ML20070D122

Concentrations of COPPER-BINDING Proteins in Livers of Bluegills from the Cooling Lake at the H.B. Robinson Nuclear Power Station

ML20070D122
Person / Time
Site:	Robinson
Issue date:	11/30/1982
From:	Harrison F, Lam J LAWRENCE LIVERMORE NATIONAL LABORATORY
To:	NRC OFFICE OF NUCLEAR REGULATORY RESEARCH (RES)
References
CON-FIN-A-0119, CON-FIN-A-119 NUREG-CR-2822, UCRL-53041, NUDOCS 8212140459
Download: ML20070D122 (40)

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NUREG/CR-2822 IICRL-5304i RE Concentrations of Copper-Binding Proteins in Livers of Bluegills from the Cooling Lake at the H. B. Robinson Nuclear Power Station Manuscript Completed: May 1982 Date Published: November 19'32 Prepared by F. L Ilarrison and J. R.12m Lawrence Livermore National Laboratory 7000 East Avenue Livermore, CA 94550 Prepared for Office of Nuclear Regulatory Research U.S. Nuclear Regulatory Commission Washington, DC 20555 NRC FIN No. A-0249 0Dk kDO K O O 2 1 P PDR

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NUREG/CR-2822 UCRL-53041 Concentrations of Copper-Binding Proteins in Livers of Bluegills from the Cooling Lake at the H. B. Robinson Nuclear Power Station F. L. Ilarrison and J. R. Lam Prepared for U.S. Nuclear Regulatory Commission l

Lawrence Uvermore

' National Laboratory

1 ABSTRACT Bluegills collected from the cooling lake of the H. B. Robinson Nuclear Power Station near the effluent discharge, near the water intake to the cooling system, and from a control population in a local pond were examined for total copper in muscle and liver tissues and metalloproteins in different compartments of liver tissues. Much lower concentrations of copper were found in muscle.than in liver tissue. Also, copper changes in the environment were reflected in liver but not t

in muscle tissue.

l Liver metalloproteins were separated into low molecular weight (LMW),

intermediate molecular weight (IMW), and high molecular weight (HMW) protein fractions using high performance liquid chromatography. Large differences in kinds and quantities of metals associated with metalloproteins were found.

Copper concentrat!ons in the LMW proteins (metallothionein-like proteins).were highest in bluegills from the discharge site and lowest in those from the control pond. Evidence of overloading of the metallothionein-like protein detoxification

. system was found in bluegills at the discharge site. These data and that from

! related studies indicate that the labile copper released from the cooling system of the H. B. Robinson Nuclear Power Station may be implicated in the increased deformities and reduced reproductive capacity found in the bluegill population in the adjacent cooling lake.

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CONTENTS Abstract ........................... iii List of Figures ........................ vi List of Tables ........................ vili Preface ........................... ix Executive Summary ...................... I introduction ......................... 2 Materials and Methods ..................... 7 Collection and Dissection of Bluegills . . . . . . . . . . . . . . 7 Reagents and Glassware ................... 7 1 solation of Metalloproteins ................. 7 Metal Analyses ....................... 9 Results ........................... 9 Tissue Metal Concentrations ................. 9 Metalloproteins ....................... 10 Discussion .......................... 23 Conclusions ......................... 25 Recommendations ....................... 25 MLP Induction in Adult Bluegills ............... 25 MLP Residence Time in Adult Bluegills . . . . . . . . . . . . 25 MLPs in Early Life-History Stages ............... 25 Direct Effects of Copper on Development . . . . . . . . . . . . 26 References .......................... 27

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LIST OF FIGURES

1. Location of Units I and 2 of the H. B. Robinson Nuclear Power Station near Florence, South Carolina .............. 3

2. Flow diagram of metalloprotein assay . .............. 8

3. Elution profiles of copper in S-Il$s of liver tissue of bluegills collected in November 1981 from the control pond. Number adjacent to each curve is the liver copper concentration in pg Cu/g dry weight ...................... 12

4. Elution profiles of copper in 5-115s of liver tissue of bluegills collected in November 1981 from the cooling lake near the intake site of the H. B. Robinson Power Station. Number adjacent to each curve is the liver copper concentration in pg Cu/g dry weight ...................... 13

5. Elution profiles of copper in 5-115s of liver tissue of bluegills collected in November 1981 from the cooling lake near the discharge site of the H. B. Robinson Power Station. Number adjacent to each curve is the liver copper concentration in pg Cu/g dry weight ..... .. ............... 14

6. (a) Elution profiles of zinc in S-115s of liver tissue of the two bluegills that had the highest concentration of copper at each site. Number adjacent to each curve is the liver copper concentration in pg Cu/g dry weight. Bluegills were collected in November 1981 from a control pond (*) and from the H. B. Robinson cooling lake near the ' intake (m) and discharge (o) of cooling water from the nuclear power station ............. .............. 15 (b) Typical UV (280-nm) absorbance chromatogram of S-115s of liver tissue of bluegills collected near the H. B. Robinson Nuclear Power Station in November 1981 ............ 15

7. Concentrations cf copper in the pellet (*) and eluted totally (a) compared to that in the liver. Bluegills were collected from the control and intake sites in November 1981.

The equation of the line for the total cluted was y = 0.758x + 7.108; R2 was 0.983. The equation of the line for the pellet was y = 0.239x + 13.554; R2 was 0.990 ......... I8

8. Concentrations of copper in the LMW (*), IMW (A), and HMW (m) protein pools compared to that eluted totally. Bluegills were collected from the control and intake sites in November 1981.

The equation of the line for LMW protein was y = 0.728x - 11.309; R2 was 0.999. The equations of the lines for the IMW and HMW protein pools were y = 0.125x - 13.27 and y = 0.10lx + 2.495, respectively; R 2 was 0.889 and 0.972, respectively ......................... 19 vi

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9. Concentrations of copper in the LMW (e) and HMW (a) protein pools and in the pellet (v) compared to that in the liver.

Bluegills were collected from the control, intake, and discharge sites in November 1981. 'Ihe solid line was drawn by eye through the points. The dashed line represents the extension of the line obtained from the regression of the compartment vs.

total liver concentration for the intake and control fish ...... 20

10. Concentrations of zinc in the intake pellet (a), discharge

• 3 pellet (o), and control pellet (a) and intake supernatant (s), i discharge supernatant (*), and control supernatant (*) compared I to that in the liver ...................... 72 l

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LIST OF TABLES

1. Copper concentrations in water samples (pg Cu/L) collected et I the H. B. Robinson Nuclear Power Station,1979 to 1980 ..... 4

2. The MLPs identified in fish tissues . ............. 6

3. Copper in muscle tissues (pg Cu/g dry weight) from bluegills collected at the H. B. Robinson Nuclear Power Station, May 1981 ......................... 9 4 Copper, cadmium, and zinc in livers (pg meta!/g dry weight) from bluegills collected at the H. B. Robinson Nuclear Power Station, November 1981 ................... I1

3. Copper in liver tissue and liver tissue compartments (10-4 pM/g wet weight) from bluegills collected at the H. B. Robinson Nuclear Power Station, November 1981 ............ 16

6. The p-values obtained from paired t-tests of the parameters measured in livers from bluegills collected from the control, intake, and discharge sites at the H. B. Robinson Nuclear Power Station, November 1981 ................... 17

7. Intercept, slope, and R2 values obtained froin the regression analyses of liver copper vs. compartment copper and total cluted copper vs. compartment copper for the control and intake samples collected at the H. B. Robinson Nuclear Power Station, November 1981. All concentrations are in 10-4pM Cu/g wet weight ......................... 19

8. Zinc in compartments of liver tissue (pg Zn/g dry weight) from bluegills collected at the H. B. Robison Nuclear Power Station, November 1981 ....................... 21

9. Zinc in compartments of liver tissue (10-4 pM Zn/g wet weight) from bluegills having the highest liver copper concentrations at each site of the H. B. Robinson Nuclear Power Station, November 1981 ...................... 22 viii

PREFACE This study is part of a larger research project that has three purposes:(1) to study the behavior of potentially toxic substances introduced to surface waters from nuclear power stations; (2) to determine the magnitude of tiie impact of these substances on representative, economically important species; and (3) to develop models to predict the behavior and the impact of these substances. The initial thrust of .the research has been investigating the impact of corrosion products from cooling systems, in particular, copper. Copper is of special interest because it is toxic to aquatic organisms.

