blood, where early reaction to physiological challenge would logically be expected. In one experiment, the test con- centrations of cadmium were 0, 3, and 24 ppm, with nine fish at each concentration; the livers were tested for aspartate aminotransferase (AAT) activity, a trans- aminase that reflects metabolic stress in vertebrates (e.g., Amador and Wacker, 1965) and invertebrates (e.g., Hammen, 1969) alike. In another experiment, only two concentrations of cadmium were used (0 and 24 ppm), with 14 fish at each concentration; the livers of these fish were tested for changes in ligand response of NAD reductase activity. The sera were subjected to electrophoresis and examined for possible changes in total-protein patterns that might reflect an increasing metal-protein fraction, for carbonic anhydrase activi- ty (a zinc-enzyme), and for esterase activity. METHODS AND MATERIALS Cunners were exposed to 0, 3, and 24 ppm cadmium for 96 hr in experiments subsequent to those described by Calabrese, Collier, and Miller (this report, Part I), under the same test conditions. The blood was drawn as described by Thurberg and Dawson (this report, Part III); the livers were excised, pooled, and placed in small plastic pouches from which as much air as possible was excluded before freezing. In several of the initial experiments that included more concentrations of cadmium (Calabrese et al., this report, Part I), the fillets were cut from the cunner frames, skinned, and packaged and frozen in the same way as the livers. Treatment of Tissue Liver.—The freshly excised livers from three fish were pooled for each sample and were frozen-stored * Abbreviations used in this report are: ME=malic enzyme, E.C.1.1.1.40; @GPdH=alpha-glycerophosphate dehydrogenase, E.C.1.1.1.8: NAD=nicotinamide-adenine dinucleotide, and NADH= the reduced form; NADR-Mg=magnesium-dependent NAD re- ductase activity: and ATT = asparate aminotransferase, E.C.2.6.1.1. until use, for no longer than 1 wk. The pooled livers were homogenized with a glass pestle in chilled, double-distilled water, 1:9 (w/v) for the AAT assay and 1:4 or 1:9 for the NADR-Mg assay. Homogenates were centrifuged for 45 min at 14,500 g and 4°C, and the supernates used as the crude enzyme preparations (E). For AAT assays of the freshly frozen livers, it was necessary further to dilute the supernates 1:9 with iced water, for a final E dilution factor of 100. Skeletal muscle.—Paired fillets, frozen-stored from 4 to 6 wk, were pounded to a rough paste with an iced mortar and pestle and centrifuged for 45 min at 14,500 g and 4°C. No suspending medium was used. The centrifuged tissue fluid (CTF) served as the en- zyme preparation for ME and a@GPdH assays. Blood.—The clotted fresh blood was clarified by centrifugation for 30 min at 1,720 g and 4° C, and the resulting serum was used for electrophoresis. Assay Procedures The water used in preparing all solutions was doubly glass-distilled; solutions of substrate and coenzyme were prepared fresh daily; and the assays were read on a double-beam, ratio-recording spec- trophotometer, in an optical cuvette with a 1-cm path length. Change in absorbance at 340 nm from 30 to 90 sec after the beginning of the reaction was taken as the unit of measurement (A A*° X 10°/min/0.10 ml EB). Aspartate aminotransferase.—The procedure used was essentially that of Bergmeyer and Bernt (1963), except for the proportions used of reagent solutions. No malic dehydrogenase was added. H, the supernate from the liver homogenate, had a dilution factor of 100. Protocol: Buffer-substrate solution: = 2.70 ml Phosphate buffer (0.1 M, pH 7.6), and K aspartate (0.25 M) NADH solution (10 mg/ml H,O) = 0.10 ml E preparation = 0.10 ml The solutions were pipetted into an optical cuvette and allowed to stand for 10 min at room temperature. Absorbance was read against a reference cuvette (con- taining medium with no initial NADH); the reference mixture was adjusted with small increments of NADH so that the difference in absorbance between sample and reference was no greater than 0.600. The reaction was not started until there was no detectable oxidation of NADH. Substrate (0.10 ml 0.2 M potassium a-ketoglutarate) was added to start the reaction. Magnesium-dependent NAD reductase. Protocol: Tris buffer, 0.1 M, pH 9.0 = 1.80 ml NAD, 13 mM, 10 mg/ml H,O = 0.10 ml MgCl.°6 H,O, 0.06 M (concn varied) H,O E to start reaction X ml (0, 0.10, 1.00) (1.00-X) ml 0.10 ml The rate of reduction of NAD was followed spectrophotometrically at 340 nm. The control cuvette contained everything but the enzyme solu- tion. Malic enzyme.—The assay for ME, a measure of the rate of NADP reduction in the presence of malate, is based on the work of Ochoa et al. (1948), and has been described elsewhere (Gould, 1965). The buffer used here was Tris, 0.1 M, pH 8.0. a-glycerophosphate dehydrogenase.