### i 

# Southern California Bight

1998 Regional Monitoring

Program

Vol IV. 

# Sediment Toxicity

### ii



## Southern California Bight 1998 Regional Monitoring Program:

**IV**

**. Sediment Toxicity** 

December 20, 2000

Steven M. Bay 1



### , David Lapota 2



### , Jack Anderson

3



### , Jeff Armstrong

4



### , Tim Mikel

5



### , Andrew W. Jirik 1



### , and Stanford Asato 6

1 

### Southern California Coastal Wate r Research Project

7171 Fenwick Lane

Westminster, CA 92683

2 

### Space and Naval Warfare Systems Center

53475 Strothe Road

San Diego, CA 92152

3 

### Columbia Analytical Services

1185 Park Center Drive Suite A

Vista, CA 92009

4 

### County Sanitation Districts of Orang e County

P.O. Box 8127

Fountain Valley, CA 92728

5 

### Aquatic Bioassay and Consulting Laboratories, Inc.

29 North Olive Street

Ventura, CA 93001

6 

### City of Los Angeles, Environmental Monitoring Division

12000 Vista Del Mar

Playa del Rey, CA 90291

### iii **Members of the Toxicology Committee** 

Anderson, Brian University of California, Davis

Anderson, Jack Columbia Analytical Services

Armstrong, Jeff Orange County Sanitation District

Asato, Stanford City of Los Angeles Environmental Monitoring Division

Bay, Stev e, (Chair) Southern California Coastal Water Research Project

Bottomley, Jay P. County Sanitation Districts of Los Angeles County

Brown, Jeff Southern California Coastal Water Research Project

Greenstein, Darrin Southern California Coastal Water Rese arch Project

Gerlinger, Tom Orange County Sanitation District

Gutoff, Dave City of San Diego

Jirik, Andrew Southern California Coastal Water Research Project

Jones, Jennifer Columbia Analytical Services

Lapota, Dave Space and Naval Warfare Syste ms Center

Lyons, Michael L.A. County Regional Water Quality Control Board

McCarthy, Mike Orange County Sanitation District

McCoy, Dan Columbia Analytical Services

Mikel, Tim (Co

- Chair) Aquatic Bioassay and Consulting Laboratories

Monji, Alan MEC Anal ytical

Nakada, Kenneth M. County Sanitation Districts of Los Angeles County

Reeve, Matt State Water Resources Control Board

Robertson, George Orange County Sanitation District

Steinert, Scott Computer Science Corporation

Weisberg, Steve Southern Cal ifornia Coastal Water Research Project

Wiborg, Lan City of San Diego

### iv **FOREWORD**

The Southern California Bight (SCB) is an important and unique ecological resource.

The diverse habitats present in the SCB allow for the coexistence of a broad spectrum of species,

including more than 500 species of fish and 1,500 species of invertebrates. The SCB is also one

of the most densely populated coastal regions in the country, which creates stress upon the

marine environment through activities such as contaminant discharge from effluents and

nonpoint sources, fishing, and habitat modification. Over $10 million is spent annually to

monitor coastal environmental quality in the SCB. These monitoring programs provide

important site

- specific information about the imp acts of individual waste discharges, but do not

describe the condition of the SCB as a whole. Regional information is needed by resource

managers to assess cumulative impacts of contaminant inputs and to evaluate relative risk among

different types of str

esses.

The 1998 Southern California Bight Regional Monitoring Project (Bight’98) is part of

an effort to provide an integrated assessment of the SCB through cooperative regional

- scale

monitoring. Bight’98 is an expansion of the 1994 Southern Californi a Bight Pilot Project

(SCBPP) regional survey (SCBPP Steering Committee 1998) and represents the joint efforts of

62 organizations (Appendix A). Bight’98 is organized into three technical components: (1)

Coastal Ecology, (2) Shoreline Microbiology, and ( 3) Water Quality. This report presents the

results of the sediment toxicity portion of Bight’98, which is a part of the coastal ecology

component. Copies of this and other Bight’98 reports are available for download at

www.sccwrp.org.

The proper citati

on for this report is: Bay, S.M., D. Lapota, J. Anderson, J. Armstrong, T.

Mikel, A.W. Jirik, and S. Asato. 2000. Southern California Bight 1998 Regional Monitoring

Program: IV. Sediment Toxicity. Southern California Coastal Water Research Project.

Wes tminster, CA.

### v **ACKNOWLEDGMENTS**

This report is a product of the dedication and hard work of many individuals who share a

common goal of improving our understanding of the environmental quality of the Southern

California Bight. The authors wish to thank the members of the Bight’98 Steering Committee

for supporting the application of sediment toxicity tests in regional monitoring. This study

would not have been possible without the exceptional skill of the field sampling personnel from

the following organ

izations: City of San Diego, Space and Naval Warfare Systems Center, MEC

Analytical, Orange County Sanitation District, County Sanitation Districts of Los Angeles

County, City of Los Angeles, Southern California Coastal Water Research Project, and Aquatic

Bioassay and Consulting Laboratories. We also wish to express our gratitude to the County

Sanitation Districts of Los Angeles for assistance with statistical analyses, to Darrin Greenstein

(SCCWRP) for coordinating sample distribution, to Lan Wiborg (City of San Diego) for analysis

of quality assurance data, and to Brian Anderson (UC Davis) for assistance with study design

and data analysis.

### vi **EXECUTIVE SUMMARY**

Sediment toxicity measurements were conducted during the 1998 Southern California

Bight Reg ional Monitoring Project (Bight’98) in order to accomplish three goals: (1) to

determine the percent of area in the SCB that contains sediments toxic to marine organisms; (2)

to compare the responses among sediment toxicity test methods; and (3) to evaluat e the

relationship between sediment toxicity and chemical contamination or changes in benthic

communities.

Sediment from 303 sites on the continental shelf between Point Conception, California,

and the United States

- Mexico international border were sampl ed between July 13 and September

16, 1998. Sites were selected using a stratified random design. Five of the strata were located

offshore (river mouths, large publicly owned treatment works [POTW] discharge areas, small

POTW discharge areas, remaining sh allow areas [5

- 30 m], and remaining mid

- depth areas [30

- 120 m]). Three additional strata were located within bays and harbors, which included marinas,

ports/industrial areas, and other harbor areas (less

- developed areas that did not serve

port/industrial or marina functions).

Subsets of the sediment samples were evaluated for toxicity using up to four methods.

Bulk sediment from 241 stations was measured for toxicity using a 10

- d amphipod

( Eohaustorius estuarius ) survival test. Sediment extracts from 268 stations were evaluated for

toxicity using the P450 human reporter gene system (HRGS) test, which measures the

concentration of organic compounds that induce the cytochrome P450 enzyme system (e.g.,

PAHs, dioxins, furans, and some PCBs). Elutriates f rom 173 samples were tested for sublethal

toxicity (bioluminescence inhibition) to phytoplankton ( Gonyaulax polyedra ) using the QwikSed

test. Interstitial water from 88 samples was analyzed for sublethal toxicity (bioluminescence

inhibition) to the marine bacterium, Vibrio fischeri (Microtox test).

Seven laboratories conducted the amphipod survival tests. An interlaboratory

comparison exercise completed prior to analysis of the Bight’98 sediment samples demonstrated

that each laboratory was capable o f meeting test performance objectives and providing similar

toxicity results. The remaining three tests were each conducted by a single laboratory.

The amphipod test detected toxicity in each of the seven strata. Amphipod toxicity was

most prevalent in

bay and harbor areas, where 13

- 37% of the area (depending upon the stratum)

was toxic. Toxicity was least prevalent in POTW outfall areas (6% of the area) and the shallow

portion of the coastal shelf (3% of the area).

Each of the other tests also detec ted sediment toxicity in selected strata. The QwikSed

test was the most sensitive of the toxicity indicators. Toxicity using QwikSed was detected in

elutriate samples from bays and harbors and also from POTW outfall areas. HRGS gene activity

was induced

by sediment extracts from 30 stations, with most of the induction produced by

samples from port/industrial or marina areas. Microtox measurements of interstitial water were

### vii

taken but were unsuitable for use because of changes in toxicity related to prolo nged sample

storage. The Microtox test data were not used for the assessment of sediment quality. The data

for the remaining three indicators were analyzed to evaluate the relative sensitivity of each test

and to provide an integrated measure of sediment quality.

While 69

- 78% agreement was observed between pairs of toxicity tests in classifying

stations as toxic/nontoxic, different patterns of response were indicated by each test. The

QwikSed test results were not correlated with either amphipod surviv al or HRGS response and

the correlation between HRGS and amphipod survival was significant but low (r = 0.285).

The three test responses were combined into an integrated assessment of sediment quality

using a weight of evidence approach that incorporat ed the relative ecological relevance and

severity of the test responses. The integrated assessment identified 19% (644 km 2 

### ) of the SCB as

areas of potential or high concern. Areas of high concern (2.7%) were almost exclusively

located within harbors and bays, while areas of potential concern were present in all strata tested.

The Bight’98 amphipod toxicity results for bay and harbor strata (13

- 37% of the area

affected) fell within the general range reported in previous local studies by the National Ocea nic

and Atmospheric Administration (NOAA) and the State Water Resources Control Board (14

- 66% of the area affected). The persistent occurrence of toxicity in port and marina areas

indicates that sediment quality in many of these areas is not improving. T hese locations are good

candidates for additional research designed to identify the cause of toxicity.