We wish to thank the scientific staff of the Carolina Power and Light Company for their excellent cooperation and advice. Special thanks gc to Dave Herlong and Carolyn Anderson for their assistance in the collection and dissection of bluegills. The authors also thank Susan Chan, George Conrad, Austin Ketcher, Josephine Wold, and David McIntyre for their assistance in sample analysis and Deborah Bennett for her assistance in statistical analyses.

The author acknowledges the continued support received from the Ecological Research Division of the Office of Health and Environmental Research of the U.S. Department of Energy for research that complemented this study and facilitated its execution.

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CONCENTRATIONS OF COPPER-BINDING PROTEINS 'N LIVER OF BLUEGILLS FROM THE COOLING LAKE AT THE H. B. ROBINSON NUCLEAR POWER STATION EXECUTIVE SUMM ARY We are attempting to establish the causes of the changes that occurred in the bluegill population in the cooling lake of the H. B. Robinson Nuclear Power Station. In the late 1970s it was noted that there were decreases in reproductive capacity and increases in structural deformities in the bluegills. /!though there was considerable effort to determine the reason for the changes, no causal factors were identified.

In a previous study of copper concentration and speciation in the intake and discharge waters of the power station, we determined that copper was present in chemical forms and amounts that could potentially cause adverse effects on some fish populations. Also, our results and those of others indicated that liver copper concentrations were high in some bluegills from the copper-impacted area of the cooling lake. We investigated the 1:inds and quantities of metals associated with the metalloproteins in livers of bluerills, because although cadmium, copper, mercury, and zinc can be detoxified by binding to metallothionein-like proteins (MLPs), the MLP-detoxification sites can be overloaded. Metals then can spill over into the metalloenzyme pool and cause deleterious effects.

Bluegills were collected from the cooling lake near the effluent discharge from the power station, near the water intake to the cooling system, and from a control pond and were examined for total metal in muscle and liver tissues. Much lower concentrations of copper were found in muscle than in liver tissue. Also, copper levels in the environment were reflected in fiver but not in muscle tissue.

Livers from bluegills from the three sampling sitas were removed, homogenized, and centrifuged and the supernatants processed on a high performance liquid chromatograph (HPLC) to separate proteins on the basis of their molecular

! weight. Eluant from the gel permeation column was monitored continuously for UV absorbance (proteins containing aromatic amino acids absorb UV light at 280 nm) and the 35 fractions collected were analyzed for copper, cadmiurn, and I zinc. Elution profiles of the supernatant showed large differences in the kinds l and quantities cf metals associated with metalloproteins present in the bluegills from the three sites. Copper concentrations in the low molecular weight (LMW)

MLP pool (sites of detoxification) were highest in bluegills from the discharge site and lowest in those from the control pond. Evidence of spillover of copper j from the LMW to the intermediate molecular weight (IMW) and high molecular

weight (HMW) metalloenzyme pool (sites of toxic action) was found in bluegills

( living in the discharge site. Also, data were obtained on these same fish that indicate that copper was displacing zinc from metalloenzymes in the HMW and IMW protein pools.

Our results indicate'that the labile copper released from the cooling system of the H. B. Robinson Nuclear Power Station accumulated in the livers of bluegills from the intake and discharge sites. Furthermore, copper was present in livers of bluegills from the discharge site at concentrations that could potentially interfere with normal metabolic activity and hence impair reproduction and development.

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Additional research is required to define the conceptrations of copper in effluents and the duration of exposure that adult and earlylife-history stages of bluegills can tolerate without adverse effects. Also, the turnover of copper an metallothioneins should be evaluated for all life-history stages. lhese ' kinds of information can be used to set emission standards that protect fisheries' resources and permit continued operation of power generating stations.

INTRODUCTION The impact on aquatic ecosystems of copper corrosion products in effluents from nuclear power stations depends on the quantities and chemical forms of copper released. Most stations that use copper alloys in cooling systems release only '

small quantities of copper when they are operating under routine conditions. This copper is present primarily in bound forms, which are considered to be of low toxicity to most aquatic organisms. However, in those ecosystems where waters of pH < 6 prevail, increased quantities of copper may be leached from cooling systems and a large fraction of this copper may be in potentially toxic labile forms (unbound).

The H. B. Robinson Nuclear Power Station is an example of a station circulating low pH water in its cooling system (Ref.1). Here water is circulated from the H. B. Robinson cooling lake, which is on Black Creek, a tributary of the Pee Dee River (Fig. 1). It is a typical blackwater stream exhibiting low pH and darkly stained water resulting from the leaching of organic material from the swampy headwater.

Environmental monitoring of the H. B. Robinson cooling lake has been conducted under cgreement with the U.S. Environmental Protecticn Agency and the South' - '

Carolina Department of Health and Environmental Control. As part of this study, the abundance of adult and larval fish populations is icilowed. A decrease in fish populations in the mid- and lower-cooling-lake areas was reported (Ref. 2). In addition, it was noticed that a number of lepomids (sunfish) collected during 1976 to 1978 had structural deformities. This was most often observed in bluegills, although some warmouth (Lepomis gulosus) exhibited the same condition. Few of the bluegills ccliected were young, suggesting very poor reproduction in the previous year.

Changes in the fish population at the H. B. Robinson cooling lake were of concern to the research staff of the Carolina Power and Light Company (CP&L). They expended a considerable effort investigating possible causes. They eliminated the following parameters: temperature, selenium, pesticides, common water-quality properties, and vitamin C storage. In addition, with the cooperation of faculty from a neighboring university, bluegills were examined for disease and pathogens, their rate of oxygen consumption, and the number of chromosomal aberrations.

Furthermore, the electrophoretic mobility of specific proteins were compared in normal and abnormal fish. None of these factors was associated with the problem.

Results of our study in 1979 and 1980 of the concentration 'and speciation of copper in the intake and d8.darge waters of the H. B. Robinson Nuclear Power Station indicated that coppe concentrations in the cooling lake were elevated and that the soluble copper was primarily in the more_ toxic, labile forms (Table 1).

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Table 1. Copper concentrations in water samples (pg Cu/L) collected at the H. B. Robinson Nuclear Power Station,1979 to 1980.

Fraction Collection Collection Soluble date site Total Particulate Labile

• Bound Total

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November 1979 Intake 57.8 11.5 26.4 19.9 46.3

' Dischargg 79.3 18.6 33.9 26.8 60.7 Control 3.1 0.5 1.5 1.1 2.6 May 1980 Intake. 33.9 10.4 12.3 11.2 23.5 Discharge 48.1 12.8 18 17.3 35.3 Control 0.7 0.3 5.8 4.8 5.1 July 1980 intake 27.4 11.5 9.1 6.8 15.9 Discharge 32.6 14.7 13~ 4.9 17.9

! - Control 3.1 0.3 0.6 2.2 2.8 a Chelex-100-tabile copper.

b Control station was located upstream from the reactor.

