PCB CONCENTRATIONS IN FISHES AND BENTHIC INSECTS … · insect taxa were caddisflies dobsonflies. an. d stoneflies an, d were sample onld y at the Cornwall station. In addition to

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PCB Concentrations in Fishes and Benthic Insects From the Housatonic River,

Connecticut, in 1984 to 1994

Report No. 95-3F

Prepared for the

General Electric Company

by the

Environmental Research Division The Academy of Natural Sciences of Philadelphia

1900 Benjamin Franklin Parkway Philadelphia, Pennsylvania 19103-1195

8 May 1995

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EXECUTIVE SUMMARY

The Academy of Natural Sciences of Philadelphia conducted a biological monitoring study in the Connecticut portion of the Housatonic River during August and October. 1994. This was the last in a series of biennial studies required by the 1990 Housatonic Raver Cooperative Agreement between the Connecticut Department of Environmental Protection and the General Electric Company.

Purpose of study

The main objective of the 1994 study was to measure PCB concentrations in several taxa offish and benthic insects, as specified in Paragraph II. B of the Cooperative Agreement, and to assess potential temporal trends by comparing these concentrations with values from previous monitoring studies in 1984—1992.

Sampling stations

The four main sampling stations are specified in the Cooperative Agreement and were the same as in the 1984-1992 studies, hi upstream to downstream order, these are (West) Cornwall, Bulls Bridge. Lake Lillinonah, and Lake Zoar (Fig. 1 of report).

Taxa monitored

The fish taxa specified by the Cooperative Agreement to be monitored were brown trout and smallmouth bass. Brown trout were sampled only at the Cornwall station, while smallmouth bass were sampled at Cornwall, Bulls Bridge, Lake Lillinonah and Lake Zoar. The benthic insect taxa were caddisflies. dobsonflies. and stoneflies, and were sampled only at the Cornwall station.

In addition to the biological monitoring required by the Cooperative Agreement, the 1994 study also included sampling in Lake Housatonic, the next Housatonic River impoundment downstream from Lake Zoar (Fig. 1 of report). This work was performed as part of the RCRA (Resource Conservation and Recovery Act) facility investigation for the river, pursuant to a USEPA corrective action permit, and to further evaluate the need for fish consumption advisories. The species sampled in this lake were smallmouth bass, American eel, white perch, yellow perch, bluegill, pumpkinseed and redbreast sunfish.

PCB analysis

Analytical method. PCB analysis was based on the method of Mullin (1985). which allows specific quantitation of over 100 individual PCB congeners. This method permits both congener-based and Aroclor-based determinations of total PCB.

THE ACADEMY OF NATURAL SCIENCES

Quantitation of total PCB. Total PCB was quantified by two procedures. The congenerbased procedure sums the concentrations of all individual congeners (up to 121) quantitated by the analytical method. The Aroclor-based procedure is based instead on the concentrations of a much smaller number of congeners that are essentially unique to Aroclor 1254 or 1260. It extrapolates from these marker congeners to Aroclor concentrations, based on the relative proportions of the markers in each Aroclor, then sums the two Aroclor concentrations. Only the Aroclor-based procedure was used in the 1984-1990 studies, while both methods were used in the 1992 and 1994 studies. The newer, congener-based procedure is believed to yield a more accurate measure of total PCB.

Data analysis and rationale

Two basic types of differences in PCB concentrations are of interest in this study: differences among years and differences among stations. Year differences were assessed for each species offish, using appropriate statistical techniques (see below). Station differences were assessed only for smallmouth bass, since no other species is monitored at all sampling stations. Only year differences were assessed for benthic insects, which are monitored at the Cornwall station. Rigorous statistical assessment of year-differences was not performed for benthic insects, because samples historically have consisted of only a single composite for each year.

PCB concentrations in an individual fish can be strongly influenced by its age (or duration of exposure), sex, and lipid content. Moreover, samples collected in different years or at different stations typically differ in their age, sex, and lipid distributions. Therefore, observed differences in PCB concentrations among years or stations may simply reflect differences in these ancillary variables (e.g., unusually high lipid levels in a particular year) rather than real differences in PCB exposure. At the opposite extreme, real differences in exposure (e.g., a declining trend among years) may be masked by variability created by differences in these ancillary variables. Therefore, to the extent that inferences regarding differences in PCB exposure are of interest, it is important to identify and remove any statistically significant influence of these ancillary variables.

Given these facts, two criteria are paramount in choosing an appropriate statistical technique for analysis of the fish data: it must permit assessment of year and station differences, and it must permit detection and removal of the effects of differences in ancillary variables (age, sex, lipid content). A standard technique satisfying both criteria is Analysis of Covariance, which was therefore chosen.

Results

Year differences. The main result of the 1994 study is a clear decrease in PCB concentrations relative to all previous years. This decrease is seen for both brown trout and smallmouth bass at all four stations that have been monitored since 1984 (e.g., Figs. 7, 8 of report). In each case, the 1994 level was either the lowest ever observed during 1984-1994, or a


member of the lowest group of statistically indistinguishable years. PCB concentrations in benthic insects also were among the lowest observed since 1978 (Fig. 9 of report).

Station differences. When data for all study years are combined, smallmouth bass show a consistent, statistically significant pattern of decreased PCB concentrations in the downstream direction. For 1994 alone, fewer statistically significant station differences were found, but concentrations at the two most downstream stations (Lakes Zoar and Housatonic) are significantly lower than those at the two most upstream stations (Cornwall and Bulls Bridge).

Fish exceeding 2 ppm total PCB. The proportion offish with PCB concentrations exceeding the 2.0 mg/kg consumption advisory limit was as low as, or lower than, in previous study years. Fish exceeding this limit were found only at Cornwall (3 smallmouth bass and 3 holdover brown trout) and Lake Housatonic (1 12-year-old smallmouth bass and 2 eel).

THE ACADEMY OF NATURAL SCIENCES in

QUALITY ASSURANCE STATEMENT

Study Number: 40910-8612-44

Study Title: PCB Concentrations in Fishes and Benthic Insects From the Housatonic River. Connecticut, in 1984 to 1994

This study was performed under the general provisions of the Division's Quality Assurance Implementation Plan (Rev. 0, November 1988). The final report was determined to be an accurate reflection of the data obtained.

The dates of Quality Assurance activities on this study are given below.

Protocol Review: 4/94

Data Review: Fisheries: 2/13/95 Chemistry: 2/10/95

Report Review: 4/11/95

ARCHIVING: Raw data and the final report are filed in the Division's archives.

Carol J. Lee Date ' Quality Assurance Unit Environmental Research Division Academy of Natural Sciences

THE ACADEMY OF NATURAL SCIENCES iv

TABLE OF CONTENTS

EXECUTIVE SUMMARY

QUALITY ASSURANCE STATEMENT iv

LIST OF TABLES viii

LIST OF FIGURES ix

INTRODUCTION 1

LOCATION OF SAMPLING AREAS 3 Cornwall 3 Bulls Bridge 4 Lake Lillinonah 4 Lake Zoar 4 Lake Housatonic 5

METHODS 6 Fish Collection and Handling 6 Benthic Insect Collection and Handling 8 Preparation of Fillet Samples 8 Analysis of PCBs 9 Aging of Fish 10 Statistical Analysis 12

RESULTS 16 Synopsis 16 Relationship Between Congener and Aroclor Quantitation Methods 16 Differences in PCB Concentrations Among Stations 20 Differences in PCB Concentrations Among Years 25 Lake Housatonic Analyses 27 Fish Exceeding Consumption Advisory Limit 29 Benthic Insect Analyses 30 Precision and Replicate Analyses 31

DISCUSSION 32

LITERATURE CITED 35

APPENDIX A. SOP for PCBs and Pesticides in Fish Tissue A-l

APPENDIX B. SOP for Quantitation of PCBs, Pesticides and Industrial Compounds B-l

THE ACADEMY OF NATURAL SCIENCES v

TABLE OF CONTENTS

APPENDIX C. PCB concentration data for 1994 fish specimens C-l

APPENDIX D. Comparison of ages offish analyzed in 1984-1994 studies of PCB in Housatonic River fishes D-l

APPENDIX E. Results of 1992 and 1994 PCB analyses of selected groups offish at five stations on the Housatonic River E-l

APPENDIX F. Comparison of results of 1984-1994 PCB analyses of smallmouth bass at five stations on the Housatonic River fishes F-l

APPENDIX G. Comparison of results of 1984-1994 PCB analyses of brown trout at Cornwall G-l

APPENDIX H. Comparison of results of 1984-1992 PCB analyses offish not analyzed in 1994 H-l

APPENDIX I. Results of replicate PCB analyses of samples from the Housatonic River in 1994 1-1

APPENDIX J. Graphs of LNTPCB and LPCBLIP by year and age for each species and station J-l

THE ACADEMY OF NATURAL SCIENCES vi

LIST OF TABLES Page

Table 1. Summary of sampling methods and locations

Table 2. Number of each target species analyzed for PCB in the Housatonic River during 1994 . 6

Table 5. Least-squares means of In-transformed total PCB, from ANCOVA and ANOVA

Table 8. Total PCB concentrations in benthic insects at Cornwall in 1994 . . . 3 0

Table 3. Average CTPCB concentrations in fish from the Housatonic River 21

Table 4 Summary of statistical analyses 22

models 23

Table 6. Summary of total PCB concentrations offish 24

Table 7. Results of 1994 PCB analyses offish from five stations on the Housatonic River 28