—This assay, with a discussion of the cation-dilution technique, is published in detail elsewhere (Gould, 1969). Electrophoretic Procedures Electrophoresis.—Electrophoresis of cunner serum (ca 3 ul/column) was performed at 4°C using a dis- continuous buffer system, on 7% polyacrylamide gel columns, pH 9.1, with sample and stacker gels of 3% polyacrylamide, pH 5.2. The electrode buffer was Tris (0.005 M)-Glycine (0.038 M), pH 8.3. Running time was 60 min at 1mA/column followed by 105 min at 3mA/column, using constant current. Both the gel for- mularies and the electrophoretic procedure are based on the work of Davis (1964) and have been fully described elsewhere (Gould and Medler, 1970). Stains.—For total-protein patterns, the gels were stained with amido schwartz 10B, 1% in 7% acetic acid. They were destained by passive diffusion in several changes of methanol-glacial acetic acid- distilled water (5:1:5), for a total of about 20 hr. For visualization of esterase sites, the gels were stained with a medium containing «a-naphthyl butyrate (50 mg in 2 ml acetone to dissolve, then 2 ml H,0), coupled with Fast Garnet GBC in 46 ml phosphate buffer, 0.1 M, pH 7.0. Polyvinylpyrrolidone (PVP) (ca 500 mg) was added to the buffer to aid solubilization of the dye. The substrate was added to the dye-PVP-buffer solution immediately prior to use, and the whole filtered through glass wool. Incubation was in the dark at room temperature for 45 min. RESULTS AND DISCUSSION In homogenates of fresh-frozen livers from cunners exposed to cadmium, AAT activity was significantly lower than in the controls (Table 1). Livers of fish ex- posed to 3 ppm Cd had only 71% of the AAT activity observed in livers of control fish and in fish exposed to 24 ppm Cd, activity dropped to 59% of the control. Whether this cadmium-induced drop in activity represents a simple enzyme block, a depression of microsomal biosynthesis, or a more involved mechanism of inhibition cannot be speculated from these few data; the observations here serve only to in- dicate possibly profitable areas for further work. Parenthetically, activity of this transaminase in fresh- frozen livers had roughly 10 times the activity of livers frozen-stored for longer than 1 wk. Livers frozen for 2- 8 wk (at ca +5°C) not only had much lower AAT ac- tivity than the fresh livers, but also had widely variable rates of loss of activity. Another fresh cunner-cadmium experimental series, with 14 fish at 0 ppm Cd and 14 at 24 ppm Cad, provided livers for a study of what appears to be a soluble, magnesium-linked NAD reductase (NADR- Mg). Initially, the assay was intended to be for a- GPdH, but it was discovered that the magnesium effect was stronger without the aGP substrate: in the presence of @GP(10 mM), 2 mM Mg produced only a 1.2% and 20 mM only a 1.8% increase in reductase ac- tivity over activity with no added magnesium; whereas with no added substrate, 2 mM Mg produced 58% and 20 mM produced 130% increase in reductase activity over that with no added magnesium (Table 2). The endogenous substrate pool might be expected to contribute a strong variable to NADR activity (although not so much in teleosts as in marine in- Table 1.—Aspartate aminotransferase activity in liver of cunner, Tautogolabrus adspersus, exposed for 96 hr to varying concen- trations of cadmium, 25 ppt salinity. Each value is the change in absorbance at 340 nm for 1 min under assay conditions and represents the average of 2 tests. Each sample is a pool of livers from three fish; the enzyme preparation (E) has a 100X dilution factor. AAT activity Test concen (A A*°X10°/min/ Cd (ppm) 0.10 ml E) 0 ppm Cd 151.5 170.0 152.5 3 ppm Cd 110.0 115.0 24 ppm Cd 84.0 93.5 101.5 Table 2.—Effect of added Mg**on NAD reductase activity with and without added @-glycerophosphate. E dilution factor is 10x. Sam- ple is pool of three control livers of cunners, Tautogolabrus adsper- sus. NAD reductase activity (A A*°X10%/min/0.10 ml E) Final concen without added with @-GP Mg**(mM) substrate (10 mM final conc) 0 5 41 2 29 48 20 65 75 vertebrates), but the assay protocol here was designed to reduce that variable as much as possible, by using the enzyme activity without added magnesium as a base from which to measure magnesium activation. Table 3 lists data showing a mild cadmium effect upon the magnesium activation of cunner liver NADR: in fish exposed for 96 hr to 24 ppm Cd, an ap- proximately 10-fold increase in magnesium concen- tration (20 mM) is required to activate NADR as much as 2 mM Mg does in the control fish. In frozen-stored cunner skeletal muscle, what appeared to be a significant difference in ME proper- ties of pooled fish exposed to cadmium, as contrasted with pooled controls, must be ascribed to individual variation. @GPdH activity was very high, but there was no apparent difference in