A preliminary study of copper concentrations in bluegill tissue was conducted.

Comparison of copper concentrations in muscle tissue of fish from the j copper-impacted and control areas showed only small differences between the two groups. Analysis of livers from these same fish showed that copper concentrations in those from the impacted area were elevated but highly variable and that the range of concentrations overlapped those from the control area.

Similar results were obtained earlier by the staff at CP&L.

j lt is well established that the liver is an important site of accumulation and

, detoxification of metals and that within liver cells, metals are bound to different classes of proteins. Results from research on mammalian livers indicate that more information about potential adverse effects of metals can be obtained from artalysis of the kinds and quantities of metals associated with the different l metalloprotein pools than of the total quantities of metal in the tissues.

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! Copper, cadmium, mercury, and zine in mammalian livers are detoxified by binding to metallothioneins. These are proteins having low molecular weights, high percentages of cysteine, and completely lacking aromatic amino acids -

(Ref. 3). A current hypothesis is that when a metal is present in excess, the total amount of that metal bound to protein in the metallothionein pool increases and eventually reaches a plateau. At this point, the metal may spill over into the metalloenzyme pool (Ref. 4). If spillover occurs, metalloenzymes are exposed to an excess of the metal required for enzymatic activity or of a competing metal, their functions become impaired, and toxic effects may occur. Toxic effects include tissue degeneration (Refs. 5, 6), reduced growth (Ref. 4), and increased mortality (Ref.1).

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Metallothioneins and proteins serving a similar function to metallothioneins have been identified in aquatic animals. These proteins have been characterized only by their molecular weight and capacity to bind metals and therefore are referred to as a metallothionein-like protein (MLP) because insufficient data are available to establish that they satisfy the criteria set forth for metallothioneins (Ref.7).

Most of the information on MLPs in aquatic animals is from research on populations exposed to cadmium under laboratory and field conditions (Refs. 8, 9). Fishes have been exposed to other metals as well as cadmium (Table 2). Data on copper MLPs in fish are available only for a few specie:.. Although no experiments to determir.e the effect of copper exposure on MLPs in fish have been performed, copper binding to MLPs has been detected in the eel Anguilla anguilla (Ref.13), in the flounder (Ref.18), in chum salmon (Ref. 4), and in the killifish (Ref. 21).

This research was performed to determine the quantities and kinds of metals associated with metalloproteins in the livers of fish from control and copper-impacted sites. The concentrations of copper, cadmium, and zinc in both the metallothionein-like and metalloenzyme-containing pools were examined.

Results from these analyses provide information on the types and quantities of metalloproteins present and indicate that copper is overloading the MLP detoxification mechanism and may be responsible for the adverse effects on reproductive and developmental processes observed in bluegills from the H. B. Robinson cooling lake.

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Table 2. The MLPs identified in fish tissues.

. Metal Reference Source Exposure conditions composition number Rock fish (liver) CdCl2, intramuscular - Cd 10 Eels (liver, kidney, Hg, chronic exposure Hg ii gill, and muscle)

Eels (gill) Hg and Cd, chronic exposure Hg,Cd 12 Eel (liver and gill) Cd, natural, acute, and chronic Cd, 2n, Cu 13 exposure Goldfish (liver and Cd, 2n, and Hg, intraperitoneal Cd,2n,Hg_ 14 kidney) injections Rainbow trout (liver) Zn, Cd, or Hg, chronic Zn,Hg,Cd 15 exposure, intragastric Rainbow trout (liver) Cd, intraperitoneal Cd 16 injections Rainbow trout (liver Zn, intraperitoneal Zn 17 and kidney) injections Flounder (liver) Nontumor- and tumor- Cd,Zn,Cu 18 bearing fish Chum salmon (liver) Hg, chronic exposure Hg,Cu 4

Coho salmon (liver, Pb or Cd, chronic exposure Cd 19 .

kidney, and gill)

Plaice (liver) Cd, intraperitoneal Cd 20 injections Killifish (liver) Cd, chronic exposure Cd,Cu 21 6

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MATERIALS AND METHODS COLLECTION AND DISSECTION OF BL*JEGILLS Bluegills were collected in May and November 1981 from Christensen's Pond (control-water of low copper concentration), near the water intake to the cooling system (water of intermediate copper concentration), and near the effluent discharge from the nuclear power station (water of high copper concentration).

In May, electrofishing was used to collect bluegills because insufficient fish were found in the traps that were set;in November, the fish were taken from traps.

Bluegills were dissected immediately after collection. All dissections were performed on ice and special care was taken in the processing of the liver.

Livers were dissected out first, as quickly as possible, and then frozen with dry ice and preserved frozen (-70*C). Liver samples from fish collected in May from each site were pooled ta form a composite sample; muscle tissue from each fish was analyzed individually. In November, ten fish were collected from each site and only the livers were analyzed; livers of two fish were pooled for a total of five samples from each site.

REAGENTS AND GLASSWARE Copper, cadmium, and zinc chloride standards were obtained from Scientific Products (Sunnyvale, CA); protein standards were purchased from BioRad (Emeryville, CA). Analytical grade cupric chloride, sucrose, and Tris-hcl were obtained from Mallinckrodt, Inc. (Paris, KY). All glassware was cleaned with MICRO (International Products Corp., Trenton, NJ) to remove adhering metals.

Only double distilled water was used. Acids used for dissolution of tissues were analytical grade. All water and reagents contained less than 1 ppb copper, unless otherwise stated.

ISOLATION OF METALLOPROTEINS Livers were placed in two volumes per weight of nitrogen-saturated, 30 mM Tris-hcl (pH 7.6) containing 10% sucrose,1 % 2-mercaptoethanol, and 200 K.I.II of Trasylol and homogenized on ice with a polytron (Fig. 2). We have found that this procedure maximizes extraction and minimizes aggregation and degradation of copper metalloproteins (Refs. 9, 22). Duplicate aliquots of each homogenate were reserved for metal analysis.

i Homogenates were centrifuged at i15,000 x g for 90 min at O'C. The final clear j supernatants (S-Il5s) were saturated witn N2 (g), frozen immediately with liquid l

nitrogen, and stored at -70'C until they were chromatographed. . Duplicate I aliquots of each S-115 and the pellets were reserved for metal analyses.

i The S-Il5s of the livers were processed on a Waters high performance liquid i

chromatograph (HPLC) fitted with a Varian TSK 3000 SW gel permeation column l

(22 x 300 mm). To separate copper metalloproteins,' aliquots of the S-ll5s were injected and eluted with a 0.15 M Nacl and 50 mM Tris-hcl buffer solution (pH 7.6 at 25'C) at a flow rate E4 mf,/ min. Molecular absorbance at 280 nm was monitored continuously and the 35 fractions collected were analyzed for metals. The column was standardized using proteins of known molecular weights.

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Liver  !

' Tris buffer Metal N atmosphere

• 2 analysis Homogenization 2 mercaptoethanol

, Trasylol Centrifugation 115,000 x g, 90 min, O C Pellet , Supernatant i f Insoluble Soluble _ Metal copper copper analysis Metal Molecular weight HPLC analysis separation TSK 3000 I I HIM IMW LMW Metal Metal Metal dnalysis analysis analysis of fractions of fractions of fractions

Fi6ure 2. Flow diagram of metalloprotein assay.