THE ACADEMY OF NATURAL SCIENCES vii

LIST OF FIGURES

Figure Page

1 Map of the study area 2

2 Comparison of average TPCB and CTPCB estimates 17

3 Relationship between TPCB and CTPCB for 1992 and 1994 samples of smallmouth bass and brown trout 17

4 Relationship between TPCB and CTPCB for 1992 and 1994 samples of smallmouth bass and brown trout, by station 18

5 Relationship between LNTPCB and In(CTPCB) for 1992 samples of species other than smallmouth bass and brown trout 18

6 Deviation between predicted and observed values of LNTPCB as a function of LNCTPCB 19

7 Mean LNCTPCB and CTPCBLIP in smallmouth bass from five stations on the Housatonic River 25

8 Average concentrations of In(CTPCB) and lipid-normalized CTPCB in brown trout and smallmouth bass from Cornwall in 1984-1994 27

9 Historical pattern of PCB concentrations in benthic insects at Cornwall, 1978-1994 31

THE ACADEMY OF 1SATURAL SCIENCES viii

INTRODUCTION

Since 1984, The Academy of Natural Sciences of Philadelphia (ANSP) has conducted biennial fish surveys in the Connecticut portion of the Housatonic River. These studies have documented a reduction of PCBs, relative to pre-1984 levels. Benthic insects have also been monitored over this period and show a similar pattern of reduced PCBs compared to pre-1984 levels. Both of these monitoring programs are part of the Housatonic River Cooperative Agreement between the Connecticut Department of Environmental Protection and the General Electric Company (hereafter, the Cooperative Agreement) and were continued by ANSP in 1994, the last year covered by the existing Cooperative Agreement.

The main objectives of the 1994 study are the following.

1. Measure PCB concentrations in selected fish of the Housatonic River for comparison with studies conducted in 1984, 1986, 1988, 1990 and 1992. As specified in Paragraph II.B of the Cooperative Agreement, the groups monitored were brown trout from Cornwall and smallmouth bass from Cornwall, Bulls Bridge Station, Lake Lillinonah and Lake Zoar (see Fig. 1).

2. Measure PCB concentrations in selected benthic insects from Cornwall for comparison with studies conducted in 1978-1981, 1984-1990 and 1992. Paragraph II.B of the Cooperative Agreement requires monitoring of benthic invertebrates at Cornwall but does not specify the taxa. For consistency with previous studies, three benthic insect taxa were sampled: filter-feeding caddisflies, and predatory stoneflies and dobsonflies.

3. Provide information on patterns of PCB bioaccumulation with respect to exposure time, lipid content and basic physiological properties offish. PCB concentrations and lipid content were analyzed in Cornwall brown trout collected at three times in August and October 1994. In addition, two brown trout from the Burlington Trout Hatcher}' were analyzed to provide information on pre-stocking PCB concentrations.

4. Measure PCB concentrations in selected fish from Lake Housatonic. Biological monitoring of Lake Housatonic (Fig. 1) is not required by the Cooperative Agreement, and fish from this lake were not analyzed in previous ANSP studies. In 1994, smallmouth bass, yellow perch. white perch, three species of sunfish, and American eel from Lake Housatonic were analyzed. These data were collected as part of a RCRA (Resource Conservation and Recovery Act) facility investigation for the river, pursuant to a USEPA corrective action permit, and to further evaluate the need for fish consumption advisories.

The 1994 study was undertaken pursuant to the requirements of Paragraph II.B of the Housatonic River Cooperative Agreement and satisfies all biological monitoring requirements for the year 1994.


^SSACHUSETTS

CONNECTICUT""

Bulls Bndge

10 miles

Figure 1. Map of the Housatonic River showing sampling stations for the 1994 fish and benthic insect collections. Approximate locations of dams forming the impoundments are also shown.


LOCATION OF SAMPLING AREAS

Fish were collected from five stations on the Housatonic River in 1994 In downstream order, these are Cornwall, Bulls Bridge, Lake Lillinonah, Lake Zoar and Lake Housatonic (Fig 1). The first four stations are specified in the Cooperative Agreement and are the same as in previous ANSP monitoring studies. Lake Housatonic was sampled for the first time in 1994

Three collecting trips were made during August and October 1994 (Table 1) Lake Housatonic was sampled during the October trip only. Additional details about each sampling trip and station are provided below.

Table 1 Summary of sampling methods and locations for 1994 ANSP fish collections on the Housatonic River, Connecticut

Sampling Location Tripl

Date

Trip 2 Trip 3 (8/1-5/94) (8/9-10/94) (10/7-12/94)

Cornwall B W* B,W*

Bulls Bridge E,G G

Lake Lillinonah E,G E

Lake Zoar E,G E

Lake Housatonic E

KEY B = Backpack electroshockmg E = Boat electroshockmg G = Gill net collection W = Walkalong (shore) electroshockmg ' = Collection with State of Connecticut gear

Cornwall

Newly stocked brown trout were collected in West Cornwall near the State Route 128 covered bridge on 4 August 1994. (Brown trout were stocked on 23 May 1994.) A backpack electroshocker was used. The specific locations sampled were just upstream of the bridge, including the mouth of Mill Brook about 20 m upstream.

A second sampling trip to the Cornwall area on 9-10 August 1994 coincided with Connecticut DEP's annual river survey. Brown trout and smallmouth bass were collected from the "Pushemup" area, approximately 3.5 km upstream of the State Route 128 covered bridge Fish were also collected from a new site on the river near the town of Cornwall Bridge. The new site was Turnip Island (west channel), an area about 5.2 km downstream from the State Route 128 covered bridge.


Two collections were made during the third trip. The first collection (7 October 1994) produced several holdover brown trout (with fin clips) in the riffle areas extending upstream from just above the "trash pool" (150 m downstream from the Route 128 covered bridge) to the mouth of Mill Brook. The second collection (12 October) produced several young adult or subadult brown trout from the areas on both banks under the Route 128 covered bridge, including the riffle areas 50 m downstream of the bridge.

Insects were collected on 8 and 9 June 1994 from the Housatonic River at US Highway 7 and the State Route 128 covered bridge.

Bulls Bridge

Fish were collected on 3-4 August 1994 by boat electroshocking and gill nets, and on 6-7 October by gill nets only. Sampling was conducted throughout the reservoir, from 1.0 km above the dam to a point just above Kent, about 0.6 km above the State Route 341 bridge. During the August collection, the reservoir level receded by 0.75 m in a 12-h period, effectively exposing shoreline and making sampling difficult. Electroshocking was conducted over parts of the rocky shoreline in areas throughout the reservoir, primarily in the upper half of the reservoir above the Route 341 bridge and in the lower section approximately 1.5 km above the dam, adjacent to the Schaghticoke Indian Reservation. Gill nets were set in the same areas covered by electroshocking, along the rock rip-rap and rock cliffs found along the shoreline in a water depth of 2-5 m.

Lake Lillinonah

Fish specimens were collected by boat electroshocking on 2-3 August and 7 October 1994. In addition to shocking, several gill nets were deployed during the August trip. Most specimens were collected by electroshocking in inlets or coves and along the shorelines from about 0.5 km below State Route 133 bridge to approximately 6 km above State Route 133. The habitats sampled included rock ledges, weed beds, coves, boat docks and brush piles.

Lake Zoar

The upper and lower end of the reservoir were sampled extensively by boat electroshocking on 1 -5 August and again on 8-9 October. Gill nets were set on 5 August in the upper reservoir area above the town of Lakeside and upstream of the Interstate 84 bridge. Boat shocking was conducted along both banks at both upper and lower ends of the reservoir and at a few places in the middle section of the reservoir. Sampling in the upper end was from the state boat ramp at Lakeside upstream to the spillway of the Shepaug Dam. Sampling for fish in the lower section of the reservoir was from about 0.6 km above Stevenson Dam to the vicinity of the cove at Kettletown State Park, about 5 km above Stevenson Dam.


Lake Housatonic

Lake Housatonic was sampled by boat electroshocking during 10-11 October 1994. Access to the reservoir was obtained through the boat ramp at Indian Well State Park. Fish samples were collected from a point 1.5 km below Stevenson Dam to just below Pink House Cove, approximately 3.0 km upstream of the State Route 8 bridge in the town of Shelton. The areas sampled included coves, shallow weed beds, rocky shoreline, snag piles and boat docks.


METHODS

Methods employed in the 1994 biological monitoring study fall into six categories: (1) collection offish and their subsequent handling up to and including storage at ANSP's Philadelphia laboratory, (2) collection of benthic insects and their subsequent handling up to and including storage at ANSP's Philadelphia laboratory, (3) preparation of fillet samples in the laboratory, (4) analysis of PCBs, (5) determining the age offish, and (6) statistical analysis of the data. We discuss each category below.

Fish Collection and Handling

Fish were collected by staff of The Academy of Natural Sciences of Philadelphia (ANSP). sometimes with the assistance of the Department of Fisheries of the State of Connecticut (at the Cornwall station). A total of 168 fish was selected to be analyzed for PCBs. The species were white perch, yellow perch, smallmouth bass, brown trout, pumpkinseed. bluegill. redbreast sunfish and American eel (Table 2).

Table 2. Number of samples of each target species analyzed for PCBs in the Housatonic River during June to October, 1994.