Temporal differences in toxicity were apparent in two areas of the SCB. Amphipod

toxicity was less prevalent in San Diego Bay compared to samples an alyzed in 1992

- 94 and

amphipod toxicity was greater in mid

- depth areas compared to samples analyzed in 1994. The

cause of these temporal differences may be related to several factors, including the use of

different amphipod test species and variations in sediment contaminant concentrations. Analysis

of sediment chemistry data (not yet available) is needed to help determine the cause of these

trends.

Sediment toxicity is just one of three types of information needed to assess coastal

sediment quality. Measures of sediment contamination and biological response (e.g., benthic

community impacts) are also needed to establish whether the toxicity patterns are ecologically

significant and associated with anthropogenic inputs.

Additional data are also nee ded to evaluate the significance of the different response

patterns between the amphipod, QwikSed, and HRGS tests. The variable responses among test

methods may reflect differences in contaminant sensitivity between species, which was the intent

of using multiple toxicity indicators. Some of the variation may also be related to differences in

exposure or contaminant bioavailability caused by different laboratory test procedures.

Comparisons of the toxicity results with sediment contamination and benthic community

characteristics are needed to help determine the predictive ability of the test methods and verify

the efficacy of the weight of evidence strategy used to integrate the toxicity results.

### viii 

## Definition of Terms:

**Benzo[a]pyrene equivalents (B[a]PE q):**  The results of the P450 HRGS assay expressed as the

concentration of benzo[a]pyrene required to produce an equivalent toxic response.

**Bioassay:** See toxicity test.

**Bioavailability:** The fraction of a chemical present in the sediment that is availa ble for uptake

by organisms.

**Control chart:** A plot of the LC50 or EC50 values from the previous reference toxicant

exposures performed by a laboratory. New tests falling between control lines representing plus

and minus two times the standard deviation are considered to be within acceptable limits.

**Cytochrome P450:** Group of enzymes that transforms the structure of organic chemicals within

the cells of an organism. The presence of organic chemicals may induce organisms to produce

these enzymes.

**Dose**

**- r**  **esponse effect:** Observed effect of different concentrations of a toxicant on bioassay test

organisms. Generally expected that effect increases with concentration.

**EC50:** Concentration of a toxicant predicted to cause a sublethal effect in 50% of test or ganisms

over the course of an exposure period.

**Elutriate:** An aqueous sample produced by mixing water with sediment, then separating the

water and sediment phases. The water phase is subsequently used for testing.

**Fold induction:** The amount of cytochro me P450 induction in the human reporter gene system

(HRGS) test, expressed relative to the control. It is calculated by dividing the luminescence of

the sample by the luminescence of the solvent control.

**Interstitial water:** Water that is between the g rains of whole sediment.

**LC50** : Concentration of a toxicant predicted to cause a lethal effect in 50% of test organisms

over the course of an exposure period.

**Microtox** ä **:** A bioassay system that utilizes luminescent bacteria to measure the toxicity of

aqu eous samples. Reduction in the ability of the bacteria to produce light indicates toxicity.

**Negative control:** A sample from a site known to be uncontaminated that is tested along with

samples of unknown toxicity.

**POTW:** Publicly owned treatment works ( wastewater treatment facility).

### ix

**P450 HRGS test:** A bioassay method in which genetically modified human cells are exposed to

extracts of a sample. The presence of certain organic chemicals induces the cytochrome P450

system of the cells, which results in a bioluminescent reaction. Increased production of light

indicates a higher concentration of the organic chemicals.

**QwikSed** ä **:** A bioassay system that utilizes luminescent algae to measure the toxicity of

sediment elutriates. Reduction in the ability of the algae to produce light indicates toxicity.

**Reference toxicant:** A single compound that is tested along with an unknown sample in order

to determine the sensitivity of the test organisms. Comparing reference toxicant results between

experiments allows for determination of the validity of each test (see control chart).

**Stratum:** A subset of stations from the stratified random sampling design. Stations within a

given stratum have some characteristic in common (e.g., location near river mouths).

**Toxici ty test:**  A laboratory experiment that measures the response (e.g., survival, growth, or

reproduction) of an organism following exposure to a sample suspected of containing harmful

substances.

### x 

## TABLE OF CONTENTS

**SECTION**

Toxicology Committee Members

................................ ................................ ........................ ii

Foreword

................................ ................................ ................................ .......................... iii

Acknowledgements

................................ ................................ ................................ ............ iv

Executive Summary

................................ ................................ ................................ ............ v

Definition of Terms

................................ ................................ ................................ ............. vii

List of Figures

................................ ................................ ................................ .................... xi

List of Tables

................................ ................................ ................................ ..................... xii

**I. INTRODUCTION**

................................ ................................ ................................ ...... 1

**II. METHODS**

................................ ................................ ................................ .............. 2

A. Sampling Design

................................ ................................ ............................ 2

B. Field Methods

................................ ................................ ............................... 4

C. Laboratory Methods

................................ ................................ ..................... 4

D. Data Analysis

................................ ................................ ................................ 7

**III. QUALITY ASSURANCE EVALUATION**

................................ ............................ 14

A. Sampling Success

................................ ................................ .......................... 14

B. Sample Storage

................................ ................................ ............................. 14

C. Test Performance

................................ ................................ .......................... 16

**IV. DESCRIPTIVE RESULTS**

................................ ................................ ..................... 25

A. Amphipod Survival

................................ ................................ ........................ 25

B. QwikSed

................................ ................................ ................................ ....... 28

C. P450 HRGS

................................ ................................ ................................ . 31

**V. COMPARISON OF INDICATORS**

................................ ................................ ....... 34

**VI. REGIO NAL ASSESSMENT OF TOXICITY** 

................................ ........................ 37

**VII. DISCUSSION**

................................ ................................ ................................ ........ 41

**VIII. CONCLUSIONS**

................................ ................................ ................................ ... 44

**IX. RECOMMENDATIONS**

................................ ................................ ........................ 46

**X. LITERATURE CITED**

................................ ................................ ............................. 48

### xi

**APPENDIX A.** Participants in the Southern California Bight 1998 Regional Monitoring

Program (Bight’98).

**APPENDIX B.** Int erlaboratory comparison of sediment toxicity tests with the Amphipod

Eohaustorius Estuarius

**APPENDIX C.** Microtox storage experiment

**APPENDIX D.** Test results by station

**APPENDIX E.** Toxicity results mapped by indicator

**APPENDIX F.** Relationship between se diment characteristics and indicator responses

### xii 

## LIST OF FIGURES

FIGURE II

- 1. Locations of all stations sampled for sediment toxicity

during the Bight’98 project.

................................ ................................ ..... 11

FIGURE III

- 1. Summary of cadmium reference toxicant test results for amphipod

.............. 1 8

survival tests.

FIGURE III

- 2. QwikSed reference toxicant results..

................................ .......................... 20

FIGURE III

- 3. Concentration of ammonia in elutriate samples stored prior to

QwikSed analysis.

................................ ................................ ................... 21

FIGURE III

- 4. Dose

- response plot of ammonia effects on G. polyedra with

regr ession curve.

................................ ................................ ................... 21

FIGURE III

- 5. Influence of ammonia correction procedure on QwikSed

luminescence value.

................................ ................................ ................. 22

FIGURE III

- 6. Reclassification of stations resulting from correction of QwikSed

results for ammonia toxicity.

................................ ................................ ..... 22

FIGURE III

- 7. Response of the P450 HRGS assay to the reference inducer (TCDD)

over the testing period for Southern California Bight’98 samples.

.............. 24

FIGURE IV

- 1. Percent of sediment samples toxic to amphipods from seven

strata in the Southern California Bight.

................................ ...................... 26

FIGURE IV

- 2. Percent of sediment samples toxic to QwikSed from seven strata

in the Southern California Bight.

................................ ............................... 29

FIGURE IV

- 3. Percent of sediment samples toxic to HRGS from seven strata in the

Southern California Bight.

................................ ................................ ........ 32

FIGURE V

- 1. Results of toxicity cl

assifications for each pair of toxicity tests.

..................... 35

FIGURE V

- 2. Comparison of indicator responses for sediment samples.

............................ 36

FIGURE VI

- 1. Percent of Southern California Bight not toxic to each indicator.

................. 37

FIGURE VI

- 2. Percent of area (+ 95% confiden ce interval) found to be toxic in

each stratum.

................................ ................................ ......................... 38

FIGURE VI

- 3. Percent of area of concern based upon joint toxicity classification.

.............. 40

### xiii 

## LIST OF TABLES

TABLE II

- 1. Number of stations selected for toxicity evaluation for each stratum.

.............. 12

TABLE II

- 2. List of participating laboratories and toxicity indicators measured.

.................. 13

TABLE III

- 1. Bight’98 toxicity sample collection success.

................................ ................. 14

TABLE III

- 2. Bight’98 toxicity sample holding time.

................................ ......................... 16

TABLE III

- 3. The HRGS assay response to Santa Monic a Bay reference sediment.

.......... 24

TABLE IV

- 1. Sediment samples toxic to amphipods from seven strata in the

Southern California Bight.

................................ ................................ ........... 26

TABLE IV

- 2. Sediment samples toxic to amphipods from nine harbors and bays in

the Southern California Bight.

................................ ................................ ..... 27

TA BLE IV

- 3. Sediment samples toxic to QwikSed from seven strata in the

Southern California Bight.

................................ ................................ ........... 29

TABLE IV

- 4. Sediment samples toxic to QwikSed from nine harbors and bays in the

Southern California Bight.