A modified procedure was used to separate the zinc metalloproteins. Because of the ionic properties of the TSK 3000 SW column, zinc bound to metalloenzymes is removed and eluted as free zine (Ref. 23). To prevent this from occurring, we saturated ionic sites ori the column with icnic cadmium by passing a 500-ppb Cd++,50 mM Tris, 0.15 M Nacl buffer solution (pH 7.6) through the column.

Next, a buffer solution with a lower concentration of ionic cadmium (20 ppb) was pumped through the column until no change in the cadmium concentration being eluted was observed. Aliquots of the S-l!5 were injected in the column and cluted with the low cadmium concentration buffer. No changes in the concentration of cadmium cluted during the sample run were observed. Also, with this procedure, no free zinc cluted from the column and the copper profiles remained the same.

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METAL ANALYSES Tissues for metal analysis were either dried at 100*C, ashed at 450*C, dissolved in a mixture of concentrated hcl and HNO3 (3:1) and brought to final volume with double-distilled water, or digested with a perchloric and nitric acid mixture and then brought to final volume with double-distilled water. A standard reference material of oyster tissue obtained from the National Bureau of Standards was analyzed with the bluegill tissues to validate analytical procedures. Unknowns and reference material were analyzed with an atomic absorption spectrophotometer; measurements were corrected for reagent blanks.

RESULTS TISSUE METAL CONCENTRATIONS Copper concentrations in muscle tissue from bluegills collected in May 1981 were lower in fish from Christensen's Pond (control site) than in those from the intake and discharge sites (Table 3). The concentrations in animals from the intake and discharge were significantly different from those frem the control site but not from each other.

Table 3. Copper in muscle tissues (pg Cu /g dry weight) from bluegills collected at the H. B. Robinson Nuclear Power Station, May 1981.

Control pond intake zone Discharge zone Fish length Copper Fish length Copper Fish length Copper (cm) (cm) (cm) 20.3 1.213 21 1.891 17.4 2.731 18.4 1.503 20 1.921 11.4(6)a 2.018 17.8 1.222 20 2.232 11.2 (6) 1.85 17.8 1.353 19 2.279 11.1 (6) 2.197

! 7.1 1.818 19 3.03 10.2 (6) 2.424 16.5 1.539 19 1.931 8.'e (l 7) I.75

( l 5.9 1.58 18 2.358 15.2 1.33 9.4 (10) 2.08

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f 5.2 1.849 9 (10) 2.849 l 15.2 1.251 8.2 (10) 2.931 l 15.2 1.923 Mean i standard deviation 16.8 1.507 16.3 2.350 11.6 2.162 17 1 1 0261 125 1 0436 1 304 1 0369

a pooled muscle sample. Va'ues in parentheses are the number of fish pooled and I

, the corresponding fish length is an average.

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Copper concentrations in. liver tissue .dif tered greatly with the collection site.

The concentrations in the pooled liver samples from animals collected in May 1981 from the control, intake, and discharge sites were 11.3,167.9, and 558.0 pg Cu/g dry weight, respectively. Analysis of the livers of bluegills collected in November 1981 also showed large differences in copper concentrations with collection site (Table 4). Copper was lowest in livers from fish from the control pond and highest in those from the discharge site. Even though the variabiliti among samples from each collection site was large, copper concentrations in livers of bluegills collected from the discharge and control sites were significantly different.

Livers of bluegills were also analyzed for zinc and cadmium (Table 4). The~mean values of the zinc concentrations followed the same pattern as those of copper, but the differences between sites were smaller. Cadmium was highest in livers from fish collected from the intake site; the reason for this larger value is unknown. There was no correlation between the three metal concentrations in the livers.

Althcugh there were large differences in the size of fish collected from different sites, no direct relationship between size and copper concentration was-observed. The average size of fish from the control site was 8% greater than that of fish from the intake site and 30% greater than that of fish from the discharge site.

METALLOPROTEINS Copper present in the livers of bluegills was resolved into four compartments. An insoluble one was recovered as the pellet from a 115,000 x g centrifugation. The soluble ones found in the S-115 were resolved by HPLC according to molecular weight. The molecular weights of the proteins eluted fell into three categories:

low molecular weight (LMW) in the 6,000. to 40,000-dalton range eluted between 73 and 93 mL, intermediate molecu.lar weight (IMW) in the 40,000- to 126,000-da_Iton range eluted between 61 and 72 mL, and _ high molecular ~ weight (HMW) in the 126,000 to >670,000 dalton range eluted between 40 and 60 mL.

Elution profiles of copper determined for S-115s of livers from bluegills taken from the control, intake, and discharge sites are shown in Figs. 3-5, respectively.

Because the elution profiles of zinc were similar for the S-Il5s from fish from l the same site, only the profiles from the livers of the two fish that had the highest copper concentration at each site are shown (Fig. 6a). Elution profiles at 280 nm were similar for the S-115s from fish from all sites and therefore only one profile is shown (Fig 6b). Cadmium concentrations in _the~ fractions of S-Il5s from all fish livers were below detection limits for almost all fractions; no elution profiles were constructed.

Quantities of copper eluted in the HMW, IMW, and LMW peak areas and present in the pellet and intact liver were determined (Table 3). In the LMW, IMW, and HMW peaks, the quantities of copper were much higher in fish from the intake and discharge sites than from the control site. The mean of the copper concentrations in the LMW peak area in S-Il5s from fish from the discharge site was 20 times greater than that from the control site; the mean from the intake site was 6 times greater. Some overlap in LMW peak size was seen in fish from the intake and discharge sites. Large differences were also observed in the HMW 10

i i

Table 4. Copper, cadmium, and zinc in livers (pg metal /g dry weight) from bluegills collected at the H. B. Robinson Nuclear Power Station, November 1981.

Sample Fish length numbera (cm) Copper Cadmium Zinc Control pond 1 16.3,15.6 7.1 0.23 47.5 2 14.8,14.9 8.9 < 0. I 62.6 3 14.0, 13.9 12.3 < 0. I 53.1 4 15.3,14.4 15.I < 0. I 62.9 5 14.5, 19.8 21.5 0.21 53.4 Mean standard deviation 15.4 13.0 <0.15 55.9 i 1.7 5.7 0.07 1 6.7 Intake site 6 15.5,17.2 29.9 3.80 60.3 7 15.3,17.3 40.6 7.78 5 I .5 8 15.8, J 4.7 66.4 0.98 58.3 9 18.4, 16.0 81.7 3.70 53.3 10 15.4, 16.9 127.9 1.63 52.6 l

Mean standard deviation 16.3 69.3 3.58 61.2 i 1.2 1 38.6 0.07 i 6.7 Discharge site 11 13.7,13.8 105.0 <0.10 78.8 12 16.4, 16.4 280.9 1.12 76.2 13 15.9,14.4 281.6 0.63 66.4 14 14.3,16.5 498.0 0.93 84.9 15 15.6,13.5 61I.9 0.80 78.6 Mean t standard deviation 15.1 355.5 0.72 77.0 1.2 i 199.9 0.39 i 6.7 a Each sample consisted of the livers from two bluegills.

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Figure 3. Elution profiles of copper in S-Il5s of liver tissue of bluegills collected in November 1981 from the control pond. Ni.!mber adjacent to each curve is the liver copper concentration in pg Cu/g dry weight.

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Figure 4. Elution profiles of copper in S-Il$s of liver tissue of bluegills collected in November 1981 from the cooling lake near the intake site of the H. B. Robinson Power Station. Number adjacent to each curve is the liver copper concentration in ug Cu/g dry weight.