Station Species

Cornwall Bulls Bridge Lillinonah Zoar Housatonic Hatcherv Total

Broun trout 36 0 0 0 0 o 38

American eel 0 0 0 0 18 0 18

Smallmouth bass 13 8 9 10 18 0 58

Bluegill 0 0 0 0 6 0 6

Pumpkinseed 0 0 0 0 6 0 6

Redbreast sunfish 0 0 0 0 6 0 6

Yellow perch 0 0 0 0 18 0 18

White perch 0 0 0 0 18* 0 18

TOTAL 49 8 9 10 90 2 168

"Includes one composite of two specimens.

Boat electroshocking was the main collecting technique employed and was used at all sites except Cornwall. A Coffelt model VVP-15 boat shocker powered by a Honda EG5000X generator was used over the following ranges, depending on the site and conditions: 110-250 volts, 20-60% pulse width, 100 pulses/sec, and 5-7 amps. Boat shocking was typically conducted at night, though some daytime and early evening shocking was performed. Daytime shocking was not productive for smallmouth bass in the upper part of Lake Zoar. The majority of the smallmouth bass was collected by boat electroshocking at night. Few smallmouth bass were collected in gill nets.


A Smith-Root model YE D.C. backpack electrofishing unit (used at 0.3-0.4 amps, 6.5 ms pulse width, 60 Hz frequency and 200-400 volts) was used at the Cornwall station to collect fish in areas accessible to wading during trips 1 and 3.

During trips 2 and 3, walkalong electroshocking was performed at the Cornwall station in cooperation with the Connecticut Department of Fisheries, using a Robin generator and Coffelt VVP unit fitted in a canoe. The target species were brown trout (stocked fish in a catch-andrelease section) and smallmouth bass. The electroshocking equipment is owned by the State of Connecticut and typically is used during their annual August surveys along the trout fishing reaches of the Housatonic River.

Arrangements were made with Burlington Trout Hatchery personnel to deliver to ANSP staff two hatchery-raised brown trout during the second sampling trip. These fish received the same field processing procedures as stocked fish caught in the river (see below). The hatchery trout were analyzed to estimate the pre-stocking level of PCB contamination.

During boat electroshocking, two people collected the stunned fish with long-handled dip nets. Specimens were held in a pre-cleaned (Alconox washed) metal tub of river water. After a shocking run was complete (approximately 45-60 min duration), the samples were measured to evaluate legal and study size limits. They were then placed in a clean stainless steel pan that was set on wet ice inside a cooler. Typically, samples were processed within 1 to 6 hours of the time of capture. Fish not needed for chemical analysis were measured and released or discarded.

At the field processing site, fish needed for chemical analysis were measured for total length in cm. A Floy tag bearing a unique serial number was attached to each fish sample by insertion into the head region with a stainless steel needle. The serial number of the tag was recorded in the field notes. Specimens were then wrapped in clean aluminum foil. The outside of the foil packs was labeled with an index card bearing information on date of capture, species, locality of capture and serial number. The foil pack and index card were secured with freezer tape and placed in clean coolers (Alconox washed) with dry ice and frozen. Chain-of-Custody forms were prepared in the field and accompanied the samples to ANSP laboratories in Philadelphia. Chain-of-Custody forms also were used to verify transfer of specimens from state collecting crews to ANSP field personnel.

Upon arrival at the ANSP laboratories in Philadelphia, fish data were logged into a computer database and the specimens were placed into the ANSP fisheries section freezer until laboratory processing. All samples were accounted for in this way. Chain-of-Custody forms were used to track the samples from the ANSP field personnel to fisheries laboratory personnel, and then to ANSP chemistry laboratory personnel for processing or storage.

Handling offish followed The Academy's Standard Operating Procedure P-14-04 (Fish Preservation, Fixation, and Curation, Rev. 2). Quality control procedures were followed during field sampling and laboratory work, with all efforts being made to minimize contamination of the specimens. Sample material was prepared using clean equipment and on clean surfaces, avoiding


contact between sample material from different specimens or contact with uncleaned laboratory surfaces.

Benthic Insect Collection and Handling

Dobsonfly larvae (Corydalus cornutus} and perlid stoneflies were collected by rapidly lifting rocks and capturing the animals with a dip net as they were swept downstream. Perlid stoneflies and hydropsychid caddisflies (Macrostemum zebratum) were picked from the surfaces of individual rocks with forceps.

Aquatic insects were placed in I-Chem Superfund Analyzed glass jars bearing a duct tape label on the outside. The label bore a unique code number with the project name, site identification, date and name of the taxon. Samples were immediately placed in a cooler with dry ice and were maintained frozen during transport to the ANSP laboratory in Philadelphia. Upon arrival, samples were transferred to a freezer and stored frozen until being ground and analyzed for PCBs.

Preparation of Fillet Samples

Fish to be analyzed for PCBs were thawed in the laboratory and measured for total length (± 0.1 cm) and weight (± 0.1 g). The field identification of each specimen was verified. For brown trout from Cornwall, fin clips, if present, were observed carefully and recorded. During sample preparation, any external or internal anomalies such as fin wear, trematodes. nematodes, etc.. were noted and recorded in the database. Laboratory methods followed The Academy's Standard Operating Procedure P-14-12 (Preparation of Tissue Samples for Contaminant Analysis. Rev. 1 Draft).

Comparisons of field and laboratory lengths indicate that laboratory lengths usually are slightly less than field lengths, indicating minor shrinkage of some fish, presumabh due to freezing.

The sex of specimens was assessed by macroscopic visual examination. As is normal, sex could not be determined with certainty in some specimens, either because they were sexually immature or because sampling occurred during a season of low gonadal development. Eels were classed as sex unknown, since they do not reach sexual maturity until just prior to or during their downstream migration to the ocean. However, virtually all eels in freshwater are expected to be (immature) females, as males tend to stay near the coast (Smith 1985). Many of the trout also could not be sexed visually, since these were immature, newly stocked fish. No male sunfish were positively identified in Lake Housatonic, but specimens of unknown sex were probably males. Lake Housatonic was sampled in October, which is a period of low gonadal development.


Each fish was given a four digit analysis number used for tracking through chemical analysis. Specimen data were entered directly into a computer database, with hard-copy backup each day.

A clean glass filleting plate and stainless steel fillet knife or scalpel blade were used for each specimen. Sterile scalpel blades were placed onto a scalpel handle and rinsed with dichloromethane prior to use in filleting and mincing. If necessary, debris and mucus were removed from the fish by rinsing in distilled water. The left fillet was taken unless both fillets were necessary to obtain sufficient sample material. Following standard practice based on typical human food-preparation customs, eels were processed with the skin and scales removed, whereas the skin and scales were left on the trout fillets. The fillets of the other species (yellow perch. white perch, smallmouth bass and sunfish species) had the skin on but the scales removed. Fillets were taken to include the flesh covering the abdominal cavity. The entire fillet was minced and placed into clean glass jars with a clean piece of aluminum foil between the jar and the lid. Fillet weight and specimen sex were recorded. Otoliths of most target specimens (except brown trout of known age) were dissected and preserved separately in small vials with 95% ethanol. The fillets were delivered to the ANSP Chemistry Section along with a Chain-of-Custody form. The remains were wrapped in aluminum foil, labeled, and refrozen, permitting examination or analysis of additional material, if necessary.

One sample of white perch was a composite of two small specimens. All other samples were of single fish.

Cleaning of the glass plates and fillet knives at the end of each laboratory session included the following steps:

1. wash with Alconox detergent and thoroughly rinse in tap water;

2. either: a) rinse in 50% nitric acid, rinse with distilled and double distilled water, and solvent-rinse with acetone and dichloromethane, or b) muffle overnight in a muffle furnace; and

3. cover with muffled aluminum foil to avoid contamination prior to use.

Analysis of PCBs

The laboratory method used for treatment of fish is based on Academy Standard Operating Procedure P-16-77, "Soxhlet Extraction and Cleanup of Tissue for PCB Congener Analysis" (Appendix A).

Fish tissues and insect samples were ground using a Tissuemizer. Each entire ground sample was desiccated by addition of six parts sodium sulfate to tissue, by mass.


An aliquot of about 70 g of this material was placed into a glass extraction thimble and extracted over approximately 20 h using 1:1 acetone:hexane in a Soxhlet extraction tube. Surrogate standards (PCBs 14, 65, 166) were added to the sample before extraction began. Following extraction, the sample volume was reduced to about 1 ml using rotary evaporation, with final volume adjusted to 10 ml. An aliquot of 1 ml was removed from this volume for gravimetric analysis of lipids. The remaining extract was concentrated to about 2 ml and washed with an equal volume of sulfuric acid. This volume was refrigerated at 3 °C overnight to separate the hexane and acid phases. The hexane phase was then transferred to another vial. The acid phase was washed with additional volumes of hexane, which were then combined with the other hexane washes. The hexane phase volume was reduced to about 2 ml.

The extract was cleaned by passing it through fully activated Florisil in a Sep-Pak cartridge, followed by four volumes of hexane. Internal standards (PCB 30 and 204) were then added to each sample.

Each sample was analyzed using capillary gas chromatography, employing electron capture detectors and DB-5 columns. The oven temperature at injection was 50 °C. ramped to 130°C at 5.0°C/min, to 203°C at 0.3°C/min. and finally to 280°C at 10.0°C/min. The compounds of interest were found in the range between 130°C and 203 °C.