properties between the cadmium-exposed and the control fish. Electrophoretic studies are as yet inconclusive. Despite considerable variation in the total-protein pattern, there is an area of tentatively labeled metal- protein complexes that appeared in most cases (8 series out of 10) to increase in fish that had been ex- posed to cadmium (Fig. 1); many more data would be necessary to establish statistical validity, however, and individual variation renders this approach questionable in an acute-static study. Detoxification mechanisms of this nature may be more prominent in a chronic study. Of the serum enzymes tested for isoenzyme varia- tion, only a-naphthyl butyrate esterase activity produced a difference in pattern between cadmium- exposed and control fish; but here again, individual variation was very strong. On the whole, the observations made in the course of this preliminary experimentation seem to point to further work with metal-activated enzymes and with the stress-indicator transaminases. The most clear- cut effects were obtained by using assays designed to measure the degree of ligand activation or inhibition. LITERATURE CITED AMADOR, E., and W. E. C. WACKER. 1965. Enzymatic methods used for diagnosis. In D. Glick (editor), Methods of biochemical analysis, Vol. 13, p. 265-356. Interscience Publ., N.Y. ANONYMOUS. 1972. ‘“‘Itai-itai byd’”’ and other views on cadmium,. Food Cosmet. Toxicol. 10:249-255. BERGMEYER, H.-U., and E. BERNT. 1963. Glutamate-oxaloacetate transaminase. In H.-U. Berg- meyer (editor), Methods of enzymatic analysis, p. 837-845. Academic Press, N.Y. CALABRESE, A., R. S. COLLIER, and J. E. MILLER. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. I. Introduction and experimental Table 3.—Mg-dependent NAD reductase activity in liver of cunner, Tautogolabrus adspersus, exposed for 96 hr to 24 ppm Cd, 25 ppt salinity. Each value is the average of two tests, and each sam- ple is a pool of three livers. Increase in activity (A A**°X10*/min) effected by addition of Mg** to assay medium 0 ppm Cd 24 ppm Cd final concen Mg**+(mM) Homogenate final concen MG*+(mM) dilution factor 0 (Init. A A) 2 20 0 (Init. A A) 2 20 10x 0 (5) 24 60 0 (13) 1 35 10X 0 (9) 29 54 0 (22) 0 26 5X 0 (21) 10 83 0 (23) 4 32 5x 0 (22) 35 56 0 (18) 12 27 2X 0 (23) 26 57 - - 5x ) (18) 20 88 = 5X 0 (20) 24 dl - = = 5x 0 (2) 10 45 12 48 24 Figure 1.—Protein pherogram of serum from cunners, Tautogolabrus adspersus, exposed for 96 hr to 0, 6, 12, 24, and 48 ppm cadmium chloride, at 25 ppt salinity. Arrow points to area tentatively considered to comprise metal-protein complexes. design. Jn Physiological response of the cunner, Tautogo- labrus adspersus, to cadmium, p.1-3. NOAA Tech. Rep. NMFS SSRF 681. DAVIS, B. J. ; 1964. Disc electrophoresis — II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404- 427. FURST, A., C. P. FLESSEL, and M. E. KELLY. 1972. Cited in J. Arehart-Treichel, Sounding out metal toxicity. Sci. News 102:223. GOULD, E. 1965. Observations on the behavior of some endogenous enzyme systems in frozen-stored fish flesh. Jn R. Kreuzer (editor), The technology of fish utilization, p. 126-128. Fishing News (Books), Lond. 1969. Alpha-glycerophosphate dehydrogenase as an index of iced-storage age of fresh, gutted haddock (Melanogrammus aeglefinus). J. Fish. Res. Board Can. 26:3175-3181. 1971. An objective test for determining whether “‘fresh”’ fish have been frozen and thawed. Jn R. Kreuzer (editor), Fish inspection and quality control, p. 72-75. Fishing News (Books), Lond. GOULD, E., and M. J. MEDLER. 1970. Test to determine whether shucked oysters have been frozen and thawed. J. Assoc. Off. Anal. Chem. 53:1237- 1241. HAMMEN, C.S. 1969. Metabolism of the oyster, Crassostrea virginica. Zool. 9:309-318. JACKIM, E., J. M. HAMLIN, and S. SONIS. 1970. Effects of metal poisoning on five liver enzymes in the killifish (Fundulus heteroclitus). J. Fish. Res. Board Can. 27:383-390. LEWIS, S. D., and W. M. LEWIS. 1971. The effect of zinc and copper on the osmolality of the blood serum of the channel catfish, /ctalurus punctatus Rafinesque, and golden shiner, Notemigonus crysoleucas Mitchill. Trans. Am. Fish. Soc. 100:639-643. NORDBERG, G. F., M. PISCATOR, and B. LIND. 1971. Distribution of cadmium among protein fractions of mouse liver. Acta Pharmacol. Toxicol. 29:456-470. OCHOA, S., A. H. MEHLER, and A. KORNBERG. 1948. Biosynthesis of dicarboxylic acids by carbon dioxide fixation. I. Isolation and properties of an enzyme from pigeon liver catalyzing the reversible oxidative decarboxly- ation of l-malic acid. J. Biol. Chem. 174:979-1000. THURBERG, F. P., and M. A. DAWSON. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. III. Changes in osmoregulation and oxygen consumption. Jn Physiological response of the cunner, Tautogolabrus adspersus, to cadmium, p. 11-13. NOAA Tech. Rep. NMFS SSRF 681. Am. nt 2 ual . oh wth? Physiological Response of the Cunner, Tautogolabrus adspersus, to Cadmium. VI. Histopathology MARTIN W. NEWMAN and SHARON A. MacLEAN' ABSTRACT The histopathological effects of acute exposure of cunner, Tautogolabrus adspersus, to water containing cadmium chloride were manifested in the kidney, intestine, hemopoietic tissue, epidermis, and gill. Few significant changes were noted in fish exposed to concen- trations less than 48 ppm for 96 hr. The results implicate renal failure as the probable cause of death after acute exposure to cadmium. INTRODUCTION The histopathological aspects of this study were undertaken in the hope of contributing to our knowledge of the effects of heavy metals at the level of the cells and tissues. Elucidation of the mechanisms of observed physiological and behavioral responses, and development of baseline information useful for in- terpretation of specimens which may be collected from naturally occurring mortalities were further goals. Only Gardner and Yevich (1970) have systematically examined blood and tissues of a teleost exposed to cadmium. While the exposure levels used in that study and the present one were similar, the fish species and the length of exposure (96 vs. 48 hr) were different. MATERIALS AND METHODS Techniques used in handling the cunner, Tautogolabrus adspersus, in this study have been described by Calabrese, Collier, and Miller (this report, Part I). Blood smears were prepared from heart blood (see Thurberg and Dawson, this report, Part III) and stained by Giemsa or Wright’s methods. Differential white cell counts were performed using the first 250 leucocytes encountered on each smear. All thrombocytes seen while counting leucocytes were also enumerated. Tissue samples were removed for physiological and biochemical studies (Thurberg and Dawson; Gould and Karolus, this report, Parts II and V respectively). The remainder of each fish was fixed in Davidson’s (AFA) fixative for 48-96 hr then transferred to 70% EtOH. Tissues were embedded in paraffin, sectioned at 6 um, and stained with a variety of techniques including Giemsa, PAS, PAS-Alcian Blue, Mallory triple-stain, and Perl’s Prussian blue ' Oxford Laboratory. Middle Atlantic Coastal Fisheries Center, National Marine Fisheries Service, NOAA, Oxford, MD 21654. 27 reaction. Tissues from each of six fish exposed to 0, 6, 12, 24, and 48 ppm cadmium, or a total of 30 fish were examined. RESULTS Intestine Pathological changes were seen in the intestinal epithelium of cunners exposed to high concentrations of cadmium. In the 24 ppm exposure group, there was some swelling of the intestinal epithelium. At 48 ppm, five of six fish exhibited varying degrees of pathological change. The columnar cells were swollen. Nuclei were hypertrophied and occupied a position farther from the basement membrane than those of unexposed fish. Nucleoli became very prominent. Numbers of mucus secreting cells appeared about equal to or slightly less than in the control animals. In two of the above five fish, the intestinal epithelium was sloughed from the basement membrane in many places and the lumen contained much cellular debris and mucus (Fig. 1). Kidney One of six fish exposed at the 24 ppm level had some cloudy swelling in a few scattered areas of the proximal tubules. At 48 ppm, the kidneys of five of six fish examined showed some degree of pathological change. Three of the fish exhibited diffuse tubular necrosis (Fig. 2), one exhibited focal tubular necrosis, and one only a few scattered necrotic lesions. The proximal segments of the tubules appeared to be most affected. The lumina of more distal areas of the tubules often contained sloughed epithelial cells or were filled with a hyaline eosinophilic material (Fig. 3). Glomeruli appeared normal. The blood spaces in kidneys of fish exposed to 24 and 48 ppm cadmium Figure 1.—Intestinal epithelium of a control fish (a) and a fish exposed to 48 ppm cadmium for 96 hr (b). Necrosis and sloughing of the intestinal epithelium is evident in the experimental animal. Note also that much cellular debris occupies the lumen. 120X; Mallory’s triple stain. contained large numbers of cells thought to be im- mature thrombocytes (Fig. 4). The occurrence of erythrophagocytosis and of hemosiderin, common in the kidneys of control fish, was reduced or absent in the kidneys of the 48 ppm group (Fig. 5). Gills The appearance of the gills of cadmium-exposed fish was quite variable. The following defects were noted in decreasing order of prevalence: epithelial hypertrophy, hyperplasia of interlamellar epithelium, and desquamation. These changes were noted at all levels of exposure. The gill tissue of some heavily ex- posed fish appeared normal and some lesions were seen in the control animals. Some of these lesions may represent postmortem changes. In view of the exten- sive variability of gill lesions which could not be cor- related with exposure levels, little emphasis was placed on the appearance of this tissue. Epidermis The epidermis appeared normal in fish exposed at levels up to 24 ppm. At 48 ppm swelling of the epithelial cells and a paucity of mucus secretion was noted (Fig. 6). Blood Obvious qualitative differences noted between smears from cunner exposed to 24 and 48 ppm cad- mium and control fish consisted of poikilocytosis and karoklasis (Figs. 7 and 8). 