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Figure 5. Elution profiles of copper in S-Il5s of liver tissue of bluegills collected in November 1981 from the cooling lake near the discharge site of the H. B. Robinson Power Station. Number adjacent to each curve is the liver copper concentration in pg Cu/g dry weight.

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Figure 6. (a) Elution profiles of zinc in S-Il5s of liver tissue of the two bluegills that had the highest concentration of copper at each site. Number adjacent to each curve is the liver copper concentration in pg Cu/g dry weight. Bluegills were collected in November 1981 from a control pond (A) and from the H. B. Robinsen cooling lake near the intake (s) and discharge (0) of cooling water from the nuclear power station. (b) Typical UV (280-nm) absorbance j

chromatogram of S-Il$s of liver tissue of bluegills collected near the l H. B. Robinson Nuclear Power Station in November 1981.

15

Table 5. Copper in liver tissue and liver tissue compartments (10-4 pMjg wet weight) from bluegills collected at the H. B. Robinson Nuclear Power Station, November 1981.

Sample Total Liver Pellet number HMWa IMWb tywc eluted tissue (%)

Control pond 1 20 12 105 161 227 76 l 2 18 12 93 143 173 39 3 20 12 103 142 205 74 4 16 12 100 160 240 86 5 25 38 254 363 445 102 Mean 20 17 131 194 258 75 Intake site 6 74 46 491 688 944 261 7 87 66 572 803 1030 232 8 125 90 783 1090 1432 348 9 150 209 938 1360 1553 399-10 146 180 1180 1590 2235 550 Mean 116 118 793 1106 1439 358 Discharge site 11 55 39 753 855 1037 192 12 506 487 1690 3130 3777 755 13 454 546 3070 4290 5507 1212 14 632 543 3120 4710 5823 1060 15 1480 1330 3530 6620 8498 1275 Mean 625 589 2433 3921 4888 899 a High molecular weight fraction was eluted between 40 and 60 mL and includes proteins in the 126,000- to >670,000-dalton range.

b Intermediate molecular weight fraction was eluted between 61 and 72 mL and includes proteins in the 40,000- to 126,000-dalton range.

c Low molecular weight fraction was eluted between 73 and 92 mL and includes proteins in the 6,000- to 40,000-dalton rar.ge.

16

peak areas; the mean from fish from the discharge site was 30 times greater and that from the intake site 5 times greater than that from the control. The quantity of copper bound to protein- in the HMW peak areas was comparatively small except in S-Il5s from four of the five pairs of fish sampled from the discharge site. The significance of the differences among sites was tested using a paired t-test (Table 6). Differences between values obtained for livers from fish co!!ected from the cascharge and control sites and between those from the intake and discharge sites were generally highly significant.

The relationships among the quantities of copper in the different compartments, including that associated with the LMW, IMW, and HMW metalloproteins and the pellet differed with the copper concentration in the liver. In fish from the control and intake sites, the quantities of copper eluted totally and in the pellet were directly related to the copper concentrations in the liver (Fig. 7). For this same group of fish (control and intake), the quantities in the LMW, IMW, and HMW protein pool were directly related to the total eluted, but the dependence of the quantity of copper in the LMW pool on the copper concentration in the liver (indicated by the slope of the LMW line) differed greatly from that of the HMW and IMW pools (Fig. 8, Table 7). The slope of the line for the LMW protein pool was about seven times greater than that for the HMW protein pool. Also there was less scatter to the data for the LMW than for the HMW and IMW protein pools; the linear regression parameter R 2 was 0.99, 0.89, and 0.97, respectively.

These data indicate that in livers of the intake and control fish, the MLPs (LMW protein pool) are by far the largest depot of copper. However, increases in soluble copper in the liver result not only in increases in the MLP pool, but also in increases simultaneously in the IMW and HMW metalloenzyme pools.

Table 6. The p-values obtained from paired t-tests of the parameters measured in livers from bluegills collected from the control, intake, and discharge sites at the H. B. Robinson Nuclear Power Station, November 1981.

Paired group Control vs. Control vs. Intake vs.

Parameter intake discharge discharge H MWa 0.6247 0.0083b 0.0211b IMWc 0.5679 0.0060d 0.0180b LM%e 0.1577 0.0002f 0.0029d Totaleluted 0.2701 0.0005f 0.0038d Total liver /g 0.2713 0.0007f 0.0052d Pellet /g 0.1194 0.0004f 0.0075d l

a High molecular weight fraction was eluted between 40 and 60 mL and includes proteins in the 126,000- to >670,000-dalton range.

b Level of significance: p < 0.05.

l

c Intermediate molecular weight fraction was eluted between 61 and 72 mL and l ingludes proteins in the 40,000- to 126,000-dalton range.

! Level of significance: p < 0.01.

l e Low n >lecular weight fraction was eluted between 73 and 92 mL and includes l proteins in the 6,000- to 40,000-dalton range.

I Level of significance: p < 0.001.

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Figure 7. Concentrations of copper in the pellet (e) and eluted totally (a) compared to that in the lier. Bluegills were collected from the control and intake sites in November 1981. The equation of the line for the total eiuted was y = 0.758x + 7.108; R2 was 0.983. The equation of the line for the pellet was y = 0.239x + 13.554; R2 was 0.990.

At the high copper concentrations measured in the livers of most discharge fish, there was a departure from the linear relationship between the quantity of copper in each compartment and in the total liver (Fig. 9). In the HMW protein pool, the quantities were greater and in the LMW protein pool and pellet they were less than expected, relative to those predicted from the copper concentrations in livers of the control and intake bluegills. These data indicate that the capacity of the MLPs (LMW protein pool) and the pellet for detoxifying or storing copper was becoming saturated and spillover into the IMW and HMW metalloenzyme pool was occurring.

The concentrations of zinc in the total liver, supernatant, and pellet of fish from all three sites were quantified (Table 8). Increases in concentration of zinc were large only in the supernatant of livers of fish from the discharge site (Fig.10).

The R2 for the regression of zinc in the supernatant vs. that in the liver was 0.967 for bluegills from the discharge site and 0.481 for those from the intake and control sites combined. Comparable values for the pellet were 0.628 and 0 341, respectively.

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Figure 8. Concentrations of copper in the LMW (*), IMW (A), and HMW (s) protein pools compared to that eluted totally. Bluegills were collected from the control and intake sites in November 1981. The equation of the line for LMW protein was y = 0.728x - 11.309; R2 was 0.999. The equations of the lines for the IMW and HMW protein pools were y = 0.125x - 13.27 and y = 0.10lx + 2.495, respectively; R 2was 0.889 and 0.972, respectively.

Table 7. Intercept, slope, and R2 values obtained from the regression analyses of liver copper vs. compartment copper and total eluted copper vs. compartment copper for the control and intake samples collected at the H. B. Robinson Nuclear Power Station, November 1981. All concentrations are in 10-4 pM Cu/g wet weight.

Independent Dependent variable (x) variable (y) Intercept Slope R2 Liver Total eluted 7.108 0.758 0.983 Liver Pellet 13.554 0.239 0.990 Liver LMWa -8.824 0.555 0.992 Liver HMWb , gywc -5.852 0.167 0.898 Total eluted LMW -11.309 0.728 0.998 Total eluted HMW 2.495 0.101 0.972 Total cluted IMW -13.270 0.125 0.889 Total eluted HMW + IMW -10.775 0.226 0.958 a Low molecular weight fraction was eluted between 73 and 92 mL and includes proteins in the 6,000- to 40,000-dalton range.

b High molecular weight fraction was eluted between 40 and 60 mL and includes proteins in the 126,000- to >670,000-dalton range.

c Intermediate molecular weight fraction was eluted between 61 and 72 mL and includes proteins in the 40,000 to 126,000-dalton range.