PCB identification was congener-specific, based on Academy Standard Operating Procedure P-16-84, "Congener-specific Quantitation of Polychlorinated Biphenyls (PCBs) by Capillary Column Gas Chromatography" (Appendix B). The method uses a reference standard of Aroclors 1232, 1248 and 1262 mixed in a ratio of 250:180:180. The identification and concentrations of individual congeners in this mixture were generated by USEPA's Large Lakes Research Station at Grosse He, Michigan, and were enumerated by Mullin (1985). Up to 121 PCB congeners are found in this mixture, containing nearly all environmentally significant PCB. The three surrogate standards and two internal standards are included in this mixture in order to make a calibration standard.

Quantitation was based on the internal standard method. Total PCB concentrations were calculated by two different procedures: by summing all 121 individual congeners analyzed, and by summing estimates of Aroclor 1254 and 1260 concentrations (see "Statistical Analysis").

Aging of Fish

Ages offish usually were estimated using otoliths, known stocking dates (brown trout), and occasionally fin clips (holdover brown trout). Examination of otoliths from several of the 36 river-captured brown trout was needed to verify their ages.

The largest pair of otoliths (sagitta) was dissected from the fish in the laboratory during the filleting procedure and placed in small vials of 95% ethanol. One of these sagitta was embedded with fast-cure epoxy resin and dried. Thin sections were cut transversely through the

THE ACADEMY OF NATURAL SCIENCES 10

otolith with a Buehler Isomet low-speed saw. Three to five of these thin sections per fish were affixed to a microscope slide with immersion oil. Sections were examined under a dissecting microscope at 12-50x magnification, with either direct viewing or viewing through a video image on a monitor. Specimens that were more difficult to age (e.g., brown trout, eel) were examined under a compound microscope (50-200x magnification) when it was necessary to examine the edge or kernel (core area) of the otolith more carefully for annuli.

When viewing the sectioned otoliths, annuli (annual marks) are visible as pronounced dark bands, containing within them faint thin bands representing other cycles of growth. Age was estimated using the pronounced bands, with the innermost band assumed to represent the first winter-spring transition (between age 0+ and 1+).

Ages were determined independently by two fisheries biologists who read the otoliths and then compared results.Except in the case of eel, discrepancies were always less than or equal to one year; exact agreement occurred for 89% of the otoliths. A mutually agreed upon determination was accomplished for these discrepancies after re-examination of the otoliths and discussion. In several cases, duplicate otoliths of the same specimen were prepared, aiding in resolution of discrepancies between the two readers.

Age estimates were made for eel on the basis of banding patterns. Though the estimates were consistent with reported ages (e.g., Ogden 1970, Hurley 1972), they are considered to be unreliable for the following reasons. Eel may produce two growth marks each year (Decider 1976, 1981), and it is frequently impossible to distinguish between summer and winter marks by standard microscopic inspection. Growth patterns may also be complicated because of upstream movement of young eel and movements from small streams into larger streams and reservoirs. As a result, the precision of eel readings was relatively low (identical readings on 17% of specimens: 56% differing by one year or less), and the age estimates therefore are not reported and were not used in statistical analyses.

In the case of brown trout, it was also necessary to determine each fish's time of residence in the river (i.e., time since stocking), since this, rather than total age. represents the duration of exposure to Housatonic PCBs. The State of Connecticut fisheries biologists discontinued fin-clipping of stocked brown trout in the Cornwall area after the 1992 stocking season. The 1994 stocked brown trout were either Burlington strain yearling (hatchery age 1+), or Quinebaug strain putative adults (hatchery age 2+). Most of the brown trout analyzed from the Cornwall station were identifiable as yearling or adult by their size at capture and otolith analysis of a select group of trout. Three of the trout sampled possessed fin clips: two trout contained a right maxillary clip corresponding to yearling trout (1+) stocked on 28 May 1992. and a third contained a left ventral clip corresponding to an adult (2+) trout stocked on 14 May 1992. The ages of these holdover trout were verified by examination of the otoliths. Information regarding fin clips, stocking dates and age at stocking was provided by the State of Connecticut Inland Fisheries Division.


The total age of each specimen was assigned by adding riverage (a fish's time of residence in the river) to age at stocking. Age at stocking was assigned assuming a hatching date of 1 January'. For example, a yearling fish stocked in April was assigned an age at stocking of 1.4 years. Riverage was the primary variable used for analysis of PCB uptake rates.

Statistical Analysis

Measures of PCB Concentrations

Several measures of PCB concentrations were used in summarizing and analyzing PCB concentrations. The primary analytical measure was total PCB concentration on a wet weight basis. This is the measure that is relevant to regulatory thresholds.

The total PCB concentration was estimated by two methods of quantitation. The first is based on measuring the concentrations of selected congeners that are essentially unique to Aroclor 1254 or 1260, extrapolating to Aroclor concentrations from the relative proportions of these congeners in each Aroclor, and then summing the two Aroclor concentrations. The resulting estimate of total PCB concentration is denoted TPCB. The second method is based on the sum of all identifiable congeners (up to 121). The resulting estimate is denoted CTPCB and is expected to provide a more accurate measure of the total PCB concentration than is TPCB.

The TPCB method was the only one used in the 1984—1990 monitoring studies, while both TPCB and CTPCB methods were used in 1992 and 1994. These two estimates of total PCB were compared using the 1992 and 1994 data. Although different, the two estimates were highly correlated in both years, allowing prediction of CTPCB from TPCB estimates for 1984-1990.

Analysis of PCB concentrations was based on the logarithmic transform of TPCB or CTPCB:

LNTPCB = In(TPCB)

LNCTPCB = In(CTPCB),

where In is the natural logarithm, and TPCB and CTPCB are in mg/kg wet weight (parts per million). The purpose of logarithmic transformation is to produce a variable whose variance is independent of the mean (homogeneous variance) and whose variation about the mean is approximately normally distributed (Gaussian residuals). These properties are desirable for standard statistical comparisons and to produce unbiased estimates of means.

Reports of the 1984 and 1986 results (ANSP 1985, 1987) used a slightly different logarithmic transformation [LPCB = ln(TPCB+l)]. LNTPCB has the advantage of being naturally related to the geometric mean, which is simply exp(LNTPCB). Unlike LPCB. however, LNTPCB attains negative values at PCB concentrations below 1.0 mg/kg.


Since PCBs partition preferentially into lipid, a fish's uptake rate and steady-state burden of PCBs are likely to be influenced by its lipid content. It is therefore accepted practice to normalize PCB concentrations to lipid content as a way of controlling for the effect of differences in lipid levels when comparing PCB concentrations among stations, species, or years. Lipid-normalization is accomplished by dividing TPCB or CTPCB by LIPID, the proportion of lipid in a tissue sample:

PCBLIP = TPCB/LIPID

CPCBLIP = CTPCB/LIPID.

In these formulas, CTPCB, TPCB and LIPID are measured variables, and the statistical distributions of PCBLIP and CPCBLIP typically are highly skewed. That is, a few specimens result in extremely high ratios of PCB to lipid, creating a long upper tail in the distribution. The arithmetic mean is unduly influenced by these extreme values, but the geometric mean is not. Therefore, the geometric means of PCBLIP and CPCBLIP provide a better measure of central tendency in lipid-normalized concentrations.

The skewed distribution of PCBLIP also makes it unsuitable for standard tests of statistical significance in analysis of variance or covariance. The logarithmic transform is more appropriate:

LPCBLIP = In(PCBLIP).

But,

LPCBLIP = ln(TPCB/LIPID) = LNTPCB - In(LIPID).

Because of this equivalence, PCB-lipid relationships were analyzed for most comparisons by analyzing statistical relationships between LNTPCB and In(LIPID). For example, In(LIPID) (hereafter referred to as LNLIP) was used as a covariate in analyses of covariance (see next section).

Statistical Comparisons of Year and Station Differences

One of the major goals of the monitoring study was to assess potential differences in PCB concentrations among years and stations. The basic statistical approach was to perform analysis of variance (ANOVA) and analysis of covariance (ANCOVA), as implemented by the General Linear Model (GLM) procedure in SAS (SAS 1985, 1988a, 1988b, 1993). Year and station were handled as discrete treatments, and the statistical significance of variation among years, of variation among stations, and of year-station interactions was assessed.

PCB concentrations in fish can be influenced by a variety of factors other than the level of exposure. Important ancillary variables include a fish's sex, age (or size), residence time in


the river (for stocked fish), and lipid content. Since samples collected in different years or at different stations usually differ in composition with respect to these ancillary variables, these differences could produce statistically significant year or station effects that are not caused by differences in PCB exposure, or at the opposite extreme, could mask the effects of real differences in exposure. It is therefore desirable to identify and remove the effects of these ancillary variables, when statistically significant.

Ancillary variables were incorporated into the ANOVA and ANCOVA models either as a discrete effect (sex) or as covariates (age, residence time, lipid content), and as interactions between the various variables. The resultant models estimate a single parameter for the grand mean of all LNTPCB variables, a single parameter for each level of the discrete variables (station, year, sex), a slope for the linear relationship between LNTPCB and each covariate. and a parameter for each combination of levels of interactions between discrete variables. Where interactions between discrete variables and covariates are included in the model, a separate slope is estimated for each level of the discrete variable. Significance of effects was assessed by the F-value of the type III sum of squares associated with that effect (SAS 1985); this assesses the contribution of each effect after all other effects in the model have been incorporated.