................................ ................................ ........... 30

TABLE IV

- 5. Sediment samples toxic to HRGS from seven strata in the

Southern California Bight.

................................ ................................ ........... 32

TABLE IV

- 6. Sediment samples toxic to HRGS from nine harbors and bays in the

Southern California Bight.

................................ ................................ ........... 33

TABLE VI

- 1. Percent of area in each stratum classified as toxic by indicator type

. ............. 38

TABLE VI

- 2. Strategy for determining levels of concern from results of three

types of sediment toxicity tests.

................................ ................................ 40

### 1 **I. INTRODUCTION**

The 1998 Southern California Bight Regional Monitoring Project (Bight’98) is organized

into three technical components: (1) coastal ecology, (2) shoreline microbiology, and (3) water

quality. The goal of the coastal ecology component of Bight’98 is to assess the condition of the

bottom environment and the health of biological resources in the SCB. Assessing th e health of

the bottom environment requires the use of multiple indicators, usually sediment contamination,

sediment toxicity, and benthic community structure. Sediment toxicity tests complement these

other indicators of sediment quality by providing a me asure of the joint effect of contaminant

mixtures. Toxicity tests use biological responses to describe sediment quality, thus providing a

mechanism to evaluate the significance of chemical contamination on marine life.

The sediment toxicity portion of Bight’98 was designed to accomplish the following

goals:

• To determine the percent of area in the SCB that contains sediments toxic to marine

organisms.

This study used four methods to measure sediment toxicity. Sediments were assessed

using the standard

ized amphipod survival test, two test methods measuring

physiological responses in marine algae and bacteria, and a method measuring the

cellular responses of specific classes of organic chemicals in sediment extracts.

Multiple test methods were included in order to ensure that the test results were not

unduly influenced by method

- specific differences in contaminant sensitivity. The

sampling plan was expanded relative to the SCBPP to include bays and harbors, where

sediment contamination concentrations ar e usually highest.

• To compare the responses among multiple sediment toxicity test methods.

The concurrent use of multiple toxicity indicators provides the opportunity to examine

their relative response to the same sample. Comparisons of the responses am ong

indicators can be used to determine which methods are most sensitive to conditions in

the SCB and whether each indicator is responding to the same or different sediment

constituents.

• To evaluate the relationship between sediment toxicity and chemical contamination or

changes in benthic communities throughout the SCB.

The relationship between sediment contamination, sediment toxicity, and benthic

community response is still poorly understood. Relatively few data have been collected

for the SCB where a

ll three types of sediment quality measures have been utilized.

Comparison of the sediment toxicity test results with chemistry and benthic community

response provides the opportunity to “calibrate” toxicity test responses to impacts on

communities and to

determine if national sediment quality guidelines based upon

chemical concentrations are applicable in the SCB.

### 2

Chapter II of this report describes the methods used to prepare the samples and measure

toxicity. A quality assurance evaluation of the te st results is provided in Chapter III, which

addresses issues of data comparability and laboratory performance during the study. Chapter IV

describes the test results for each of the indicators separately, illustrating patterns in the

prevalence and sever

ity of toxicity among the sampling strata. A comparison of the results for

three of the tests is presented in Chapter V. Chapter VI uses the toxicity results to provide a

regional assessment of sediment toxicity based upon the percent of area affected. This chapter

also integrates the results from each indicator. Discussion of the results is contained in Chapter

VII. Conclusions from the study are presented in Chapter VIII and recommendations for future

studies are presented in Chapter IX.

Evaluati on of the relationships between sediment toxicity, chemistry, and benthic

community responses is not included in this report. These comparisons will be incorporated into

a future Bight’98 integrative report, scheduled for completion in Fall 2001.

### 3 **II. METHODS** 

**A. Sampling Design**

Three hundred and three sites on the continental shelf between Point Conception,

California, and the United States

- Mexico international border (Figure II

- 1) were sampled

between July 13 and September 16, 1998. Sites were se lected using a stratified random design.

Five of the strata were located offshore (river mouths, large publicly owned treatment works

[POTW] discharge areas, small POTW discharge areas, remaining shallow areas [5

- 30 m], and

remaining mid

- depth areas [30

- 1 20 m]). Three additional strata were located within bays and

harbors, which included marinas, port/industrial areas, and other harbor areas (less

- developed

portions of bays and harbors that did not serve port/industrial or marina functions). In addition

to the stratified random sites, 13 non

- random sites were located in offshore areas, distant from

known point sources. These 13 sites had been sampled at periodic intervals since 1977

(Thompson

et al. 1987, Thompson

et al. 1993, Word et al. 1979) and were selected for historical

comparison. Results for the historical comparisons are not presented in this report.

Sites were selected randomly within each stratum, rather than by investigator pre

selection, to ensure that they were represen tative and could be extrapolated to the response of the

entire stratum. Within the small POTW stratum, four samples were randomly placed in an

approximately 3 km 2 

### area around each outfall. For the river mouth stratum, 2

- 4 random samples

were placed withi n a 3 km radius of each river mouth, with the number of samples proportional

to the area contained within 3 km (different areas resulted from the differing shape of the

coastline near each outfall). For all other strata, a systematic component was added t o the

selection process to minimize clustering of sample sites. The systematic element was

accomplished by using an extension of the sampling design used in the SCBPP and in the

Environmental Protection Agency’s (EPA’s) Environmental Monitoring and Assess ment

Program (EMAP) (Stevens 1997) . A hexagonal grid was placed over a map of the sampling

area, a random subsample of hexagon cells was chosen from this population, and one sample was

obtained at a randomly selected site within each gri d cell. The hexagonal grid structure ensures

systematic separation of the sampling effort, while the random selection of sites within grid cells

ensures an unbiased estimate of ecological condition. Additional details of this site selection

process are p rovided in the Coastal Ecology Study Plan (Bight’98 Steering Committee 1998)

.

Samples were collected for four types of toxicity measures: (1) amphipod survival, (2)

QwikSed, (3) Microtox, and (4) P450 Human Reporter Gene System (HRGS). T he number of

sites sampled within each stratum (Table II

- 1) varied among measures because of limitations in

laboratory capacity and available processing effort. The amphipod survival test was the primary

indicator of toxicity in the study. Samples from 2 47 stations were selected for amphipod toxicity

measurement, which included all bay/harbor stations and a subset of stations from the offshore

stratum. Stations sampled for the QwikSed and Microtox indicators were a subset of those

sampled for amphipod to xicity, with the primary emphasis on stations within bays and harbors

(Table II

- 1). The largest number of stations was selected for the P450 HRGS, which shared the

level of sampling effort used for sediment chemistry measurements.

### 4

**B. Field Methods**

Sed iment samples were collected with a 0.1 m 2 

### modified van Veen grab. Up to 2.5 L of

sediment were collected for measurement of solid phase or elutriate toxicity using amphipod

survival and QwikSed

 tests, respectively. A plastic (high

- density polyethylene [HDPE],

polycarbonate, or Teflon) scoop was used to collect sediment from the top 2 cm of the

undisturbed surface material in the grab. Contact with sediment within 1 cm of the side of the

grab was avoided in order to minimize cross

- contamination. The se diment was placed in clean

HDPE containers and distributed as follows: 2.25 L for amphipod test and 0.25 L for

QwikSed  . Following collection, samples were stored on wet ice or refrigerated at 4 ° C.

Samples for the Microtox tests, which were based upon interstitial water analysis, were

collected in the same manner, but were placed in a 0.25 L polycarbonate centrifuge bottle. These

samples were also processed for acid volatile sulfide analysis, so the containers were filled

completely, leaving no headsp ace. They were kept on wet ice or refrigerated at 4 ° C prior to

centrifugation.

Organic solvent extracts for P450 HRGS analysis were obtained from surface sediment

samples collected for hydrocarbon analysis. These samples were collected with a plastic,

stainless steel, or Teflon

- coated metal scoop and placed in a 0.25 L plastic or glass container

with a Teflon

- lined lid. The samples were initially placed on wet ice and then frozen within 24 h

of collection.

**C. Laboratory Methods**

Selection of sedimen

t toxicity test methods was made by the Toxicology Committee, a

technical committee comprised of representatives of the agencies participating in Bight’98. The

Toxicology Committee also specified sample holding times and established performance criteria

f or each of the tests. Four laboratory indicators were used to evaluate sediment quality.

Assessment of solid phase toxicity using the amphipod test was a cooperative effort involving

seven laboratories (Table II

- 2). Each of the other indicators was anal yzed by a single laboratory.

Amphipod Survival

Toxicity to amphipods was determined using a 10

- d survival test (U.S. EPA 1994) with

Eohaustorius estuarius . Sediment samples were distributed among the test laboratories based

upon the l ocation of the laboratories. Amphipods and negative control sediment were collected

from Beaver Creek, Oregon, a non

- contaminated estuarine site, and held in the laboratory at least

4 d, but no longer than 14 d, prior to the test. Testing was conducted i n 1 L glass test containers.

Sediment was added to the test containers 1 d prior to the start of the test. Sediment samples

were thoroughly mixed and then added to the test containers to form a sediment layer

approximately 2 cm deep. Filtered seawater ( 20 g/kg salinity) was added slowly until a final

volume of 800 mL was reached. Pipettes connected to an air source provided aeration.

Sediments were allowed to equilibrate overnight. Each sample consisted of five randomly

arranged replicates, along with an extra container for water quality. A negative control

(amphipod collection site sediment) was included with each batch of samples tested.