28 Differential leucocyte counts revealed throm- bocytopenia and lymphocytopenia. However, the percentage of neutrophils increased (Table 1). DISCUSSION The histological and hematological response of an estuarine teleost to cadmium has been studied by Gardner and Yevich (1970) using mummichog, Fun- dulus heteroclitus. Their experiments involved acute exposure to 50 ppm cadmium for up to 48 hr. The findings of the present study are very like those of Gardner and Yevich. Discrepancies which did arise might be related to difference in the fish species used, or length of exposure. Changes in the intestine of cunners were similar to those reported in mummichogs. Gardner and Yevich (1970) found an increase in mucus cell activity while the present study indicates a normal or slightly depressed level of activity. This difference may be at- tributable to the increased exposure time of cunner. Lymphocytic infiltration of the submucosa was not observed in the present study. Kidney Morphologic changes in the kidney of teleosts exposed to cadmium have also been documented by Gardner and Yevich (1970). Pathological changes in the kidneys of mammals exposed to cadmium are well known. Foster and Cameron (1963) produced renal lesions in rabbits with two subcutaneous injections of CdCl, (9 mg Cd*/kg). These lesions were limited to Figures 2 and 3.—Kidney of control fish (a) and of fish exposed to 48 ppm cadmium for 96 hr (b). Note diffuse tubular necrosis in 2b. Tubular epithelium in various stages of degeneration can be seen in 3b. Note the swollen nuclei of still intact epithelial cells and cellular debris in the dilated lumina. 120 (2), 300 (3); Azure-eosin stain. Table 1.—Effects of 96-hr exposure to cadmium chloride on differential leucocyte counts of cunners, Tautogolabrus adspersus (figures are averages, standard deviation in parenthesis). Leucocytes (%) Cadmium exposure No. Mature Small Medium (ppm) sampled thrombocytes Neutrophils lymphocytes lymphocytes Eosinophils Blasts Monocytes 0 8 69.3 (410.8) 15.6 (+9.1) 11.2 (+3.9) 2.2 (+0.7) 0.7 (+0.7) 1.1 (+1.2) 0 3 9 65.3 (£10.1) 18.5 (+10.3) 10.3 (+4.9) 2.4 (41.4) 0.6 (40.4) 2.9 (438.3) 0 6 8 60.5 (£16.3) 23.7 (+19.1) 9.3 (+5.6) i 3 (42.3) 1.2 (0.5) 2.3 (1.7) <0.1 12 9 65.2 (416.5) 24.1 (+16.3) 7.5 (£4.9) 3 (41.0) 0.6 (+0.5) 1.6 (40.5) <0.1 24 9 56.3 (420.8) 32.1 (419.3) 6.4 (+4.6) i 6 (+0. 9) 0.6 (40.6) 3.0 (+2.6) <0.1 48 10 35.9 (412.4) 50.2 (412.4) 4.5 (44.8) 1.5 (+1.1) 1.4 (41.1) 6.1 (+4.8) 0 29 Figure 4.—Kidney of control fish (a) and of fish exposed to 24 ppm cadmium for 96hr(b). Note difference in the cell populations occupying the vascular spaces. 480; Azure-eosin stain. . a” Lee ; owes BEA SS - ‘ ee ee SS ‘ ‘ ~- Fat + ees we eu © ey - ve : g. . : Jf x = a ; P é nS > ai ae * Race So ; > 5 < . i tiled ~y Re : WP is oid * > a4 Yr [ =. < we \ “ty mek ‘\ * “Ky * x . ws Rite . wr. Soe 3 . ; - LJ 2 oe \ > ‘ * \ > ¥ & ss a * s i + , . bs - ‘ = é ‘ . ~ \ ‘ < . - ~ . ‘ . a * 2 ? . s ras . a | 7 Prd t ¥ ’ ‘ . ° ~ * < 5 -- * ¥ eq Ca Sry > we Wes w+ ‘ . Loman | . nN wt os . i ’ \ * * ; \ - a abe < ole Figure 5.—Kidney of control fish (a) and of fish exposed to 48 ppm cadmium for 96 hr. An abundance of hemosiderin can be seen in the control fish. 120; Perl’s stain. the proximal tubules. Similar experimental results centrations in renal cortex reach approximately 200 were obtained by Dalhamn and Friberg (1957); ppm wet-weight, functional and morphological Ahlmark et al. (1961) found decreased glomerular change occurs in the kidneys of rabbits, rats, and men filtration rates in human workers exposed to cad- (Friberg, Piscator, and Nordberg, 1971). After ad- mium. Proteinuria is another common finding in ditional exposure, the functional changes result in the © human cadmium workers (Friberg, 1950; Piscator, failure of the kidney to reabsorb the metallothionein, — 1962). Proteinuria occurs subsequent to tubular and increased excretion of cadmium and a lowering of dysfunction because of decreased protein reabsorption the concentration of the metal in the kidney occurs. in the proximal tubule. The effect of cadmium poisoning on the amount of During chronic exposure or early in acute exposure, hemosiderin in the teleost kidney has not been cadmium is concentrated in the renal cortex in the previously described. Unlike most homeotherms, form of metallothionein, a cadmium- or zinc- many teleosts normally have a great deal of pigment containing protein with a molecular weight of about in the kidney, composed of both lipofuscin and 10,000 (Kagi and Vallee, 1960). When cadmium con- hemosiderin. The observation of reduced pigment in 30 Figure 6.