Tests were made for each station separately. For smallmouth bass, tests were also performed with stations combined, including the four Cooperative Agreement stations for 1984—1994 (Cornwall, Bulls Bridge, Lillinonah and Zoar) and the Cooperative Agreement stations plus Lake Housatonic for 1994 alone. Tests were also made for some subsets of the data, mainly to provide more balanced designs. For example, some models were run excluding specimens whose sex could not be determined.

A set of models was run for each species or species-station test, including different groups of main effects (station, year, sex, age, lipid) and interactions. Typically, models were first run using a number of main effects and interactions, and subsequent models dropped effects or interactions which were not found to be statistically significant. Finally, a model was run using only significant effects and interactions. This model was the basis for all reported levels of significance, estimated treatment effects, and so on.

The removal of non-significant terms from a statistical model pools variance associated with the removed effects with residual error. Since this procedure increases both the sums of squares and degrees of freedom of the residual error, it can either increase or decrease the mean squares error. While alpha levels higher than 0.05 have been suggested as a means of removing non-significant terms (cf. Sokal and Rohlf 1969), this pooling did not greatly affect significance of other effects in the analyses performed. In general, once significant main effects were included in models, the significance of interactions did not depend on which other interaction terms were included (e.g., significance of a station-year interaction did not depend on inclusion of station-sex, year-sex or lipid-station interactions, although they did depend on the inclusion of year and station main effects).


ANOVA and ANCOVA were used to estimate model parameters (including slopes of the age or lipid relationships). The LSMEANS option (SAS 1985) was used to estimate the least squares means associated with each treatment level. These are the mean values of LNTPCB for each treatment level (e.g., each year, station or sex) at the mean value of the covariate(s) (age and lipid) over all observations. The least squares means adjust for the covariate effects and provide estimates of LNTPCB independent of the age composition or lipid contents within each set of samples.

Where there were more than two levels for a treatment variable, multiple range tests were used to determine which pairs of levels were significantly different (e.g., pairs of stations or years). When ANOVA or ANCOVA indicated a significant station or year effect, the REGWQ tests (SAS 1985) were used to identify significant differences between pairs of means (the least significant difference in the REGWQ test). Multiple range tests are normally done on treatment means. This would not adjust for differences in distribution of covariate values among treatment levels (e.g., differences in age composition of samples between stations or years), which were frequent in these data. To avoid this problem, the least significant difference in the REGWQ test was used on the least squares means (which adjust for the covariate values) rather than on the treatment means.

Sex was indeterminate in a small number of smallmouth bass specimens, complicating the statistical analysis. Excluding these individuals reduces statistical power, but inclusion of a third sexual group with so few members yields a highly unbalanced statistical design. This dilemma was resolved as follows. Statistical models initially were run with indeterminate sex included as a third sexual group. If the sex effect was found to be statistically significant, individuals of indeterminate sex were deleted and the models were rerun with only male and female sexual groups. If the sex effect was not significant, individuals of indeterminate sex were retained but the models were rerun without sex groups.

Statistical analyses were conducted using data from both methods of PCB quantitation: Aroclor-based (TPCB or LNTPCB) and congener-based (CTPCB or LNCTPCB). The Aroclorbased method was used in all previous studies, while the congener-based method was used only in 1992 and 1994; congener-based estimates for the remaining years were calculated from regressions derived from the 1992-1994 data. Results based on the two types of estimates were generally very similar. For this reason, and also because direct Aroclor-based estimates are available for all years, tests of statistical significance are reported only for the Aroclor-based method. However, because the congener-based concentrations are believed to be more accurate at least for 1992 and 1994, means are reported for this method, as well.


RESULTS

Synopsis

The 1994 data indicate a decrease in PCB concentrations relative to all previous years. This decrease is seen for brown trout as well as smallmouth bass, and at all four stations that have been monitored since 1984. In addition, the proportion offish with PCB concentrations exceeding the 2.0 mg/kg consumption advisory limit was as low as, or lower than, in previous study years, with concentrations exceeding the limit found only at Cornwall (3 smallmouth bass and 3 holdover brown trout by both methods of estimation, and one additional smallmouth bass for the Aroclor-quantitation) and Lake Housatonic (1 12-year-old smallmouth bass and 2 eel).

When data for all study years are combined, there is a consistent, statistically significant pattern of decreased PCB concentrations across stations in the downstream direction. For 1994 alone, fewer statistically significant station differences were found, but concentrations at the two most downstream stations (Lakes Zoar and Housatonic) are significantly lower than those at the two most upstream stations (Cornwall and Bulls Bridge).

We now present these and other results in detail, beginning with the relationship between Aroclor- and congener-based PCB estimates (which was used to calculate congener-based estimates from previous Aroclor-based estimates for 1984-1990) and proceeding to spatial and temporal trends. A complete listing of data from the 1994 study in tabular and graphic forms can be found in Appendices C-J.

Relationship Between Congener and Aroclor Ouantitation Methods

The 1994 CTPCB estimates were consistently lower than the corresponding TPCB estimates (Fig. 2), but the two were highly correlated (Figs. 3, 4). These data apply only to brown trout and smallmouth bass (the only species monitored at the Cooperative Agreement stations in 1994), but the relationship for these species was similar to that for the other species analyzed in 1992 (Fig. 5).

The relationship differed somewhat between 1992 and 1994 (Fig. 3): the two estimates were more similar in 1994 (Fig. 2), and there was a slight difference in the slope of the TPCBCTPCB relationship between the two years. While other group effects were also weakly significant (e.g., station, In(age)), these added essentially no predictive power to the overall regression (e.g., r2 increased from 0.9961 to 0.9980 by the addition of year and further increased to 0.9984 by the addition of station, species, In(lipid) and In(river-age)). Examination of deviations between predicted and observed In(TPCB) values in plots of In(TPCB) versus In(CTPCB) shows very good agreement for almost all data (Figs. 3, 4, 5). There are a few points with moderate deviation, and some of the observed group and covariate effects may reflect the influence of these points.


9 v

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txl / î 10 w [92 84 ! 92 94 ! 92 94 1 82 84 ! 92 94 | 84

Com Com BB Lill. Zoar Hous

TROUT SMALLMOUTH BASS

: i TPCB !777/\ CTPCB

Figure 2. Comparison of geometric mean TPCB and CTPCB estimates Values are shown for 1992 and 1994, and for Cornwall (Corn.), Bulls Bridge (B.B.), Lake Lillinonah (Lill), L Zoar (Zoar) and L Housatonic (Hous.).

-3 -2 -1 0 1 2 LN(CTPCB)

Figure 3. Relationship between TPCB and CTPCB for 1992 and 1994 samples of smallmouth bass and brown trout.


-3 - 2 - 1 0 1 2 LN(CTPCB)

O Cornwall a Bujls Bridge A Lillinonah v Zoar o Housatonic

Figure 4. Relationship between TPCB and CTPCB estimates for 1992 and 1994 samples of smallmouth bass and brown trout, by station.

T~

- 2 - 1 0 1

LN(CTPCB) mg/kfl wet wt

Figures. Relationship between LNTPCB and In(CTPCB) for 1992 samples of species other than smallmouth bass and brown trout.


The high correlation between the two estimates of total PCB allows prediction of one from the other. Congener-based concentrations were predicted for the historical data (19841990) using the combined regression for the 1992-1994 trout and bass data. Predictions were based on a regression of In(CTPCB) versus In(TPCB). No year difference was included in the model, since the goal was to estimate concentrations in past years. Even without a year difference, the two measures were very highly correlated (r = 0.996). Nevertheless, plotting the regression residuals against In(CTPCB) values reveals that residuals from the two years tend to cluster separately, indicating that the data are not entirely homogeneous (Fig. 6).

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CTPCB = 0.882TPCB09376.

The exponent of TPCB is close to unity, indicating that CTPCB estimates are almost directly proportional to TPCB estimates. This property was also observed when separate regression equations were developed for 1992 and 1994. Similar results were obtained by Draper et al. (1991). Average estimated CTPCB concentrations for 1984-1994 are displayed in Table 3.

Differences in PCB Concentrations Among Stations

It is well known that different species offish at a single site typically exhibit different PCB concentrations. It is therefore difficult to interpret patterns of PCB concentrations among different sites when the sites are characterized by different species, since site differences and species differences are confounded. For this reason, the assessment of potential upstream-downstream patterns in the Housatonic River is based on data for smallmouth bass, the only species analyzed at all stations.

When data for all years are analyzed together, among-station variation is highly statistically significant (Table 4), and pairwise comparisons show each station to be significantly different from all others (Table 5). The overall station difference is also highly significant when 1994 data are analyzed alone, but pairwise comparisons reveal fewer statistically significant differences. Concentrations at Zoar and Housatonic were, however, significantly lower than concentrations at Cornwall, Bulls Bridge and Lillinonah, after adjustment for differences in age and lipid (Table 5). This is consistent with the trend of decreasing PCB concentrations in the downstream direction, which was observed in previous monitoring studies.

Visual inspection of the Aroclor- and congener-based means corroborates this downstream trend of decrease (Tables 3, 6 and 7, Fig. 7). For example, the least squares mean TPCB level in smallmouth bass in 1994 decreased 53% between Cornwall and Lake Lillinonah, and decreased 52% between Lake Lillinonah and Lake Zoar (Table 6).