### 5

At the start of the test, amphipods were added randomly until a total of 20 animals per

container was present. Tests were conducted at 15 ° C under constant illumination. Test animals

were exposed to the sediment samples for 10 d, with daily checks for air and mortality. Any

floating animals were submerged by gently pushing them beneath the surface with a probe. At

the end of the exposure period, the sediment was screened through a 0.5 mm screen and the

number of surviving amphipods was recorded.

A laboratory intercalibration exercise was conducted prior to the start of testing in order

to document the compar ability of the results. This exercise demonstrated that comparable data

were produced by each of the laboratories. Details of this exercise are presented in Appendix B.

A cadmium reference toxicity test was conducted concurrently with each sediment

tox icity test. The reference toxicant test consisted of three replicates of five concentrations of

dissolved cadmium, plus a control. No sediment was included in the reference toxicant tests.

Ten amphipods were added to each replicate and exposed to the re ference toxicant for 4 d. At

the end of 4 d, the total number of surviving animals was recorded and the concentration causing

50% mortality (LC50) was calculated. The Spearman Karber method was used to calculate the

LC50, which was then compared to a cont rol chart prepared from the results of past reference

toxicant tests. A test result within two standard deviations of the mean control chart LC50 was

considered acceptable. A test not falling within two standard deviations was evaluated for

acceptability

by employing the best professional judgment of the laboratory.

Samples of overlying water and interstitial water were obtained from the extra test

container for measurement of initial water quality (temperature, pH, dissolved oxygen, salinity,

and tot al ammonia). Overlying water quality was also measured at the end of the exposure

period. Water quality measurements made for the reference toxicant test were conducted using a

similar methodology to the sediment phase of the test.

QwikSed Elutriate Test

The QwikSed elutriate test is a measure of light production from the bioluminescent

dinoflagellate Gonyaulax polyedra following exposure to a sample. A reduction in

bioluminescence relative to the control reflects an adverse effect of the sample on the test

organisms.

QwikSed tests were conducted on sediment elutriates, which were prepared by mixing

approximately 75 grams of wet sediment with 300 mL of filtered seawater. The mixture was

stirred for 30 min and settled for 60 min; then the supernatant was poured off for use in the test

(U. S. EPA 1991) . All elutriates were passed through an 8 μ m membrane filter prior to mixing

for all dilutions. Five elutriate concentrations were prepared from the initial sediment

- water

mixture: 100, 50, 25, 12.5, and 6.25% (ASTM 1998). These dilutions were then mixed in 125

mL Erlenmeyer flasks with an equivalent volume of dinoflagellate cell stock to a final

concentration of approximately 200 cells/mL. Dinoflagellates were obtained from laboratory

cultures maintained in a filtered (0.45 μ m) enriched seawater medium (ESM) under 40

- watt

### 6

cool

- white fluorescent bulbs on a 12:12 h (light:dark) cycle at 19 ° C. A control sample (ESM

only) was included with each elutriate sample tested.

Three mL from ea

ch of the elutriate dilution flasks were pipetted into each of five

disposable spectrophotometer cuvettes (five replicates per concentration). The cells were then

incubated for 24 h at 19 ° C at a light intensity of 4,000 lux on a 12D:12L h photoperiod.

Total mechanical stimulable light (TMSL) was measured from each of the cuvettes after

24 h, using the QwikSed photometer system. The contents of the cuvette were stirred while in

the photometer, stimulating the dinoflagellates to emit light that was det ected by a

photomultiplier tube (PMT). The PMT counts were accumulated during stirring and compared

to the control response.

The salinity of the prepared elutriates was checked with a refractometer and maintained

at ~33 g/kg. Total ammonia was measur ed with an Orion ammonia electrode; the temperature

and pH of the elutriate were measured concurrently. Total ammonia was measured in all 100%

elutriates and then converted to unionized ammonia. Unionized ammonia concentrations were

then back

- calculated for all dilutions in each sample.

Four reference toxicity tests with copper sulfate were conducted during the testing period.

Two tests were conducted on August 25, 1998, a third test was conducted on September 16,

1998, and a fourth test was conducte d on January 8, 1999. The copper sulfate reference toxicant

data were examined to verify that all tests fell within ±2 standard deviations of the mean. If

outliers were observed, the test data were flagged to indicate that the health of the cells was

que stionable. Several toxicity tests were also conducted using ammonium chloride dissolved in

seawater to determine the sensitivity of G. polyedra to ammonia.

Microtox Interstitial Water Test

Sediment interstitial (pore) water was assessed using the Mi crotox TM 

### Rapid Toxicity

Testing System, in which a change in light production from a luminescent bacterium relative to a

control sample is used to measure toxicity. Interstitial water was obtained by centrifuging the

sediment at 3,000 g for 20 min. The r esulting interstitial water samples were stored frozen in

borosilicate glass vials with Teflon

- lined lids. The samples were analyzed using a Microtox

M500 analyzer following the Microtox Comparison Test Method (Microbics Corp. 1995)

. Bac teria ( Vibrio fischeri strain NRRL B

- 11177) were added to triplicate cuvettes containing 1.5

mL of 100% interstitial water and incubated at 15 ° C for 15 min. The luminescence of each

replicate was measured and compared to a matching control (seawater) sam ple. The percent of

difference in mean light production in the samples relative to the control sample was then

calculated. Reproducibility of the test organism response was determined by testing a phenol

reference toxicant solution at a single concentratio n.

P450 HRGS Solvent Extract Test

The P450 human reporter gene system (HRGS) test measures changes in luminescence of

human hepatoma cells transfected with a luciferase reporter gene following exposure to solvent

### 7

extracts of sediment. An increase in luminescence indicates that the sample contains organic

compounds that induce the production of cytochrome P450 (CYP1A1), such as PAHs, dioxins,

furans, and coplanar PCBs (Anderson et al. 1995) . Sediments from over 1,000 stations located

in numerous U.S. coastal areas have already been tested by this method in NOAA investigations

(Anderson et al. 1999)

.

The details of the HRGS method (EPA Method 4425) have been previously published

(APHA 1998, ASTM 1999, EPA 1999); there fore, only a short summary follows. Hexane

extracts of sediments were prepared by the Southern California Coastal Water Research Project

(SCCWRP) and exchanged into dichloromethane (DCM) before testing. Extracts were applied

at a volume of 2 μ L to single or duplicate wells in six

- well plates containing 2 mL of culture

media. After 16 h incubation, the cells were washed with Hank’s Balanced Salt Solution

(Mediatech, Herndon, VA) and lysed with 200 μ L of a solution containing 1% Triton, 25 mM

Tricine, pH 7 .8, 15 mM MgSO 4 

### , 4 mM EDTA, and 1 mM dithiothreitol (DTT). Cell lysates

were centrifuged at 6,000 rpm for 10 s, and 50 μ L of the supernatant was applied to a 96

- well

plate, followed by addition of luciferase assay reagents. Luminescence in relative light units

(RLUs) was measured using a ML2250 Luminometer (Dynatech Laboratories, Chantilly, VA)

and divided by the RLUs measured for a solvent blank sample to determine the fold induction of

the sample.

Extracts supplied by SCCWRP also included a method blan k for each extraction batch, as

well as extracts of the Santa Monica Bay (SMB) reference sediment used for interlaboratory

calibration studies by the Bight’98 analytical chemistry laboratories. These samples were tested

by HRGS concurrently with the sedim ent samples.

A reference toxicant, 1 ng/ mL Tetrachlorodibenzo

- p

- dioxin (TCDD), was included with

each test batch. The fold induction response of the TCDD was compared to a control chart; if a

result deviated by more than two standard deviations from the running mean (106 ± 17), then the

samples were reanalyzed. The HRGS fold induction response to all inducing compounds is

linear to approximately 100, and then plateaus. For this reason, any sample producing a fold

induction >100 was diluted 1:10 and rete sted.

The HRGS results were reported in terms of benzo[a]pyrene equivalents on a dry weight

basis ( μ g/g B[a]PEq). These values were calculated from the mean fold induction responses,

based upon the HRGS concentration

- response curve for benzo[a]pyrene, t he amount of extract

applied, the volume of the extract, and the sample dry weight.

**D. Data Analysis**

Data were analyzed using two methods: (1) Calculation of mean parameter response

(e.g., average survival) in the SCB and in various strata (e.g., mar inas), and (2) assessment of the

fractional area within each stratum exceeding selected parameter thresholds.

### 8

Confidence intervals were calculated by multiplying the standard error by the two

- tailed t

value corresponding to α = 0.05. Statistical differences between populations of interest were

defined on the basis of non

- overlappi ng confidence intervals. Use of the ratio estimator for the

standard error approximates joint inclusion probabilities among samples and assumes a negligible

spatial covariance, an assumption that appears warranted based upon preliminary examination of

the data. The assumption is conservative in that its violation would lead to an overestimation of

the confidence interval (Stevens and Kincaid 1997)

.

The percent of area exceeding a selected threshold was calculated after converting the d ata

to its binomial form. For any sample observation, p i 

### was 1 if it exceeded the threshold value and 0

otherwise. The proportion of area that exceeded the selected threshold value was taken as the

weighted mean of the indicator variable p i 

### . The toxicit y thresholds used were specific to each test

and are described below.