—Epidermis of control fish (a) and of fish exposed to 48 ppm (b). Note nuclear hypertrophy and paucity of mucus secreting cells in 6b. The separation of the epidermis from the deeper layers is probably sectioning artifact. 120; PAS-hematoxylin stain. the cunners exposed to 48 ppm cadmium may be caused by the metal’s effect on the hemopoietic system. Friberg (1950) found no effects in human bone marrow after respiratory exposure. Wilson, DeKds, and Cox (1941) found that rats fed a diet containing as low as 31 ppm cadmium would be anemic. By ad- ministering iron to rats receiving cadmium in their diet, anemias were reduced (Friberg, 1955). This in- dicated that cadmium was not directly blocking hemoglobin synthesis, but might be interfering with the uptake of iron by the intestine. If a reduction of iron absorption was occurring in the cunner, this, along with the observed decrease in erythrophagocytic activity, might account for the observed reduction in hemosiderin in exposed animals. Gill The lack of correlation between exposure level and observed pathologic changes in the gill tissue can be explained by Gardner and Yevich’s observations on mummichogs. They noted that gill lesions were focal in that all gill filaments were not involved, and the lesions were of a random nature, not limited to specific areas of the branchial arches. The importance of the effects of heavy metals on the respiratory epithelium of fishes is not clear. Skid- more (1964) believed that death of rainbow trout, Salmo gairdneri, exposed to acutely toxic solution of zinc sulphate was caused by tissue hypoxia, resulting from the damaging effect of the metal on gill epithelium. Subsequent experimental studies seem to confirm this hypothesis (Skidmore, 1970). Burton, _ Morgan, and Cairns (1972) studied levels of lactic and _ pyruvic acid in muscle and liver of rainbow trout ex- . posed to acute zinc toxicity and reached conclusions ! similar to Skidmore. Schweiger, cited in Eisler (1971), concluded that the toxic action of cadmium on several 3] freshwater fish and invertebrates was due to suffoca- tion. Friberg et al. (1971) cite examples of respiratory exposure to cadmium producing lung damage in mammals at levels which were insufficient to cause kidney damage. Doudoroff and Katz (1953) are cited by Mount and Stephan (1967) as listing nine references attributing the death of fish in solutions containing heavy metal salts to coagulation of mucus on the gill or damage to gill tissue. Bilinski and Jonas (1973) found that exposure of rainbow trout to 11.2 ppm Cd’** for 72 hr resulted in a 66% mortality, but there was no significant change in the ability of the gill tissue to oxidise lactate. Evidence from the present study implicates renal failure as a cause of death in cunner after acute ex- posure to cadmium. Although pathological changes were observed in gill tissue and gills exposed to even low levels of cadmium seemed to sustain some physiological damage (Thurberg and Dawson, this report, Part III), these changes do not seem to be asociated with mortality. On the other hand, those fish exposed to 48 ppm cadmium for 96 hr all died within a few days after being returned to clean seawater (Calabrese, Collier, and Miller this report, Part I). Therefore, as regards the effects of cadmium on the cunner, mortality seems to be associated with severe pathological changes of an apparently irreversi- ble nature taking place in the kidney. Epidermis The destruction of the mucus cells of the epidermis could have important consequences as it would eliminate the fish’s first line of defense against infec- tious microorganisms. The possibility that heavy metals would destroy mucus cells with exposures too short to cause renal damage should be investigated. In the present study, the fish had already received a Figures 7 and 8.—Blood smear of control fish (a) and fish exposed to 48 ppm cadmium for 96 hr (b). Note poikilocytosis, karyolkasis, and abundant smudge cells in 7b, 8b. 185 (7), 750X (8); Wright’s stain. lethal exposure to the metal so that destruction of the mucus cells was not an important factor in prognosis. Blood An understanding of the changes taking place in the blood of the cunner must await further study. The in- crease in eosinophils noted by Gardner and Yevich (1970) was not seen, possibly because of the longer ex- posures used in this study. Wilson et al. (1941) noted an increase of eosinophils of rats after dietary ex- posure to cadmium, and Friberg (1950) found an in- crease of eosinophils in rabbit blood from a normal 3% level to 25% after exposure to cadmium oxide dust. Thrombocytopenia, lymphocytopenia, and neutrophilia in the present study parallel the changes found by Gardner and Yevich (1970) in mummichogs and have also been found in other species under stress or after adrenal corticoid administration (Weinreb, 1958). 