Table 3 Average CTPCB concentrations in fish from the Housatonic River, Connecticut Results for 1992 and 1994 are based on actual quantified CTPCB values Results for 1984-1990 were estimated from TPCB data, using regressions between In(CTPCB) and In(TPCB) that were established with data from 1992 and 1994

Species

Brown Trout

Rainbow Trout

Smallmouth Bass

Bluegill

Brown Bullhead

Carp

Largemouth Bass

Pumpkinseed

Redbreast Sunfish

Yellow Perch

Smallmouth Bass

Bluegill

Brown Bullhead

Carp

Largemouth Bass

Pumpkinseed X Redbreast

Pumpkinseed

Redbreast Sunfish

White Catfish

White Perch

Yellow Perch

Smallmouth Bass

Bluegill

Brown bullhead

Carp

Eel

Largemouth bass

Pumpkinseed

Redbreast sunfish

White catfish

White perch

Yellow perch

Smallmouth bass

Smallmouth bass

Station

C

C

C

B

B

B

B

B

B

B

B

L

L

L

L

L

L

L

L

L

L

L

Z

Z

Z

Z

Z

Z

Z

Z

Z

Z

Z

H

1984

2.75

-

1.99

078

072

095

1 16

-

1 31

1 14

161

0.48

1.99

1.85

1.13

-

-

1.26

4.76

1.89

0.58

1.02

089

038

388

-

039

-

009

222

084

007

045

-

1986

5.27

-

2.61

-

1 54

-

-

-

-

072

134

-

-

•

-

-

-

-

6.27

1.86

-

1.33

.

-

-

-

-

-

-

255

-

-

-

-

Year

1988

4.06

2.63

3.77

185

168

517

209

027

166

087

233

0.47

1.42

5.61

1.15

0.27

0.03

0.03

4.33

1.53

0.22

1.20

019

062

1207

104

1 15

0 11

015

340

126

021

084

-

1990

4.41

-

.

-

-

-

-

-

-

084

210

0.47

-

-

-

-

0.20

0.37

-

-

0.35

0.95

013

-

-

236

-

0 16

020

-

087

024

059

-

1992

7.25

-

2.78

-

-

-

-

-

-

056

1 35

0.45

-

-

-

-

0.18

0.47

-

-

0.32

141

025

-

-

530

-

022

024

-

1 01

026

1 13

-

1994

1.31

-

141

-

-

-

-

-

-

-

1 23

-

-

-

-

-

-

-

-

-

-

051

-

-

-

-

-

-

-

-

-

-

043

0.51


Table 4. Summary of statistical analyses of differences in PCB concentrations among groups of fish from the 1984-1994 studies on the Housatonic River. Results are based on ANCOVA analyses LNTPCB is the dependent variable, r2 is the total vanance explained for models containing only significant terms. The remaining columns show the significance level for discrete effects (station, year, sex), covariates [In(age), In(nver-age), In(lipid)], and slope differences for age or lipid relationships (between station, year or sex), ns indicates an effect that is not statistically significant. See text for explanation.

Significance of Effects in ANOVA/ANCOVA Model 1Years Station r

Year Station Sex Lnage or Lnnvage

Lnlipid Slope Difference

Brown Trout

AM C 077 00001 not not me 00001 00001 Lnage*\ear 0 0001 app

All (Rivage

Table 5. Least-squares means of In-transformed total PCB (LNTPCB), from ANCOVA and ANOVA models with significant year or station differences (see Table 4 and text) Where there are significant or nearly significant covanate effects (age or lipid), least-squares means are the means for the various years or stations after adjustment for covanate effects. Years or stations joined by dashes are not significantly different.

Years Station Least Squares Means for Year or Station Terms in Model

Brown Trout

All Cornwall 92 2 12

90 1 89

88 1 58

Distinct years 92-90 88 86 84 94

86 1 30

84 088

94 047

Lnhpid Lnmage V ear Lnmage*\ ear

All (Rivage< 1)

Cornwall 92 1 97

90 159

88 1 40

Distinct years 92 90-88 88-86 84 94

86 1 13

84 036

94 0063

Lnl ip id Lnnvage \ear

All (Rivage > 1 )

Cornwall 92 238

90 234

84 191

94 1 80

86 1 79

88 1 77

Lnhpid Vear

Distinct years 92-90-84-94-86-88

Smallmouth Bass

All Bulls Bridge

90 1 12

88 085

84 066

Distinct years 90-88 88-84-92

92 043

86 021

84-92-86 92-86-94

94 0062

Lnhpid \ ear Sex

All Cornwall 92 1 25

88 1 208

90 1 207

86 091

Distinct years 92-88-90-86 86-84 94

84 073

94 0074

\ear Sex Lnhpid'Sex

All Lillmonah 92 -0055

86 -0325

84 -0327

88 -0384

90 -046

94 -096

Vear 1 nage'Sex

Distinct years 92-86-84-88-90 88-90-94

All Zoar 92 -0 11

90 -026

88 -050

84 -084

94 -1 23

\ ear Lnhpid*Sex

Distinct) ears 92-90-88-84 88-84-94

\l\ CBLZ 90 034

92 0315

88 0303

Distinct years 90-92-88 86-84 94

C 073

B 040

L -029

86 0082

Z -076

84 0042

94 -05"

1 nhpid L nage Station > ear Sex 1 nage*Station

Distinct stations C B L Z

1994 CBLZH C 021

B 0 18

Distinct stations

L -0026

C-B-L H-Z

H -097

Z -1 136

Lnhpid Lnage Station

THE ACADEMY OF 1SATURAL SCIENCES 23

Table 6. Summary of total PCB concentrations (mg/kg wet weight) of fillets of fish collected in ANSP surveys of the Housatonic River. Least squares means are means adjusted for age and/or lipid effects.

Smallmouth Bass Brown Trout Year

C C B L Z H

Least squares Mean TPCB1

_

1994 1.60 1.27 0.92 0.60 0.29

1992 8.33 3.08 2.23 1.45 0.70 _

1990 6.62 3.16 2.28 1.48 072 _

.1988 4.85 3.04 2.20 1.43 0.69 _

1986 3.67 2.44 1.76 1.14 _

.1984 2.41 2.34 1.70 1.10 0.53

Geometric Mean CTPCB

1994 1.11 1.27 1.19 0.41 0.34 042

1992 6.33 2.49 1.29 1.11 0.88 _

Geometric Mean TPCB

1994 1.22 1.40 1.33 0.44 0.35 043

1992 8.07 3.30 1.69 1.45 1.12 _

1990 5.30 3.14 2.32 1.02 0.59 _

1988 4.80 3.88 2.59 1.20 0.73 _

_

1986 5.51 2.64 1.41 1.13 _

_1984 2.30 200 1.80 1.07 0.39

Percent < 2.0 mg/kg TPCB (CTPCB)

1994 86 (92) 69 (77) 100(100) 100(100) 100(100) 94 (94)

1992 0(2) 14(21) 75 (88) 75 (88) 71 (71) _

1990 0 17 17 100 100 _

1988 0 8 21 88 88 _

_ .1986 4 31 58 77

1984 50 38 50 92 100

For smallmouth bass, calculated from model of all years, stations C, B, L and 2, and at overall sex ratio (48 1% female, 50 82% male, 0.99% not determined)


SMALLMOUTH BASS

8 Q.

-1

-2 B L Z

STATION

700

600

500

400

300 o

200

100

0

B L Z

STATION

Figure 7 Mean LNCTPCB and CTPCBLIP in smallmouth bass samples from five stations on the Housatonic River. For 1984-1990, CTPCB was estimated from regressions of CTPCB versus TPCB using 1992 and 1994 data Symbols: C = Cornwall, B = Bulls Bridge, L = Lake Lillmonah, Z = Lake Zoar, H = Lake Housatonic.

Differences in PCS Concentrations Among Years

Temporal trends were assessed by analyzing among-year differences in LNTPCB after adjusting for age, lipid and sex effects (Tables 4, 5). Brown trout and smallmouth bass were treated separately, and smallmouth bass from different stations were treated separately as well as cumulatively. We now consider these species in turn.


Brown Trout

ANCOVA revealed highly significant variation in LNTPCB among years (Table 4). Pairwise comparisons indicated that concentrations were highest in 1992 and 1990 (these years were not statistically different) and decreased in the order 1988, 1986, 1984, and 1994 (Table 5). Thus, brown trout PCB concentrations at Cornwall were significantly lower in 1994 than in any other year of the biological monitoring studies. This is true regardless of whether the concentrations are normalized to lipid content.

ANCOVA also revealed highly significant relationships between LNTPCB and In(lipid) and In(riverage) (Table 4). (Riverage is a fish's residence time in the river, which is more meaningful than actual age for stocked fish such as trout.) Analyses also indicate statistically significant differences in slopes of the LNTPCB-riverage relationship between years (Table 4): the same was noted in analyses of the 1984-1992 data (ANSP 1993).

Visual examination of the Aroclor- and congener-based means indicates that the unadjusted mean PCB concentration (expressed as LNCTPCB) in brown trout at Cornwall was low in 1984, higher and approximately the same from 1986 to 1990, highest in 1992, and lowest in 1994 (Table 6, Fig. 8 top). When normalized to lipid (Fig. 8 bottom), the mean PCB level shows an apparent increasing trend from 1984 through 1990, with sharp decreases in 1992 and 1994. The 1994 mean is the lowest value observed in any year. This is the same pattern indicated by the above ANCOVA results for the Aroclor-based estimates.