For the amphipod test, two events had to occur before a sample could be classified as

toxic: (1) a statistically significant reduction in survival compared to the control and (2) a

mi nimum percent reduction in survival between the sample and control. Statistical significance

was determined by performing a t

- test between the sample and control with a 0.05 level of

significance. Samples with a significant reduction in mean survival tha t was greater than 20%

relative to the control (i.e., a control normalized survival of less than 80%) were determined to

be moderately toxic. This measure of toxicity has been used in previous surveys (Bay et al.

1998, U. S. EPA 1994) and has been shown to represent a 90% power to determine statistical Mean parameter values were calculated using a weighted mean:



# ∑ ∑

= = 

### =

n

i i n

i i i



### w w p

m

1

1



### where:

m = Mean value for population j

p i 

### = Parame ter value (e.g., concentration) at station i

w i 

### = Weighting for station i, equal to the inverse of the inclusion probability for the site

n = Number of stations sampled in population j

Standard error of the mean response was calculated as:

Standard Error = 

# ( )

( )

2

1 1 2



### *

      −



# ∑ ∑

= =

n

i i n

i i

i



### w w m p n

### 9

significance in survival between control and sample (Thursby et al. 1997) . Samples with mean

survival rates of less than 50% of the control were classified as highly toxic.

The QwikSed test responses were corrected for the influence of ammonia toxicity by

using the dose

- response relationship calculated from experiments using seawater spiked with

known concentrations of ammonia. A nonlinear logistic regression was used to describe the

influence of unionized ammonia on QwikSed luminescence. The resulting best

- fit equation was:



# ( )      

+ + =

3

2 1

0



### / 1 a a x a

a y

where:

y = QwikSed luminescence

x = Unionized ammonia concentration

a 0 

### =

- 402.553

a 1 

### = 517.485

a 2 

### = 2.737

a 3 

### = 0.706

The QwikSed data (expressed as a percent of the control luminescence) were corrected for

ammonia toxicity using a two

- step process. First, the net change in luminescence due to

ammonia ( y a 

### ) was determined by subtracting the predicted luminescence ( y ) from th e control ( y a 

### = 100

- y ). Second, the corrected value ( y c 

### ) was obtained by increasing the measured

luminescence (

y m 

### ) by the amount due to ammonia ( y c 

### = y m 

### + y a 

### ).

Example calculation:

If: Measured luminescence ( y m 

### ) = 70.2% of control

Unionized ammonia (

x ) = 0.0527 mg/L

Then: Predicted luminescence due to ammonia ( y ) = 85.0

Net change due to ammonia ( y a 

### ) = 100.0

– 85.0 = 15.0

Corrected value (

y c 

### ) = 70.2 + 15.0 = 85.2%

Samples with <84% of control bioluminescence were classified as being moderately

tox ic. This value represents the level at which there is a 90% power to detect a significant

difference in response from the control. Highly toxic samples were defined as those with <50%

of control bioluminescence.

For the Microtox test, samples were class ified as toxic if there was a 5% reduction in

luminescence relative to the control. This value corresponds to the level at which there is a 90%

power to detect a significant difference in response relative to the control. Samples producing

<50% of the co ntrol luminescence were classified as highly toxic.

### 10

Two thresholds were used for the classification of the P450 HRGS test responses.

Samples containing ≥ 32 μ g/g B[a]PEq were classified as potentially toxic; this value

corresponds to the upper 99% confide nce interval of samples analyzed in previous surveys

(Anderson et al. 1999) . Values less than 11 μ g/g B[a]PEq are not likely to be associated with

any adverse biological effects, but impacts on organisms are unknown at concentrations betw een

11 and 32 μ g/g B[a]PEq. The classification of highly toxic was applied to samples with ≥ 60

μ g/g B[a]PEq, a level associated with the occurrence of degraded benthic communities (Fairey et

al. 1996)

.

### 11

FIGURE II

- 1. Locations of all stations sampled for sediment toxicity during the Bight’98 project.

### 12

TABLE II

- 1. Number of stations selected for toxicity evaluation for each stratum.

Number of Stations per Indicator Type

Stratum Amphipod QwikSed Microtox P450 HRGS

Offshore

River Mouths 44 1 1 44

Shallow 30 12 12 30

Mid

- depth 30 17 17 30

POTW 30 30 30 66

Bays/Harbors

Marinas 39 39 39 39

Ports/Industrial 35 35 35 35

Other 39 39 39 39

Historically

Sampled

Shallow 6

Mid

- depth 7

Total 247 173 173 296

### 13

TABLE II

- 2. List of participating laboratories and toxicity indicators measured.

A=Amphipod test, M=Mictotox test, Q=QwikSed test, P=P450 HRGS test.

A M Q P

Aquatic Bioassay and Consulting Laboratories Ö

City o f Los Angeles (EMD) Ö

City of San Diego (Metro) Ö

Columbia Analytical Services Ö

Marine Pollution Studies Laboratory Ö

MEC Analytical Systems Ö

Orange County Sanitation District (OCSD) Ö Ö

SCCWRP Ö

U.S. Navy (SPAWAR) Ö

### 14 **III. QUALITY ASSURANCE EVALUATION**

**A. Sampling Success**

Bight’98 field crews maintained a good sampling record. Samples were successfully

collected at 92

- 100% of the total sites planned for each toxicity indicator (Table III

- 1). For some

strata, the number of samples collected exceeded the nominal number planned; this situation

resulted from the inclusion of 10

- 20% more stations in the sampling plan in order to compensate

for the inability to collect samples at some stations. Shallow and mid

- depth o ffshore locations

were nearly all collected successfully. The lowest completion rate was near river mouths and in

areas of San Diego Bay, where the presence of coarse sediments hindered grab operations.

TABLE III

- 1. Bight’98 toxicity sample collection success.

Amphipod QwikSed & Microtox P450 HRGS

Strata Number

Sampled Percent

of Target Number

Sampled Percent of

Target Number

Sampled Percent of

Target

Offshore

River Mouths 31 70 1 100 31 70

Shallow 33 100 13 100 33 97

Mid

- d epth 34 100 21 100 34 100

POTW 30 100 30 100 48 73

Bays/Harbors

Marina 39 100 39 100 39 100

Ports/Industrial 37 100 37 100 37 100

Other 37 95 37 95 37 95

Historically

Sampled 13 100

TOTAL 241 98 178 100 272 92



### B. Sam **ple Storage**

An objective of 14 d of storage time between collection and test initiation was established

for the amphipod and QwikSed tests. This guideline was met for 89% of the amphipod test

samples (Table III

- 2). Samples for QwikSed were usually stor ed for a longer period of time.

Only 31% of the QwikSed samples were tested within two weeks, but all of the samples were

tested within two months. Delays in testing were due to two factors: (1) the rapid collection of

sediment samples by multiple agenc ies, which overwhelmed laboratory capacity; and (2) the

need to retest some QwikSed samples that failed test performance criteria. The impact of

extended sample storage time on the QwikSed results has not been documented, but analysis of

the data indicate s that the effects, if any, were minor.

### 15

Microtox analysis of interstitial water samples was completed within the six

- month

interval established by the Toxicology Committee. An extended holding time was needed for

this test in order to accommodate other laboratory activities. The planned storage conditions

were not maintained for these water samples, however. Interstitial water samples were held

under refrigeration after extraction from the sediments, instead of being frozen, for

approximately four mon

ths until they were transported to the testing laboratory. The samples

were then frozen for up to two months and then thawed for analysis. A post

- survey study

conducted to evaluate the effect of extended sample storage indicated that prolonged storage

un der refrigeration produced significant and unpredictable changes in Microtox results

(Appendix C). The Microtox data were therefore declared invalid and were not used in assessing

sediment toxicity.

Sediment samples for HRGS testing were stored frozen f or approximately 12 months

before extraction. While this period is longer than the 6 months specified by the Toxicology

Committee, the types of compounds detected by this method (high molecular weight PAHs,

coplanar PCBs, dioxins, furans) have demonstrate d stability under frozen conditions. A sample

storage study conducted during Bight’98 indicated that concentrations were stable during the

storage period. The HRGS tests were completed and the data were reported within three months

of extraction, within the objective specified in the Bight’98 Quality Assurance Plan.

### 16

TABLE III

- 2. Bight’98 toxicity sample holding time.

Time Amphipod QwikSed Microtox P450 HRGS

Number Percent Number Percent Number Percent Number Percent



### ≤ 2 weeks 214 89 55 31

3 weeks 27 11 40 23

2 months 83 46

6 months 86 100

9

- 12 months 272 100

**C. Test Performance**

Amphipod Survival

All of the 241 samples collected were successfully tested. The mean contr ol survival rate

was 96% (values ranged from 75

- 100%).

All but two of the amphipod sediment toxicity tests met all necessary acceptability

criteria. The two cases where test performance criteria were not met were judged to have little

influence on the outcome of the test. In one case, one control replicate had a percent survival of

75%, less than the 80% minimum specified in the protocol. In the other case, the experiment

was terminated 1 d early (after 9 d). Examination of the results for both of t hese tests indicated

that the outcome of the test was unlikely to have changed had all of the test criteria been met.

Consequently, these tests were not repeated and the data were used in this report.

Water quality parameters (pH, dissolved oxygen [DO], salinity, and ammonia) were

generally within acceptable limits (U.S. EPA 1994) . The desired range for salinity was 20 ±3

g/kg. Salinity in the overlying water ranged from 18

- 27 g/kg during the Bight’98 tests.