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Figure 9. Concentrations of copper in the LMW (*) and HMW (a) protein pools and in the pellet (v) compared to that in the liver. Bluegills were collected from the control, intake, and discharge sites in November 1981. The solid line was drawn by eye through the points. The dashed line represents the extension of the line obtained from the regression of the compartment vs. total liver concentration for the intake and control fish.

The quantities of zinc in the various compartments of the liver were quantified for the pairs of fish having the highest copper concentration at each site (Table 9, Fig.10). The quantities of zinc present in the HMW and IMW peak areas were smaller in S-Il5s from bluegills from the discharge site than from the inuke and control sites. These data indicate that copper may be displacing zint-from metalloenzymes in the HMW and IMW protein pools.

The total quantities of zine and copper associated with the LMW protein pool (MLPs) in livers of the two fish that had the highest concentrations of copper at each site were calculated. The quantities of metals in the fish from the control pond was 1.4 pM, the intake site was 2.2 pM, and the discharge site was 4.1 x 10-3pM. In the HMW pool the comparable values for the same animals were 2,1.8, and 2.8 x 10-3pM. These data confirm that increased concentrations of copper and zinc in the liver are associated primarily with the LMW protein fractions.

20

l l

I Table 8. Zinc in compartments of liver tissue (pg Zn/g dry weight) from bluegills collected at the H. B. Robinson Nuclear Power Station, November 1981. '

Sample number Liver Supernatant Pellet Control pond l 1 60.4 69.4 45.5 2 73.I 109.2 43.2 3 90.3 122.3 48.5 4 62.3 71.6 44.3 5 32.8 68.5 43.4 Intake site 6 60.3 72.3 47.7 7 52.6 58.1 38.0 8 58.I 77.I 36.5 9 56.5 67.4 37.1 10 51.1 58.6 45.6 Discharge site l

11 78.8 90.3 40.5 12 73.1 I28.3 45.0 13 72.3 70.9 34.3 14 106.7 96.5 56.3 15 79.6 80.8 47.0 t

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Figure 10. Concentrations of zinc in the intake pellet (a), discharge pellet (o),

and control pellet (a) and intake supernatant (s), discharge supernatant (*), and control supernatant (a) compared to that in the liver.

pM Zn Table 9. Zinc in compartments of liver tissue (10-4 t each bluegills having the hig site of the H. B. Robinson Nuclear Power Station, November 1981.

Sample Collection number site HMWa IMW b ggwc 5 Control 2,026 1,194 1,170 10 Intake 1,624 1,148 999 15 Discharge 1,423 876 559 a High molecular weight fraction was eluted between 40 and 48 mL and includes proteins in the 360,000 to >670,000-dalton range.

b Intermediate molecular weight fraction was eluted between 52 and 68 mL and includes proteins in the 55,000- to 360,000-dalton range.

c Low molecular weight fraction was eluted between 72 and 92 mL and includes proteins in the 6,000- to 35,000-dalton range.

22

DISCUSSION There is considerable information on copper concentrations in tissues of freshwater fish (Ref. 24). In general, the data support our results indicating that muscle tissues contain low concentrations of copper and do not reflect increases of copper in the environment, whereas liver tissues are high in copper and do reflect such changes.

Liver copper concentraticns have been determined for bluegills from different sites (Refs. 25-28). Data obtained by Benoit are of special interest because the analyses were performed on fish exposed chronically in the laboratory to known concentrations of copper (Ref. 28). Although the copper concentrations in the livers of fish exposed to 12, 21, 40, 77, and 162 pg Cu/L increased with exposure concentrations, only the difference between those exposed to 162 pg Cu/L and controls were significant because of the large variability within each sample group. Variability appeared to increase with exposure concentration; the fractional standard des;ation of the mean copper concentration was higher in copper-exposed than in control fish. These data indicate that there are large differences in response of fish to the same concentration of copper.

Our data on inetalloproteins, like that of others, support the concept that MLPs play an important role in the detoxification of metals. We found much higher concentrations of copper MLPs in livers of bluegills from copper-impacted sites than in those from the control site. Although no data are available on the induction of MLPs in fish exposed chronically to known concentrations of copper, the results available on exposure to cadmium indicate that MLPs are induced in quantities related to exposure concentrations (Refs.10,13). Because the concentrations of copper MLPt. in livers of fish from the discharge site (higher copper) were greater than in those from the intake site (lower copper), it appears that copper MLP induction in bluegills is also related to exposure concentrations.

Information on the partitioning of copper in different compartments of the liver as a function of liver copper concentration is limited. A number of investigations have measured copper concentrations in MLPs and metalloenzymes in fish livers, but these were not related to total liver concentrations (Refs. 4,13,18, 21). We measured the quantities of copper not only in the MLPs (LMW protein pool) and metalloenzymes (IMW and HMW protein pools), but also in the insoluble fraction of the liver (pellet). Our data on partitioning of copper indicate that in bluegills i having copper liver concentrations <2500 pM/g wet weight, there were increased l quantities of copper in these compartments with increased quantities of copper in the liver. However, the rate at which copper increased was much higher in the MLPs than in the other compartments. Departure from the linear relationship was seen in livers containing >2500 pM Cu/g wet weight. The data indicate that the rate of increase of copper associated with the MLPs and pellet was lower in these fish, which were all from the discharge site, than in fish with livers containing < 2500pM copper, whereas in the metalloenzyme pool, the rate was higher. Factors controlling the partitioning of copper appear to differ in animals with high and low concentrations of copper. Because liver concentrations are demonstrated to reflect exposure concentrations, it is suggested that partitioning dif fers with exposure concentrations.

l 23 l

l

Results from previous studies of metalloproteins in livers of vertebrates and invertebrates indicate that when the capacity to produce MLPs is exceeded, spillover of metal into the metalloenzyme pool occurs. Our results on the partitioning of copper among liver compartments indicate that in the fish from the discharge site, disproportionate amounts of copper are present in the meta!!oenzyme pool. It is of interest to note that the spillover is indirated at the same total liver concentrations that Benoit found bluegills to have decreased reproductive capacity and increased mortality (Ref. 28).

Changes may occur at the subcellular level when the capacity to synthesize MLPs is surpassed. In the presence of a large excess of a metal, zinc may be displaced from metaltoenzymes. Our data indicate that copper was displacing zinc in the metalloenzyme pool in livers from fish from the discharge site. This may result in conformational changes in enzymes that interfere with the binding of the substrate to the enzyme and reduce the rate at which essential metabolic reactions occur. Also, the displacement of a required metal from a metalloenzyme may result in the splitting of enzymes into subunits that may prevent regulation of enzymatic activity.

Deleterious effects of metals are related not only to current exposure concentrations, but also to past exposure conditions. Evidence is available that fish preexposed to sublethal doses of metals are more tolerant of metals in subsequent exposures. Bouquegneau showed that preexposure to a sublethat dose of HgCl 2, which induces the synthesis of metallothioneins in the gills, reduces the toxicity of mercury (Ref. 29). Furthermore, he stated that preexposure with a sublethal dose of CdCl2 was more effective than HgCl 2 in reducing the toxicity of mercury. Dixon and Sprague found that rainbow trout preexposed to 94pg Cu/L for 21 d had lower mortality rates when exposed to 570pg Cu/L than control fish not preexposed (Ref. 30). Furthermore, the whole-body copper concentrations did not increase significantly in preexposed fish, but did in control fish. Preexposure also resulted in significant increases in the quantities of LMW hepatoproteins in the soluble fraction of the liver.