The among-year differences primarily reflect differences in PCB concentrations of newly stocked fish (i.e., riverage less than one year), which show essentially the same pattern (Table 5). Adjusted mean concentrations for holdover trout (i.e., riverage greater than one year) are relatively similar over the study period, and no significant pairwise differences were seen. Riverage of newly stocked trout varies among study years because of different stocking and sampling dates, but the sharp decrease in PCB concentrations in 1994 cannot be attributed to unusually low riverages.

Two hatchery brown trout were sampled just prior to stocking to determine baseline PCB levels before uptake in the river. The concentrations in both specimens were very 1cm (0.03-0.04 mg/kg). indicating that pre-stocking contamination is only a minor component of the PCB burden of brown trout collected from the river in the 1994 study.

Smallmouth Bass

ANCOVA revealed statistically significant variation among years in LNTPCB, whether stations were lumped or analyzed separately (Table 4). Pairwise comparisons were therefore used to rank years according to their mean LNTPCB (Table 5). These rankings differed somewhat, depending on the specific station or stations analyzed, but 1994 was always either the lowest year or a member of the lowest group of statistically indistinguishable years.


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CORNWALL

I E 1

8 Q_

BROWN TROUT SMALLMOUTH BASS

SPECIES CODE

BROWN TROUT SMALLMOLTDH BASS

SPECIES CODE

Figure 8. Average concentrations of In(CTPCB) and lipid-normalized CTPCB in brown trout and smallmouth bass from Cornwall in 1984-1994 studies. For 1984-1990, CTPCB was estimated from regressions of CTPCB and TPCB for 1992 and 1994 data.

Visual examination of the Aroclor- and congener-based means indicates that unadjusted mean total PCB was variable over years at each station (Table 6, Fig. 7 top). In even' case, however, the 1994 mean was the lowest of all study years. This was also true of the 1994 lipid-normalized mean (Fig. 7 bottom).

Lake Housatonic Analyses

Lake Housatonic was sampled only in 1994, so that no temporal comparisons can be made. Results for smallmouth bass from Lake Housatonic are reported above and compared with results from the other stations. American eel, white perch, yellow perch and three species of sunfish also were analyzed to provide information on PCB concentrations in these sport species (Table 7).


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PCB concentrations in American eel varied most closely with lipid content, which differed greatly among specimens.. The PCB-lipid relationship was highly significant (p < 0.0020 for CTPCB) by ANCOVA analysis, with the highest PCB concentrations occurring in high-lipid specimens.

For white and yellow perch, males had significantly higher PCB concentrations than females (for CTPCB, p < 0.0005 for white perch and 0.0076 for yellow perch), and there was an increase in PCB concentration with age (p < 0.0065 for white perch and p< 0.0066 for yellow perch). Only 4 of the 18 specimens of white perch and 3 of the 18 yellow perch were males. All male white perch were of similar ages (6-8 years old), while ages of females varied from 1 to 13. As a result. PCB-age relationships among sexes can't be compared. There was no significant relationship between PCB and lipid for either species.

For all sunfish analyzed together, specimens of unknown sex (probably males, see methods) had significantly higher PCB concentrations than females (p < 0.02 for CTPCB) and PCB increased with age (p < 0.02). There was no significant PCB-lipid relationship, and no clear difference in concentrations among the three species.

Fish Exceeding Consumption Advisory Limit

Total PCB concentrations in fillet samples were compared to the FDA consumption advisory limit of 2 mg/kg using congener-based estimates (CTPCB), which are considered to be more accurate than Aroclor-based estimates. None of the 33 1994-stocked brown trout had PCB concentrations exceeding 2 mg/kg, though all 3 of the holdovers exceeded this limit (Table 6, Appendices C. F. G; 2 mg/kg total PCB corresponds to 0.69 when In-transformed). In the case of smallmouth bass. 3 of the 13 fish from Cornwall and 1 of the 18 fish from Lake Housatonic exceeded 2 mg/kg by both quantitation methods, with the Lake Housatonic specimen being relatively old (12 years). An additional bass from Cornwall exceeded 2 mg/kg by the Aroclor quantitation (TPCB = 2.08), but not by the congener quantitation method (CTPCB = 1.82 mg/kg). The only other fish exceeding 2 rng/kg were 2 of the 18 eel from Lake Housatonic.

The percentage offish exceeding the FDA limit in 1994 was relatively lo\\ compared to previous years. This was particularly true of brown trout at Cornwall. In 1992. for example. 35 of the 36 1992-stocked brown trout had concentrations exceeding 2 mg/kg, whereas none of the 1994stocked brown trout exceeded this limit in 1994.

The percentage offish exceeding 2 mg/kg in samples from each station may exceed the actual proportion in the field population, since samples are small and nonrandom. Moreover, specimens for analysis are chosen to include a range of sizes, so that larger specimens tend to be over-represented relative to the population. This is likely to result in over-representation of high PCB concentrations if concentration increases significantly with age and size. This effect may have occurred in brown trout at Cornwall (where the only fish exceeding 2 mg/kg were holdovers), since the percentage of holdover trout among the specimens analyzed (8%) was much higher than the


percentage of holdover trout encountered in the field. It may also have occurred in Lake Housatonic smallmouth bass (where the only fish exceeding 2 mg/kg was 12 years old), since a significant PCB-age relationship was detected there.

Benthic Insect Analyses

Results of the 1994 Cornwall benthic insect analyses are displayed in Table 8 Field densities of two taxa (caddisflies and dobsonflies) were high enough to permit collection of replicate samples. As in 1992, the level of variation in total PCB among replicate samples demonstrates the desirability of collecting replicates when feasible. Congener-based estimates were consistent!} lower than Aroclor-based estimates, as with the fish samples.

The 1994 Aroclor-based estimates of total PCB are compared with those for previous years in Fig. 9. Concentrations in 1994 were among the lowest since 1978; only 1990 and 1985 showed similar or possibly lower levels.

Table 8. Total PCB concentrations in benthic insects at Cornwall in 1994 Caddisflies are filter feeders, while dobsonflies and stoneflies are predators.

Taxa

Hydro psych idae (Caddisflies)

Sample 1

Sample 2

Sample 3

Geometric mean

Arithmetic mean

Corydalidae (Dobsonflies)

Sample 1

Sample 2

Geometric mean

Arithmetic mean

Perlidae (Stoneflies)

(one sample)

Total PCB

Aroclor-based

142

242

2.07

1 92

1 97

224

3 83

293

303

1.09

(mg/kg wet)

Congener-based

1.34

2 4 1

1.81

1 80

1 85

1 86

3 34

249

260

1 01


25

D Filter feeders 20 • Predators

I15

CD O 10 Q. n n

Ml . • . • ! • • • ,1 1978 79 '80 '81 '82 '83 '84 '85 '86 '87 '88 '89 '90 '91 '92 '93 '94

YEAR

Figure 9. Historical pattern of PCB concentrations in benthic insects at Cornwall, 1978-1994.

Precision and Replicate Analyses

Analytical precision was measured by replicate analysis (i.e., separate extraction and analysis) of subsamples from each of 10 specimens. Only fish samples were employed for this purpose, since benthic insect samples contained insufficient material.

Standard deviations of replicate analyses tend to be proportional to the mean (Appendix I), so precision is best expressed as the coefficient of variation of TPCB, or the standard deviation of LNTPCB. The median coefficient of variation of TPCB for 1994 replicate analyses was 0.106. and the median standard deviation of LNTPCB was 0.107. The precision of a pair of replicate analyses may be calculated as the relative percent difference (RPD):

RPD= 100* - R2 /MR

= 100* v/2* CV,

where Rl and R2 are the two replicates, and MR is their mean. The median RPD for the 1994 samples was 1 5%, which is excellent.


DISCUSSION

The most striking result of the 1994 biological monitoring study is that total PCB levels in both species offish were the lowest observed in any year during the period 1984-1994. This was true for brown trout at Cornwall (the only sampling station for this species) and for smallmouth bass at all four Cooperative Agreement sampling stations, whether or not PCB concentrations were normalized to lipid content. PCB levels in benthic insects at Cornwall were also among the lowest observed since 1978, with only 1985 and 1990 showing potentially lower (but similar) levels.

Historically, PCB inputs to the Housatonic River likely decreased following elimination of PCB usage in upstream manufacturing in the late 1970s. Effects of this reduction are evident in the historical record for benthic insects at Cornwall, in which a pronounced decrease in PCB concentrations can be seen between 1978 and 1979 (Fig. 9). Subsequent levels remained well below those of 1978 but showed substantial variation rather than a consistent trend of continuing decline. Extensive biennial monitoring data for various species offish show a similar pattern of variation over the period, 1977-1992 (e.g., Table 3; see LMS 1987 for pre-1984 data). Most species showed a decrease in PCB concentrations between the late 1970s and early 1980s, with essentially trendless variation thereafter. In 1994, however, the data show a clear decrease in PCB concentrations in insects and both species offish that were monitored. And in the latter case (where tests of statistical significance are possible), concentrations for each species and station are the lowest, or a member of the lowest statistically distinct group, in any study year.