Interstitial water salinit y ranged from 17

- 35 g/kg. Although above the desired limits, the test

salinities were within the tolerance range of E. estuarius (1

- 35 g/kg). Temperatures in overlying

water ranged from 13.5

- 16.7 ° C and were within the desired range of 15 ±3 ° C. In over lying

water, pH ranged from 7.01

- 9.63, while DO ranged from 4.4

- 10.8 mg/L. Interstitial water pH

and DO ranged from 6.79

- 8.20 and 3.9

- 8.2, respectively.

The concentration of unionized ammonia in the overlying water ranged from 0.00 to 0.49

mg/L, exce

pt for a single value of 1.33 mg/L that exceeded the ammonia acceptability criterion

of 0.8 mg/L. The high value was measured at the end of the experiment, so no corrective action

could be taken.

A total of 19 reference toxicant tests with cadmium were conducted by the laboratories

during the survey. The purpose of reference toxicant tests is to determine whether test organism

### 17

response and test procedures are comparable among different testing periods and laboratories.

Reference toxicant test results i ndicated that the laboratory test organisms and individual

laboratory performances were similar. The mean LC50 values from participating laboratories

ranged from 5.20

- 8.56 mg/L (with a mean value of 6.74). Control survival ranged from 90

- 99%

(with a mean survival rate of 96%). All laboratories submitted acceptable reference toxicant

results and all values were within two standard deviations of the calculated mean LC50 (Figure

III

- 1).

### 18 Laboratory 1 1 1 1 1 2 3 3 3 4 5 5 5 6 6 6 6 7 7EC50 (mg Cd/L)



### 0 5 10 15

+2 SD

Mean

-2 SD



### FIGURE III

- 1. Summary of cadmium reference toxicant test resul ts for amphipod

survival tests. Dashed lines indicate the value corresponding to two standard

deviations from the mean.

### 19

QwikSed

Most toxicity tests met the acceptability requirements established for the respective test.

Out of a total of 178 sedim ent samples collected, 173 met the appropriate American Society for

Testing and Materials (ASTM) 1998 criteria (ASTM 1998) . The failed tests had poor survival

of the dinoflagellates in the control samples. Additional tests showing poor control performance

(less than 1 million counts per 30 seconds) were re

- run several days later. A total of 53 samples

were retested either because of low controls and/or poor test precision.

A copper sulfate reference toxicant was analyzed three times d uring the testing period

and once after completion of the analyses. All test results fell within two standard deviations of

a mean IC50 of 156 ug/L (Figure III

- 2). These results indicate that the sensitivity of the

laboratory culture of G. polyedra did n ot change throughout the sediment test period.

Elutriate pH, temperature, and salinity were within acceptable ranges for all tests.

Temperature was maintained at 19 ±1° C and the pH of 100% elutriates ranged from 7.32

- 8.74.

Salinity ranged from 32

- 35 g /kg.

An increase in elutriate ammonia concentration with storage time was observed (Figure

III

- 3). Samples stored longer than 30 d tended to have higher ammonia concentrations. Most of

the 100% elutriate samples contained unionized ammonia concentrati ons above 0.04 mg/L, the

level associated with toxic effects (Figure III

- 4). Two actions were taken to minimize the

influence of ammonia toxicity on the results. First, data from only the 25% elutriate

concentration, which contained one

- fourth of the amm onia concentration, were evaluated in the

study. In addition, all data were corrected for ammonia toxicity by increasing the luminescence

value by the amount that was lost due to ammonia. The regression procedure used to perform

the correction is describ ed in Section II (Methods).

Correction for ammonia toxicity had a minor impact on most of the data for the 25%

elutriate. Little or no change was observed for 80% of the samples (Figure III

- 5). However,

correction increased the measured value by more t han 10 percentage points in 16 stations

(approximately 10% of the stations tested). Fourteen stations were reclassified as a result of the

correction, either from moderately toxic to nontoxic or from highly toxic to moderately toxic.

Most of the reclassi

fied stations were located within bays and harbors (Figure III

- 6); eight of the

reclassified stations were located in San Diego Bay.

### 20 Date of TestCopper IC50 (μg/L)



### 0 50 100 150 200 250

+ 2SD

Mean

- 2SD



### 8/25/98A 8/25/98B 9/16/98 1/8/99



### FIGURE III

- 2. QwikSed reference toxicant results. Symbols represent copper

reference toxicant samples analyzed dur ing or after batches of Bight’98 samples.

Dashed lines indicate the value corresponding to two standard deviations from

the mean.

### 21 

# Storage Time (days) 0 10 20 30 40 50 60Unionized Ammonia (mg/L)



## 0.0 0.1 0.2 0.3 0.4 0.5

FIGURE III

- 3. Concentration of ammonia in elutriate samples stored prior to

QwikSed analysis. Values are the mean of a ll samples stored for the indicated

number of days.

FIGURE III

- 4. Dose

- response plot of ammonia effects on G. polyedra with

regression curve.



## 0 20 40 60 80 100 120

0 0.1 0.2 0.3 0.4 0.5



## Unionized Ammonia (mg/L)Bioluminescence (% of Control)

### 22 

## Change in Luminescence (Percent of Control) 0 10 20 30 40 50 60Percent of Samples

0 20 40 60 80 100



### FIGURE III

- 5. Influence of ammonia correction procedure on QwikSed

luminescence value.



## Stratum Shallow Mid-depth POTW Ports Marinas Other Harbor

River MouthsNumber of Samples



### 0 1 2 3 4 5 6



### FIGURE III

- 6 . Reclassification of stations resulting from correction of QwikSed

results for ammonia toxicity. All changes reflected a reduction in the severity of

effect.

### 23

P450 HRGS

The HRGS assay was performed on 12 different days. The mean response to the

re ference inducer, TCDD, was a fold induction of 106.3 with a coefficient of variation of 16%

(Figure III

- 7). This response was always within the control chart acceptable limits of 55

- 151,

based upon prior data. The coefficient of variation among duplicate sediment extracts was

always less than 10%.

In three cases, (Stations 2263, 2251, and 2360) the HRGS fold induction response to the

samples was higher than 100, which was beyond the linear range of the assay. These samples

were diluted 1:10 in DCM and retested on the following test date. The fold induction response to

the diluted sample was acceptable (less than 100) in each case and the data were included in the

database.

Five samples of the SMB reference material were tested to examine the combined

variability of the extraction and test procedure. Results for four of the samples had low

variability, with HRGS responses of 96

- 170 μ g B[a]PEq/g (Table III

- 3). The HRGS results were

approximately three times higher for the last sample tested (387 μ g B[ a]PEq/g). Since the

reference toxicant results (Figure III

- 7) indicated that the assay was performing normally during

analysis of the last SMB extract, it is assumed that non

- homogeneity of the sediment was the

cause of this high variation.

### 24 Date of TestFold Induction



### 0 50 100 150 200

+ 2SD

Mean

- 2SD

5/3 5/7 5/11 5/21 5/24 5/27 6/8 6/11 6/15 6/18 6/22 7/20



### FIGURE I II

- 7. Response of the P450 HRGS assay to the reference inducer (TCDD)

over the testing period for Southern California Bight’98 samples. Dashed lines

indicate the value corresponding to two standard deviations from the mean. All

tests were conducted in 1 999.

TABLE III

- 3. The HRGS assay response to Santa Monica Bay reference sediment.

B[a]PEq

Analysis Date Dilution ( μ g/g)

3

- 31

- 99 1:1 104.4

4

- 7

- 99 1:10 95.8

4

- 9

- 99 1:1 169.5

4

- 13

- 99 1:1 139.9

4

- 19

- 99 1:10 386.7

Mean 179.3

Standard

Dev iation 119.6

### 25 **IV. DESCRIPTIVE RESULTS**

**A. Amphipod Survival**

Among 241 sediment samples collected throughout the SCB, 42 (17% of the total)

showed some degree of toxicity (Table IV

- 1, Figure IV

- 1). Eight samples (3%) were classified

as highly tox

ic (survival less than 50% of the control value), while 34 samples (14%) were

classified as moderately toxic (survival less than 80% of the control).

Amphipod survival was closely related to proximity to bays and harbors, where 25% of

the samples were cl

assified as toxic. All of the highly toxic sediments detected in this study

were collected from within harbors, or rivers that drain into harbors (i.e., the Los Angeles River).

Within harbors (Table IV

- 1), samples from marinas had the highest frequency o f toxicity (38%).

Samples from port and industrial areas were toxic in 22% of the cases. Finally, 14% of samples

collected from other areas of bays and harbors were toxic to amphipods. For many harbors,

samples collected in inner areas were more toxic t han those collected near entrances (Appendix

E).

The shallow stratum had the lowest percentage of toxic samples (3%). Samples from

POTW outfall areas were toxic in 6% of the cases, while 13% of samples taken near river

mouths produced a toxic response . The mid

- depth stratum had the highest frequency of toxicity

among the offshore strata, with 21% of sites showing moderate toxicity to amphipods.

Among individual harbors (Table IV

- 2), the largest number of highly toxic samples (5)

came from Newport B

ay, and the largest number of moderately toxic samples (9) came from San

Pedro Bay (including the Los Angeles River). Newport Bay was the only harbor in which toxic

samples exceeded nontoxic samples (9 toxic versus 2 nontoxic, or 82% toxicity). No toxici ty

was found in Dana Point Harbor, Mission Bay, or Ventura Harbor, although sampling was less

extensive in these areas (1

- 3 samples each). San Diego Bay sediments also showed relatively

infrequent toxicity (11%).