32 LITERATURE CITED AHLMARK, A., B. AXELSSON, L. FRIBERG, and M. PISCATOR. 1961. Further investigations into kidney function and pro- teinuria in chronic cadmium poisoning. Proc. 13th Int. Congr. Occup. Health, p. 201-203. BILINSKI, E., and R. E. E. JONAS. 1973. Effects of cadmium and copper on the oxidation of lac- tate by rainbow trout (Salmo gairdneri) gills. J. Fish. Res. Board Can. 30:1553-1558. BURTON, D. T., E. L. MORGAN, and J. CAIRNS, JR. 1972. Mortality curves of bluegills (Lepomis macrochirus Rafinesque) simultaneously exposed to temperature and zi stress. Trans. Am. Fish. Soc. 101:435-441. CALABRESE, A., R. S. COLLIER, and J. E. MILLER. 1974. Physiological response of the cunner, Tautogolab adspersus, to cadmium. J. Introduction and experimental design. Jn Physiological response of the cunner, Tauto- golabrus adspersus, to cadmium, p. 1-3. NOAA Tech. Rep. NMES SSRF 681 DALHAMN, T., and L. FRIBERG. 1957. Morphological investigations on kidney damage in chronic cadmium poisoning. Acta Pathol. Microbiol. Scand. 40:475-479. DOUDOROFF, P., and M. KATZ. 1953. Critical review of literature on the toxicity of industrial wastes and their components to fish. II. The metals, as salts. Sewage Ind. Wastes 25:802-839. EISLER, R. 1971. Cadmium poisoning in Fundulus heteroclitus (Pisces: Cyprinodontidae) and other marine organisms. J. Fish. Res. Board Can. 28:1225-1234. FOSTER, C. L., and E. CAMERON. 1963. Observations on the histological effects of sub-lethal doses of cadmium chloride in the rabbit. II. The effect on the kidney cortex. J. Anat. 97:281-288. FRIBERG, L. 1950. Health hazards in the manufacture of alkaline accumu- lators with special reference to chronic cadmium poisoning. Acta Med. Scand. Suppl. 240, 124 p. 1955. Iron and liver administration in chronic cadmium poisoning and studies of the distribution and excretion of cadmium. Experimental investigations in rabbits. Acta Pharmacol. 11:168-178. FRIBERG, L., M. PISCATOR, and G. NORDBERG. 1971. Cadmium in the environment. CRC Press, Cleveland, 166 p. GARDNER, G. R., and P. P. YEVICH. 1970. Histological and hematological responses of an estu- arine teleost to cadmium. J. Fish. Res. Board Can. 27:2185-2196. GOULD, E., and J. J. KAROLUS. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. V. Observations on the biochemistry. 33 In Physiological response of the cunner, Tautogolabrus adspersus, to cadmium, p. 21-25. NOAA Tech. Rep. NMFS SSRF 681. KAGI, J. H. R., and B. L. VALLEE. 1960. Metallothionein: A cadmium- and zinc-containing pro- tein from equine renal cortex. J. Biol. Chem. 235:3460-3465. MOUNT, D.I., and C. E. STEPHAN. 1967. A method for detecting cadmium poisoning in fish. J. Wildl. Manage. 31:168-172. PISCATOR, M. 1962. Proteinuria in chronic cadmium poisoning. I. An electrophoretic and chemical study of urinary and serum proteins from workers with chronic cadmium poisoning. Arch. Environ. Health 4:607-621. SKIDMORE, J. F. 1964. Toxicity of zinc compounds to aquatic animals, with special reference to fish. Q. Rev. Biol. 39:227-248. 1970. Respiration and osmoregulation in rainbow trout with gills damaged by zinc sulphate. J. Exp. Biol. 52:481-494. THURBERG, F. P., and M. A. DAWSON. 1974. Physiological response of the cunner, Tautogolabrus adspersus, to cadmium. III. Changes in osmoregulation and oxygen consumption. Jn Physiological response of the cunner, Tautogolabrus adspersus, to cadmium, p. 11-13. NOAA Tech. Rep. NMFS SSRF 681. WEINREB, E. L. 1958. Studies on the histology and histopathology of the rain- bow trout, Salmo gairdneri irideus. 1. Hematology: under normal and experimental conditions of inflamation. Zoo- logica 43:145-153. WILSON, R. H., F. DeEDS, and A. J. COX. 1941. Effects of continued cadmium feeding. J. Pharm- acol. Exp. Ther. 71:222. % U.S. GOVERNMENT PRINTING OFFICE: 1974—697- 788/i9 REGION I0 oO - . | 5. » = © a —— 648. Weight loss of pond-raised channel catfish (Ictalurus punctatus) during holding in processing plant vats. By Donald C. Greenland and Robert L. Gill. December 1971, iii + 7 pp., 3 figs., 2 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 649. Distribution of forage of skipjack tuna (Euthynnus pelamis) in the eastern tropical Pacific. By Maurice Blackburn and Michael Laurs. January 1972, iii + 16 pp., 7 figs., 3 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 650. Effects of some antioxidants and EDTA on the development of rancidity in Spanish mackerel (Scomberomorus maculatus) during frozen storage. By Robert N. Farragut. February 1972, iv + 12 pp., 6 figs., 12 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 651. The effect of premortem stress, holding temperatures, and freezing on the biochemistry and quality of skipjack tuna. By Ladell Crawford. April 1972, iii + 23 pp., 3 figs.. 4 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 653. 