Prior to 1994, PCB concentrations in fish and insects of the Connecticut portion of the Housatonic River appear to have been following a two-phase temporal pattern consisting of an initial sharp drop after elimination of PCBs from upstream manufacturing processes, and subsequently essentially trendless variation about much lower levels. This same pattern has been observed in other large-scale systems contaminated by PCBs, such as Lake Ontario (e.g.. Suns et al. 1993). Whether the significant reductions observed in 1994 represent the onset of another declining phase or a transient fluctuation about the existing plateau cannot be determined at this time.

It is likely that the total mass of bioavailable PCB in the Housatonic River has continued to decline throughout the 1980s and 1990s. But with the exception of an initial sharp decline in the late 1970s, it has been difficult to detect a corresponding declining trend in tissue PCB concentrations. Broadly speaking, the reason for this seems to be that major sources of variability exist, affecting both the transport of PCB-laden sediment (and therefore exposure levels for biota) and the uptake and depuration of PCB by biota. Only a rather strong "signal" (declining trend in tissue PCB concentration) can be distinguished from this relatively high level of "noise" (natural variability).

The Academy's studies of biota in the Connecticut portion of the Housatonic River have revealed several important sources of biological variability. For example, analysis of 1984-1992 data (ANSP 1993) suggested a seasonal cycle of PCB concentrations in trout at Cornwall, linked to changes in lipid content. It was hypothesized that decreases in lipid content in late summer or early fall (possibly due to high summer temperatures) are followed by net losses of PCBs in late fall and winter, but that PCB concentrations subsequently increase in the same fish the following spring.


Newly stocked trout start out with very low PCB levels but, in typical years, rapidly bioaccumulate over a period of roughly two months to levels found in holdovers. Thus, newly stocked trout quickly wind onto the same cycle that holdovers exhibit. This hypothesis explains the facts that in 1992, lipid-adjusted concentrations in holdover and newly stocked trout were similar in August, and that PCB concentrations do not continue to increase with age in holdover trout that have been in the river 2, 3 and 4 years (cf. Appendix Fig. J-l).

A similar pattern is present among newly stocked trout collected in 1994. The average lipid concentration in fish collected in October (1.79%, river-age 0.4, Appendix E) is lower than in August (2.74%, river-age 0.2), and PCB concentrations in October likewise tend to be lower than the August concentrations. Lipid-adjusted PCB concentrations are very similar among the two months (Appendix E), indicating that the difference in PCBs between August and October can be explained by the change in lipid over the summer and fall. Unlike 1992, however, PCB concentrations in newly stocked fish were clearly lower than those of holdover fish for both unadjusted and lipid-adjusted PCB concentrations. This probably indicates that PCB exposure was unusually low in 1994, preventing newly stocked trout from bioaccumulating sufficiently to reach holdover levels.

In addition to the apparent seasonal pattern of uptake and loss, there have been differences among years in PCB concentrations in newly stocked trout in the first few months after stocking. These patterns are demonstrated by the ANCOVA analyses of newly stocked trout (Tables 4, 5). The}' are seen after adjustment for lipid concentrations and may reflect differences in PCB exposure. Notably, concentrations in newly stocked trout in 1994 and 1984 were relatively low, particularly when compared to concentrations in 1992.

These patterns have not been as evident in smallmouth bass. However, no increase in PCB concentration with age has been noted for this species at Cornwall or Bulls Bridge (Table 4). Since almost all bass analyzed are greater than 2 years old (Appendix D). this pattern is analogous to the absence of a PCB-age relationship among holdover trout. The absence of an age relationship could be due to cycles in lipid and PCB, as hypothesized for trout, or to equilibration of uptake and depuration rates. Because the forage fish base at Cornwall is limited compared to that at Bulls Bridge, there may be less of a switch to piscivory by large bass, mitigating the increase in bioaccumulation with size and age.

Temporal patterns of PCBs in the larger, downstream reservoirs (Lillinonah and Zoar) differ somewhat from those in the upper two stations. There was no increasing trend through 1992 (ANSP 1993). although there was a decrease in 1994. In the two lower stations, PCB has increased with age of fish. While this trend was weak at Lillinonah and Zoar in 1994, it was more evident at Lake Housatonic. This trend may reflect lower variation from factors such as storm-related transport that tend to mask an age relationship. The greater depth of the lakes and their larger forage base may also prevent summer temperature stress and lipid loss.

In summary, the 1994 results indicate that PCB concentrations in brown trout and smallmouth bass are the lowest that have been observed in any of The Academy's biological monitoring studies conducted on the Connecticut portion of the Housatonic River since 1984.


Moreover, concentrations in benthic insects at Cornwall are among the lowest that have been observed since 1978. The 1994 results for smallmouth bass also confirm the spatial pattern of decreasing PCB concentrations from upstream to downstream, as indicated in previous Academy reports. To determine whether the marked decrease in tissue PCB concentrations at Cornwall, Bulls Bridge, Lake Lillinonah, and Lake Zoar persists, it is recommended that additional sampling be performed in 1996.


LITERATURE CITED

Academy of Natural Sciences of Philadelphia (ANSP). 1985. Interim report on PCB concentrations in fish from the Housatonic River, Connecticut, for General Electric. Rept. No. 85-1 OF. Acad. Nat. Sci. Phila. 35 pp.

. 1987. PCB concentrations in fishes from the Housatonic River, Connecticut, in 1986. Rept. No. 87-15. Acad. Nat. Sci. Phila. 64 pp.

. 1990. PCB concentrations in fishes from the Housatonic River. Connecticut in 1984. 1986 and 1988. Rept. No. 89-30F. Acad. Nat. Sci. Phila. 182 pp.

. 1991. PCB concentrations in fishes from the Housatonic River. Connecticut in 1984 to 1990. Rept. No. 91-20. Acad. Nat. Sci. Phila. 132 pp.

. 1993. PCB concentrations in fishes from the Housatonic River. Connecticut, in 1984 to 1992. Rept. No. 93-12F. Acad. Nat. Sci. Phila. 146 pp.

Decider, C.L. 1976. The problem of the supemumary zones in otoliths of the European eel (Anguilla anguilla (Linnaeus, 1758)); a suggestion to cope with it. Aquaculture 9:373-379.

Decider. C.L. 1981. On the age and growth of cultured eels (Anguilla anguilla (Linnaeus, 1758)). Aquaculture 26:13-22.

Draper. W.M.. D. Wijekoon and R.D. Stephens. 1991. Speciation and quantitation of Aroclors in hazardous wastes based on PCB congener data. Chemosphere 22:147-163.

Hurlev D. A. 1972. The American eel (Anguilla rostratd) in Eastern Lake Ontario. J. Fish. Res. Bd. " Can. 29:535-543.

Lawler. Matusky & Skelly Engineers (LMS). 1987. Chapter 6 of Housatonic River PCB Sediment Management Study Program for Monitoring the Natural Recover, of the River Project No. 337-017.

Mullin. M. D. 1985. PCB Workshop, USEPA Large Lakes Research Station, Grosse He. MI. June 1985.

Ogden. J.C. 1970. Relative abundance, food habits and age of the American eel. Anguilla rostrata (LeSueur), in certain New Jersey streams. Trans. Amer. Fisher. Soc. 99:54-59.

SAS. 1985. SAS User's Guide: Statistics. Version 5 Edition. SAS Institute Inc. Can'. N.C. 956 pp.

SAS. 1988a. SAS Language Guide for Personal Computers. Release 6.03 Edition. SAS Institute Inc. Cary, NC.558 pp.


SAS. 1988b. SAS Procedures Guide. Release 6.03 Edition. SAS Institute Inc. Gary, NC. 441 pp.

SAS 1993. SAS Companion for the Microsoft Windows environment. SAS Institute, Inc. Version 6. First edition. Gary, NC. 356 pp.

Smith, C.L. 1985. The Inland Fishes of New York State. The New York State Department of Environmental Conservation. 522 pp.

Sokal, R.R. and F.J. Rohlf. 1969. Biometry. W.H. Freeman and Co. San Francisco. 776pp.

Suns, K., G.G. Hitchin and D. Toner. 1993. Spatial and temporal trends of organochlorine contaminants in spottail shiners from selected sites in the Great Lakes (1975-1990). J. Great Lakes Res. 19:703-714.


APPENDICES


APPENDIX A

SOP No. P-16-77: Extraction and Cleanup of Fish Tissue for PCB and Pesticide Analysis

ACADEMY OF NATURAL SCIENCES ENVIRONMENTAL RESEARCH DIVISION

Procedure No. P-16-77 Rev. 1 (4/95)

EXTRACTION AND CLEANUP OF FISH TISSUE FOR PCB AND PESTICIDE ANALYSIS

Prepared by: Michelle Donne

Approved by: ( M/W\—\ /OL£-~- Date: Carol Lee/ / / / Quality Assurance Unit

Procedure No. P-16-77 Page 1 of 4 Rev. 1 (4/95)

EXTRACTION AND CLEANUP OF FISH TISSUE FOR PCB AND PESTICIDE ANALYSIS

Prerequisite: Use of this method requires a working knowledge of the inherent hazards and possible routes of contamination in working with organic solvents. Also, a working knowledge of glassware cleaning and standard residue analysis techniques is required.

1.0 METHOD

This method includes instructions for extracting PCBs and pesticides from fish tissue. Also, specific criteria for gas chromatography (ECD-capillary) and quantitation on a congener and compound specific basis is included. For basic instructions on gas chromatography see SOP No. P-16-84.

2.0 SUMMARY

The fish tissue is combined with sodium sulfa

Documents

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