Since the numbers of toxic versus nontoxi c samples do not indicate the magnitude of

toxicity among samples, comparisons of strata by the mean percent of controls is also presented

for each toxicity classification (Table IV

- 1). For highly toxic samples, one sample from the Los

Angeles River produ

ced the lowest survival rate (7% of the control), followed by one sample

from a ports/industrial area (16%), followed by the average of six marina samples (29%).

Among moderately toxic samples, mean percents of controls were similar (57

- 73%), as were the

nontoxic samples (91

- 98%). Within the SCB as a whole, the mean percent of control for highly

toxic sediments was 22%, for moderately toxic sediments 68%, and for nontoxic sediments 93%.

### 26

TABLE IV

- 1. Sediment samples toxic to amphipods from seven strata i n the

Southern California Bight.

Highly Toxic Moderately Toxic Nontoxic

No. % Mean 95% CI No. % Mean 95% CI No. % Mean 95% CI

Offshore

Shallow 0 0 na na 1 3 68 na 32 97 98 96

- 99

Mid

- Depth 0 0 na na 7 21 73 71

- 7 6 27 79 93 90

- 96

River Mouths 1 3 7 na 3 10 64 60

- 67 27 87 96 94

- 98

POTW Outfall Areas 0 0 na na 2 7 57 na 28 93 97 95

- 100

Bays/Harbors

Ports/Industrial 1 3 16 na 7 19 73 67

- 80 29 78 90 87

- 92

Marinas 6 15 29 18

- 41 9 23 68 61

- 74 24 62 94 90

- 98

Other 0 0 na na 5 14 65 54

- 76 32 86 91 89

- 94

All Stations 8 3 22 16

- 28 34 14 68 66

- 71 199 83 93 92

- 94

Shallow Mid-Depth River POTW Ports/Industrial Marinas Other Harbor All StationsPercent of Samples

0 5 10 15 20 25 30 35 40

Highly Toxic

Moderately Toxic



### FIGURE IV

- 1. Percent of sediment samples toxic to amphipods from seven strata

in the Souther n California Bight.

### 27

TABLE IV

- 2. Sediment samples toxic to amphipods from nine harbors and bays in

the Southern California Bight. San Pedro Bay includes stations located near the

Los Angeles River and the Terminal Island Wastewater Treatment Plant.

High ly Toxic Moderately Toxic Nontoxic

Number Percent Number Percent Number Percent



### Ventura Harbor 0 0 0 0 1 100

Channel Islands Harbor 1 33 1 33 1 33

Marina del Rey 0 0 3 43 4 57

San Pedro Bay 2 4 9 20 34 76

Anaheim Bay 0 0 1 33 2 67

Newport Bay 5 45 4 36 2 18

Mission Bay 0 0 0 0 3 100

Dana Point Harbor 0 0 0 0 3 100

San Diego Bay 0 0 5 11 41 89

All Harbors 8 7 23 19 91 74

### 28

**B. QwikSed**

Results for the 25% elutriate concentration were used to describ e the magnitude and

spatial patterns of toxicity to G. polyedra . This concentration was selected in order to provide a

conservative measure of toxicity that would minimize the effects of interferences from

noncontaminant factors such as ammonia. Toxicity results were subdivided into three

categories: highly toxic (<50% of controls), moderately toxic (51

- 83% of controls), and nontoxic

(>84% of controls).

Out of a total of 173 samples tested, 37 samples (20%) were classified as toxic (11 highly

toxic an d 26 moderately toxic). All toxic samples were located within POTW or bay/harbor

strata. Seventy

- nine percent of all samples tested were classified as nontoxic (Table IV

- 3).

Four (14%) of the samples from the POTW stratum were classified as highly to xic

(Figure IV

- 2). No toxic samples were present within the shallow and mid

- depth strata, which

included many of the stations offshore of Ventura and Santa Barbara counties that were found to

be toxic to amphipods (Appendix D).

Of 116 sediment samples collected from bays/harbors, 7 samples (6%) were classified as

highly toxic and another 25 samples (22%) were classified as moderately toxic. The most

prevalent stratum of toxicity was marina areas, where 36% of the stations were classified as

moderately

or highly toxic. The QwikSed test classified 84% of all harbor/bay samples from the

SCB as nontoxic (Table IV

- 4).

Within individual harbors, 1 out of 7 samples in Marina del Rey was classified as highly

toxic, while 1 out of 42 samples in San Pedro B ay, 2 out of 11 samples in Newport Bay, and 3

out of 43 samples in San Diego Bay were also classified as highly toxic (Table IV

- 4). Forty

- five

percent of the samples (5 out of 11) collected in Newport Bay and 28% of the samples (13 out of

43) collected in

San Diego Bay were moderately toxic.

### 29

TABLE IV

- 3. Sediment samples toxic to QwikSed from seven strata in the

Southern California Bight.

Highly Toxic Moderately Toxic Nontoxic

No. % Mean 95% CI No. % Mean 95% CI No. % Mean 95% CI

Offshore

Shallow 0 0 na na 0 0 na na 13 100 98 95

- 101

Mid

- Depth 0 0 na na 0 0 na na 21 100 98 97

- 100

River Mouths 0 0 na na 0 0 na na 1 100 100 na

POTW Outfall Areas 4 14 7 0

- 21 1 4 51 na 23 82 98 96

- 100

Bays/Harbors

Ports/Industrial 0 0 na na 5 14 79 76

- 82 30 86 97 95

- 99

Marinas 3 8 39 20

- 60 11 28 76 73

- 81 25 64 98 96

- 99

Other 4 11 29 5

- 54 9 25 66 60

- 73 23 64 98 96

- 99

All Stations 11 6 11 0

- 22 26 15 64 55

- 74 136 79 98 97

- 100

Shallow Mid-Depth River POTW Ports/Industrial Marinas Other Harbor All StationsPercent of Samples

0 5 10 15 20 25 30 35 40

Highly Toxic

Moderately Toxic



### FI GURE IV

- 2. Percent of sediment samples toxic to QwikSed from seven strata in

the Southern California Bight.

### 30

TABLE IV

- 4. Sediment samples toxic to QwikSed from nine harbors and bays in

the Southern California Bight. San Pedro Bay includes stations located near the

Los Angeles River and the Terminal Island Wastewater Treatment Plant.

Highly Toxic Moderately Toxic Nontoxic

Number Percent Number Percent Number Percent



### Ventura Harbor 0 0 0 0 1 100

Channel Islands Harbor 0 0 1 33 2 67

M arina del Rey 1 14 0 0 6 86

San Pedro Bay 1 2 4 10 37 88

Anaheim Bay 0 0 1 33 2 67

Newport Bay 2 18 5 45 4 36

Mission Bay 0 0 1 33 2 67

Dana Point Harbor 0 0 0 0 3 100

San Diego Bay 3 7 13 30 27 63

All Harbors 7 6 25 22 84 72

### 31

**C. P450 HRGS**

The HRGS assay identified markedly elevated concentrations of CYP1A1

- inducing

compounds (PAHs, coplanar PCBs, dioxins, and furans) in 30 of 259 samples (11%) tested from

the seven strata (Table IV

- 5). Responses in 14 of the toxic s amples were classified as highly

toxic ( ≥ 60 μ g/g B[a]PEq), while the remainder contained 32

- 59 μ g/g B[a]PEq and were

classified as potentially toxic. From previous NOAA studies (Anderson et al. 1999) , it appears

that sediments producing a

response of 11 μ g/g B[a]PEq or less would not cause biological

effects, but insufficient data were found to determine whether levels between 11 and 32 μ g/g

B[a]PEq would produce any impacts on the biota.

Few of the toxic stations were located in the off shore or river mouth strata. Potential

toxicity was detected in 4 samples (8%) from POTW outfall areas. Toxicity was detected in 1

- 3

stations from each of the other offshore strata. A very high response (903 μ g/g B[a]PEq) was

measured in 1 sample, locat ed near Coal Oil Point in Santa Barbara County. This response was

more than four times greater than the highest concentrations measured in bay/harbor area

samples ( ≤ 182 μ g/g B[a]PEq).

Ports/industrial areas contained the largest number of toxic stations ; 36% of the stations

within this stratum were classified as toxic (Figure IV

- 3), with the majority of these stations

classified as highly toxic. A lower frequency of toxicity was detected in marina areas (15%) and

no samples from other locations within b ays/harbors contained toxic concentrations of inducing

compounds.

Aside from the extremely high concentration in the Coal Oil Point sample, the largest

individual HRGS responses were measured in samples from port/industrial areas in San Pedro

Bay and San Diego Bay. Mean concentrations of HRGS

- inducing compounds in each

classification were not significantly different among strata (Table IV

- 5). Stations classified as

highly toxic in each stratum contained an average of 88

- 115 μ g/g B[a]PEq, while potentiall y

toxic stations contained an average of 35

- 52 μ g/g B[a]PEq.

Toxic samples were located in five of nine individual bays or harbors (Table IV

- 6). San

Diego Bay contained the largest number of toxic stations, with 11 of 46 (24%) stations from this

bay bei ng classified as potentially or highly toxic. Relatively high percentages of toxic stations

were also identified for Marina del Rey (43% of 7 stations) and San Pedro Bay (16% of 45

stations). Only 1 of the 11 stations in Newport Bay was found to be toxic

.

### 32

TABLE IV

- 5. Sediment samples toxic to HRGS from seven strata in the Southern

California Bight.