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For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 657. Making fish protein concentrates by enzymatic hydrolysis. A status report on research and some processes and products studied by NMFS. By Malcolm B. Hale. November 1972, v + 32 pp., 15 figs., 17 tables, 1 appendix table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 658. List of fishes of Alaska and adjacent waters with a guide to some of their literature. By Jay C. Quast and Elizabeth L. Hall. July 1972, iv + 47 pp. For sale by the Superinten- dent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 659. The Southeast Fisheries Center bionumeric code. Part I: Fishes. By Harvey R. Bullis, Jr., Richard B. Roe, and Judith C. Gatlin. July 1972, xl + 95 pp., 2 figs. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 660. A freshwater fish electro-motivator (FFEM)-its characteristics and operation. By James E. Ellis and Charles C. Hoopes. November 1972, iii + 11 pp., 9 figs. 661. A review of the literature on the development of skipjack tuna fisheries in the cen- tral and western Pacific Ocean. By Frank J. Hester and Tamio Otsu. January 1973, iii + 13 pp., 1 fig. For sale by the Superintendent of Documents, U.S. Government Printing Of- fice, Washington, D.C. 20402. 662. Seasonal distribution of tunas and billfishes in the Atlantic. By John P. Wise ana Charles W. Davis. January 1973, iv + 24 pp., 13 figs., 4 tables. For sale by the Superinten- dent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 663. Fish larvae collected from the northeastern Pacific Ocean and Puget Sound during April and May 1967. By Kenneth D. Waldron. December 1972, iii + 16 pp., 2 figs., 1 table, 4 appendix tables. For sale by the Superintendent of Documents, U.S. Government Print- ing Office, Washington, D.C. 20402. 664. Tagging and tag-recovery experiments with Atlantic menhaden, Brevoortia tyran- nus. By Richard L. Kroger and Robert L. Dryfoos. December 1972, iv + 11 pp., 4 figs., 12 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 665. Larval fish survey of Humbolt Bay, California. By Maxwell B. Eldridge and Charles F. Bryan. December 1972, iii + 8 pp., 8 figs., 1 table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 666. Distribution and relative abundance of fishes in Newport River, North Carolina. By William R. Turner and George N. Johnson. September 1973, iv + 23 pp., 1 fig., 13 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 667. An analysis of the commercial lobster (Homarus americanus) fishery along the coast of Maine, August 1966 through December 1970. By James C. Thomas. June 1973, v + 57 pp., 18 figs., 11 tables. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 668. An annotated bibliography of the cunner, Tautogolabrus adspersus (Walbaum). By Fredric M. Serchuk and David W. Frame. May 1973, ii + 43 pp. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 669. Subpoint prediction for direct readout meteorological satellites. By L. E. Eber. August 1973, iii + 7 pp., 2 figs., 1 table. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 670. Unharvested fishes in the U.S. commercial fishery of western Lake Erie in 1969. By Harry D. Van Meter. July 1973, iii + 11 pp., 6 figs., 6 tables. For sale by the Superinten- dent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 671. Coastal upwelling indices, west coast of North America, 1946-71. By Andrew Bakun. June 1973, iv + 103 pp., 6 figs., 3 tables, 45 appendix figs. For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402. 672. Seasonal occurrence of young Gulf menhaden and other fishes in a northwestern Florida estuary. By Marlin E. Tagatz and E. Peter H. Wilkins. August 1973, iii + 14 pp., 1 fig., 4 tables. For sale by the Superintendent of Documents, U.S. Government Printing Of- fice, Washington, D.C. 20402. 673, Abundance and distribution of inshore benthic fauna off southwestern Long Island, N.Y. By Frank W. Steimle, Jr. and Richard B. Stone. December 1973, iii + 50 pp., 2 figs., 5 appendix tables. 674. Lake Erie bottom trawl explorations, 1962-66. By Edgar W. Bowman. January 1974, iv + 21 pp., 9 figs., 1 table, 7 appendix tables. UNITED STATES DEPARTMENT OF COMMERCE NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION POSTAGE AND FEES PAID NATIONAL MARINE FISHERIES SERVICE U.S. DEPARTMENT OF COMMERCE SCIENTIFIC PUBLICATIONS STAFF Goring ROOM 450 1107 N.E. 45TH ST SEATTLE. WA 98105 OFFICIAL BUSINESS Library iS) Division of Fishes U. S. National Museum Washington, D.C. 20560 nig