Little information is available on the turnover of MLPs in the liver of fish.

Reichert et al. exposed Coho salmon to increased aqueous cadmium for 15 d and then measTre~d cadmium concentrations in the gill, liver, kidneys, and blood af ter 8- and 37-d depurations (Ref.19). During depuration, cadmium concentrations decreased in the gills and blood, stayed constant in the liver, and increased in the kidney. However, because they did not separate the metalloproteins in the liver and kidney, it is not known whether the cadmium was associated with MLPs. In another study, brook trout were exposed to cadmium and it was found that metal concentrations in the liver and kidney continued to increase after 12-wk depuration (Ref. 31).

The MLPs in the bluegill livers appear to bind copper, cadmium, or zinc. We have no information on the relative binding affinities for the three metals in bluegills. Noel-Lambert found that zinc, but not copper, could be displaced by cadmium on the MLPs in liver of eels (Ref.13). This was not unexpected because it is well known that copper is bound much more firmly to metallothioneins than either cadmium or zinc.

24

b CONCLUSION S i

l Our results indicate that characterization of the molecular weights of the proteins that metals associate with provides more sensitive and accurate mdications of metal stress than does measurement of total liver concentrations.

However, data from bluegills exposed to met;'s in the field are more difficult to

interpret than those from fish exposed in ae laboratory because concentration and duration of exposure are unknown and may contribute to.the variability in the results.

Our analyses further indicate that labile copper released from the cooling system

of the H. B. Robinson Nuclear Power Station may be implicated in the increased deformities and reduced reproductive capacity found in the bluegill population in the adjacent cooling lake. We determined that copper was a causai factor because concentrations were high in.most fish from the discharge site and it was

! present in the metalloenzyme pool at concentrations that could potentially interfere with normal metabolic activity.

Additional research is required to define the concentrations of copper in effluents and the duration of exposure that adult and early life-history stages of bluegills can tolerate without adverse effects. Also, the turnover of copper in metallothioneins should be (. valuated for all life-history stages. These kinds of information can be used to set emission standards that protect fisheries' resources and permit continued operation of power generating stations.

RECOMMENDATIONS MLP INDUCTION IN ADULT BLUEGILLS Information is needed to assess the concentrations of copper in effluents and the durations of exposure that bluegills' can tolerate without adverse effects.

l Concentrations of copper in effluents are dependent on plant operational

procedures and on seasonal changes in the environments. Fish may be exposed to (1) continuous low levels of copper, (2) alternate periods of high and low levels of l copper that reflect seasonal changes in after flow in the system, or (3) a pulse of l copper during start-up of water flow through the cooling system after a l shutdown. Controlled laboratory experiments that take these variables into co~ideration should be performed.

MLP RESIDENCE TIME IN ADULT BLUEGILLS The turnover of MLPs in bluegills should be evaluated. The time required for depuration of the copper bound to MLPs will determine the frequency of intermittent high concentrations of copper in the water that can be tolerated with a minimum of adverse effects on bluegill populations.

MLPS IN EARLY LIFE-HISTORY STAGES The role of MLPs in early life stages should be determined. No information is available regarding now the MLP detoxification system is activated and how the capacity of the system changes with development. These kinds of data are required to schedule pulse releases from nuclear power stations so that the impact to eggs, larvae, and fry of bluegills is minimized.

25 i.

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, , , . , _ - . - . ~ _ , . , _ _ . _ . , . , _ - . . , . . _ . . . _ -,_m., , _ _ _ _ _ , . . .._,--,_y __

- . , , . . . _ . _ , _ - _ . - . , m.,- ,.

DIRECT EFFECf5 OF COPPER ON DEVELOPMENT Experiments are required to determine how chronic, sublethat exposures of copper affect embryonic development. Continuously exposing . embryos to low levels of copper may be deleterious. They may grow slowly or become -

anatomically malformed. This could result in decreased survival of early and adult stages because of increased predation of those fish that were severely affected and in stunted and deformed adults of those fish that were moderately affected.

A T

26

REFERENCES

1. F. L. Harrison, J. Lam, and R. Berger, " Sublethal Responses of Mytilus edulis to increased Dissolved Copper," in Proc. Symp. Biological Availability of Trace Metals, October 4-8, 1981, Hanford, Washington (in press).

2. Carolina Power and Light Company, "H. B. Robinson Steam EJectric Plant, 1976-78, Environmental Monitoring Program Results," Carolina Power and Light Company, Florence, South Carolina, Vol.1,1979.

3. B. L. Vallee, "Metallothionein: Historical Review and Perspectives," in Metallothioneins, J. H. R. Kagi and M. Nordberg, Eds. (Birkhauser Verlah Basel, Stuttgart, Germany,1979), pp.19-40.

4. D. A. Brown and T. R. Parsons, " Relationship Between Cytoplasmic Distribution of Mercury and Toxic Effects of Zooplankton and Chum Salmon (Oncorhychus keta) Exposed to Mercury in a Controlled Ecosystem," Journal of the FisherieTResearch Board of Canada 35, 880-884 (1978).

5. F. L. Harrison and R. Berger, " Effects of Copper on the Latency of 15 Lysosomal Hexosaminidase in the Digestive Cells of Myti!us _ edulis," Marine Biology (in press).

6. J. S. Young and G. Rosijadi, Pacific Battelle Northwest Laboratory, Sequimn, WA, private communication (1982). Available from PNL.

7. J. H. R. Kagi and M. Nordberg, " Biochemical Properties," in Metallothioneins, J. H. R. Kagi and M. Nordberg, Eds. (Birkhauser Verlah Basei, Stuttgart, Germany,1979), pp. 56-64.

8. T. L. Coombs and S. G. George, " Mechanisms of Immobilization and Detoxification of Metals in Marine Organisms," in Physiology and Behavior of Marine Organisms, D. S. McLusky and A. J. Berry, Eds. (Pergamon Press, New York,1978), pp.179-187.

9. G. Roesijadi, " Influence of Copper on the Clam Protothaca staminea: Effects on Gills and Occurrence of Copper-Binding Proteins," Biological Bulletin 158, 233-247 (1980).

! 10. R. W. Olafson and J. A. J. Thompson, " Isolation of Heavy Metal Bin 6.'g Proteins from Marine Vertebrates," Marine Biology 28, 83-86 (1974).

I1. J. M. Bouguegneau, Ch. Gerday, and A. Disteche, " Fish Mercury-Binding

! 'thionein Related to Adaptation Mechanisms," FEBS Letters 55, 173-177 l (1975).

l

12. J. M. Bouguea,neau, " Evidence for the Protective Effect of Metallothioniens Against Incrganic Mercury Injuries in Fish," Bulletin of Environmental Conta,mination and Toxicology 23, 218-219 (1979).

I 27 1

(

13. F. Noel-Lambot- C. Gerday, and A. Disteche, " Distribution of Cadmium, Zinc and Copper in the Liver and Gills of the- Eel Anquilla anguilla with Special Reference to Metallothioneins," Comparative Biochemistry and

- Physiology (Part) C: Comparative Pharmacology 61(1),177-188 (1978).

14. E. Marafante, " Binding of Mercury and Zinc to Cadmium-Binding Protein in Liver and Kidney of Goldfish (Carassins auratus L.)," Experientia 32, 149-150 (1976).

15. R. Vaasjoki and 3. K. Miettinen, "Zn/Cd and Zn/Hg Metalloprotein Interactions in Rainbow Trout Studies by Gel Chromatography and Isoelectric Focusing," in Clinical Chemistry and Chemical Toxicology of Metals, S. 5. Brown, Ed. (Elsevier, N.>rth Holland Biomedical Press, New York,1977), pp. 71-74.

16. 3. H. Beattie and D. Pascoe, "A Cadmium-Binding Protein in Rainbow Trout," Toxicology Letters. 4, 241-246 (1979).

17. K. B. Pierson, " Characterization and in Vivo Responses of Zinc Induced Metallothionein in Rainbow Trout Saliso gairdneri," Amer.ican Zoologist 20(4), 797 (1980).

I 8. D. A. Brown, " Increases in Cd and Cd:Zn Ratio in the High Molecular Weight Protein Pool from Apparently Normal Liver of Tumor-Bearing Flounders (Parophrys vetulus)," Marine Biology 44, 203-209 (1977).

19. W. L. Reichert, D. A. Federighi, and D. C Malins, " Uptake and Metabolism of Lead and Cadmium in Coho Salmon (Oncorhynchuskistch)," Comparative Biochemistry and Physiology (Part) C: Comparative Pharmacology 63C, 229-234 (1979).

20. 1 Overnell, I. A. Davidson, and T. L. Coombs, "A Cadmium-Binding Glymorotein from the Liver of the Plaice," Piochemical Society _

Transact 6ns 5, 267-269 (1977).

21. R. 3. Pruell and F. R. Engelhardt, " Liver Cadmium Uptake, Ce .a inhibition and Cadmium Thionein Production in the Killifish (Funoulus l heteroclitus) Induced by Experimental Cadmium Exposure," Marine Environmental Research 3, 101-111 (1980).

22. L. Ryden and h. F. Deutsch, " Preparation and Properties of the Major

( Copper-Binding Compartment in Human Fetal Liver," Journal of Biological Chemistry 253(2), 519-524 (1978).

23. K. T. Suzuki, " Direct Connection of High-Speed Liquid Chromatograph (Equipped with Gel Permeation Column) to Atomic Absorption Spectrophotometer for Metalloprotein Analysis: Metallothionein," Analytica_1 Biochemistry 102, 31-34 (1980).

24. P. M. Stokes, " Copper Accumulations in Freshwater Biota," in Copper in the Environment, 3. O. Nriagu, Ed. (John Wiley and Sons, Inc., New 'ork, 1979),

pp. 356-381.

28

+

25. Carolina Power and Light Company and Lawler, Matusky and Skelly Engineers," Investigation of Deformities and Lowered Recruitme.nt of Bluegill (Lepomis macrochirus) in Robinson impoundment," Carolina Power & Light Company, Florence, South Carolina,1981.

26. 3. P. Giesy and 3. G. Wiener, " Frequency Distributions of Trace Metal Concentrations in Five Freshwater Fishes," Transactions of the American Fisheries Society 106(4), 393-403 (1977).

I

27. B. R. Murphy, G. 3. Atchison, A. W. McIntosh, and D. 3. Kolar, " Cadmium and Zinc Content of Fish from an Industrially Contaminated Lake," Journal of Fish Biology 13, 327-335 (1978).

28. D. A. Benoit, " Chronic Effects of Copper on Survival, Growth, and Reproduction of the Bluegill (Lepomis macrochirus)," Transactions of the American Fisheries Society 104, 333-358 (1975).

29. 3. M. Bouquegneau, " Evidence for the Protective Effect of Metallothioneins Against Inorganic Mercury injuries to Fish," Bulletin of Environmental Contamination and Toxicology 23, 218-219 (1979).

30. D. G. Dixoa and 3. B. Sprague, " Copper Bioaccumulation and Hepatoprotein Synthesis During Acclimation to Copper by Juvenile Rainbow Trout,"

Aquatic Toxicology 1, 69-81 (1981).

31. D. A. Benoit, E. N. Leonard, G. M. Christensen, and 3. T. Fiandt, " Toxic Effects of Cadmium on Three Generations of Brook Trout (Salvelinus fontinalis)," Transactions of the American Fisheries Society 4, 550-560 (1976).

3AS/af 29

U.S. NUCLE AR REGULATORY COMMISSION 022 BIBLIOGRAPHIC DATA SHEET l

a. T1TLE AND SUBTITLE (Add Vosume No.. sf acprcorrate) 2. (Leave biat'kl Concentrations of Copper-Binding Proteins in Livers of Bluegills from the Cooling Lake at the H.B. Robinson Nuclear 3 RECIPIENT'S ACCESSION NO.

Power Station

7. AUTHORIS) 5. DATE REPORT COMP 8.ETED

^

F.L. Harrison and J.R. Lam MoNT" May I'1'98"2

9. Pt:MoRMiNG ORGANIZATION N AME AND MAILING ADDRESS IInclud- ?p Code! DATE REPORT ISSUEO MONTH Lawrence Livermore National Laboratory November l YEAR 1982 P.O. Box 5507, Mail Code L-453 e . (tese, olan * /

Livermore, CA 94550 B (Leave Diank)

12. SPONSo8ING ORGANIZATION NAME AND MAILING ADDRESS (ine,uoe lip Cooe>

10. PROJECT-TASK / WORK UNIT No.

Offic of Nuclear Requiatory,Research FIN A0119 U.S. kuclear Regulat6ry Commission

n. CONTRACT NO.

Washington, DC 20555 DOE /NRC Interagency Agreement

'3. TYPE OF REPORT PE RIOD COV E RE D (inclusive aa:est Topical

15. SUPPLEMENTARY NOTES 14. (Leave etant)

16. ABSTR ACT 000 words or less)

Bluagills collected from the cooling lake of the H.B. Robinson Nuclear Power Station near the effluent discharge, near the water intake to the cooling system, and from a control population in a local pond were examined for total copper in muscle and liver tissues and metalloproteins in different compartments of liver tissues. Also, copper changes in the environment were reflected in liver but not in muscle tissue. Liver metalloproteins were separated into low molecular weight (LW), intermediate molecular weight (IMW), and high molecular weight (HMW) protein fractions using high performance liquid chromatography.

Large differences in kinds and quantities of metals associated with metalloproteins were found. Copper concentrations in the LW proteins (metallothiorein-like proteins) were highest in bluegills from the discharge site and lowest in those from the control pond.

Evidence of overloading of the metallothionein-like protein detoxification systein was found in bluegills at the discharge site. These data and that from related studies indicate that the labile copper released from the cooling system of the H.B. Robinson Nuclear Power Station may be implicated in the increased deformities and reduced reproductive capacity found in the bluegill population in the adjacent cooling lake.

17. KEY WORDS AND DOCUMENT ANALYSIS 17a. DESCRIPTORS

[

metal binding, protein, bluegills, effluents, detoxification, copper 17h. IDENT?FIE RS/OPEN-ENDE D TERMS

18. AVAILA8tLeTY STATEMENT 19. SE CURITY CLASS (Th,s report) 21. NO. OF P AGES UNCLASSIFIED 20 SECURITY CLASS (Thes page) 22. PRICE UNLIMITED s UNCLASSIFIED _

NRC FORM 335 (7-771

-. . , . . - - - , . . . - --- .,~ - - . . - . - - . , _ - - .- , . - - - . .