Highly Toxic Potentially Toxic Nontoxic

N % Mean 95% CI N % Mean 95% CI N % Mean 95% CI

Offshore

Shallow 1 3 90 na 0 0 na na 32 97 4 3

- 5

Mid

- Depth 2 6 490 na 1 3 35 na 31 91 8 6

- 10

River Mouths 1 3 88 na 2 6 44 na 28 90 4 3

- 6

POTW Outfall Areas 0 0 na na 4 8 53 47

- 61 44 92 8 6

- 9

Bays/Harbors

Ports/Industrial 8 22 112 80

- 144 5 14 42 30

- 54 24 65 17 16

- 21

Marinas 2 5 90 na 4 10 43 32

- 55 33 85 12 10

- 14

Other 0 0 na na 0 0 na na 37 100 10 9

- 12

All Stations 14 5 392 0

- 897 16 6 39 32

- 46 229 88 7 5

- 8

Shallow Mid-Depth River POTW Ports/Industrial Marinas Other Harbor All StationsPercent of Samples



### 0 5 10 15 20 25 30 35 40

Highly Toxic

Potentially Toxic



### FIGURE IV

- 3. Percent of sediment samples toxic to HRGS from seven s trata in the

Southern California Bight.

### 33

TABLE IV

- 6. Sediment samples toxic to HRGS from nine harbors and bays in the

Southern California Bight. San Pedro Bay includes stations located near the Los

Angeles River and the Terminal Island Wastewater Tre atment Plant.

Highly Toxic Potentially Toxic Nontoxic

No. Percent No. Percent No. Percent



### Ventura Harbor 0 0 0 0 1 100

Channel Islands Harbor 0 0 0 0 3 100

Marina del Rey 1 14 2 29 4 57

San Pedro Bay 3 7 4 9 38 84

Anaheim Bay 0 0 0 0 3 100

Newport Bay 1 9 0 0 10 91

Mission Bay 0 0 0 0 3 100

Dana Point Harbor 0 0 0 0 3 100

San Diego Bay 6 13 5 11 35 76

All Harbors 11 9 11 9 100 82

### 34 

## V. COMPARISON OF INDICATORS

Pairwise comparisons of toxicity classifications based u pon the amphipod, QwikSed, and

HRGS test results indicated that the tests were in agreement for most of the samples (Figure V

1). The amphipod survival and HRGS induction tests had the highest percentage of agreement,

with 78% of samples classified as non toxic or toxic by both methods. Classifications based upon

the QwikSed test had a similar level of agreement (69%) with either the amphipod or HRGS test

results.

Most of the agreement among tests occurred in the designation of samples as nontoxic.

Re latively few samples were classified as toxic by any two test methods. Approximately 18

- 26% of the samples identified as toxic based upon amphipod survival were also classified as

toxic by the HRGS or QwikSed tests, respectively (Figure V

- 2). Agreement o n toxicity

classification was lower between HRGS and QwikSed (8%).

When toxicity was present in a sample, the magnitude of response was variable among

the three test methods (Figure V

- 2). Relatively few samples produced a strong toxic response in

any two tests. No samples were identified as toxic by all three methods. Fifty

- six percent of the

samples were identified as nontoxic by all three methods, however.

A weak significant correlation was found between the magnitude of response for

amphipod surviv al and HRGS induction (Figure V

- 2). This correlation had a negative sign,

indicating the expected trend of reduced amphipod survival in sediments containing elevated

contaminant concentrations. No correlation was present between QwikSed and either amphip od

or HRGS results.

Stronger correlations were present between sediment total organic carbon (TOC) or grain

size (percent fines) and toxicity (Appendix F). Amphipod survival was significantly correlated

with TOC (r =

- 0.49) and grain size (r =

- 0.56). Although survival tended to be lower in some

organically enriched or fine sediments, many of the samples containing the highest TOC

concentrations or the finest sediments were classified as nontoxic. The HRGS test responses

were also found to be signific antly correlated with increased TOC (r = 0.70) or increased

sediment fines (r = 0.53). The QwikSed test was not significantly correlated with either

sediment parameter.

### 3 5 75% Both Toxic

Neither Toxic



### Amphipod vs. P450

3%

P450 Only

8% Amphipod Only

14% 

### QwikSed vs. P450

1% P450 Only

11%

QwikSed Only

20%

68% 

### Amphipod vs. QwikSed

Amphipod Only

14%

5%

QwikSed Only

16%

64%



### FIGURE V

- 1. Results of toxicity classifications for each pair of toxicity tests.

Each pie diagram shows the percentage of stations for which both tests yield

either the same result (either toxic or not toxic) or disagree (only one test

detected toxicity).

### 36

FIGURE V

- 2. Comparison of indi cator responses for sediment samples. Dashed

reference lines indicate toxicity classification thresholds. The P450 plots do not

include one high sample (903 m g/g). Asterisks indicate a significant correlation. 0 20 40 60 80 100 120

0 10 20 30 40 50 60 70 80 90 100 110

QwikSed Response (% of Control)Amphipod Survival (% of Control)

Toxic to

Amphipods

Only Toxic to QwikSed Only Nontoxic

to Both

Toxic to Both

r = -0.018

0 20 40 60 80 100 120

0 20 40 60 80 100 120 140 160 180 200

P450 Response (ug B(a)P Eq/g)Amphipod Survival (% of Control)

Toxic to

Amphipod

s Toxic to

P450 Only Nontoxic

to Both

Toxic to Both

r = -0.285**

0 20 40 60 80 100 120

0 20 40 60 80 100 120 140 160 180 200

P450 Response (ug B(a)P Eq/g)QwikSed Response (% of Control)

T oxic to

QwikSed

Only Toxic to

P450 Only Nontoxic

to Both

Toxic to

Both

r = -0.081Amphipod Survival (% of Control

### 37 **VI. REGIONAL ASSESSMENT OF TOXICITY**

Sedim ents from most areas of the SCB were of good quality. Sediments were nontoxic in

85

- 99% of the area studied, depending upon the indicator used (Figure VI

- 1). The amphipod test

detected the largest area of toxicity, 504 km 2 

### (Table VI

- 1). The QwikSed test identified the

smallest area of toxicity, 48 km 2



### .

Each of the indicators were consistent in detecting the smallest extent of toxicity in the

shallow offshore stations. The amphipod and HRGS tests each identified 3% of the shallow area

as toxic, which

represented a single station, while no toxicity was found using the QwikSed test.

Estimates of the spatial extent of toxicity varied among indicators for the other strata, with no

single indicator being most sensitive for all strata (Figure VI

- 2).

St rata located within bays and harbors contained the largest relative area of toxicity

compared to offshore strata for each indicator. The amphipod and HRGS tests detected the

highest percentage of toxic area within the port/industrial (20 and 38%, respecti vely) and marina

strata (38 and 12%, respectively), while the QwikSed test identified the largest toxic area within

the marina (38%) and other category strata (31%). The tests identified toxicity in 6

- 23% of

POTW outfall areas and in 9

- 12% of the areas ne ar river mouths. Areal estimates of toxicity

within the mid

- depth stratum varied markedly between indicators, from 0% (QwikSed) to 21%

(amphipod). The incidence of amphipod toxicity in mid

- depth areas represented 390 km 2



### , accounting for most of the diffe rence in bight

- wide estimates of toxic area relative to QwikSed

and HRGS.



## Indicator

Amphipod QwikSed HRGSPercent of Area



### 0 20 40 60 80 100

FIGURE VI

- 1. Percent of Southern California Bight not toxic to each indicator.

### 38

TABLE VI

- 1. Percent of area in each stratum classified as toxic by indicator type.

Area Amph ipod QwikSed HRGS

Stratum Km 2 

### Moderate High Moderate High Potential High

Shallow 1,066 3 0 0 0 0 3

Mid

- depth 1,838 21 0 0 0 3 6

River Mouths 165 10 3 na na 6 3

POTW Outfall

Areas 186 6 0 5 18 8 0

Ports/Industrial 27 18 2 15 0 15 23

Marinas 17 21 17 31 7 8 4

Other 108 13 0 22 10 0 0

SCB 3,407 14.3 0.1 0.8 0.6 2.6 4.9



# Stratum Shallow Mid-depth River POTW Ports/Industrial Marinas Other HarborPercent of Area



## 0 20 40 60 80 100

Amphipod

QwikSed

HRGS



## *

FIGURE VI

- 2. Percent of area (+ 95% confidence interval) found to be toxic in

each stratum. *Only one sample from river s tratum analyzed for QwikSed.

### 39

The results for each of the three toxicity indicators were combined to provide an

integrated assessment of sediment quality. The results were weighted relative to the severity of

response and perceived ecological relevance of the indicator. The greatest weight was given to

the amphipod survival results because the amphipod test used test conditions that were most

similar to the habitat of interest (benthic species, direct sediment exposure) and a response with

high biologic al significance (survival). Lesser weight was assigned to the QwikSed and HRGS

test results because these methods used sublethal responses to a test matrix that had been

manipulated by elutriation or extraction procedures. The weighted results were combi ned to

classify each station into one of three levels of concern (Table VI

- 2).

The high concern category included stations where reduced survival of some benthic

animals was expected. All stations that were highly toxic to amphipods (<50% survival) wer e

classified as areas of high concern. Stations were also placed into this category if they produced

moderate amphipod toxicity and were also identified as toxic in either the QwikSed or HRGS

test. If the QwikSed and P450 tests were classified as highly t oxic, a station was defined as an

area of high concern even though the amphipod test showed no toxicity.

Areas of potential concern included stations where a less severe (sublethal) toxic

response was measured or the results were inconsistent between th e survival and sublethal tests.

Stations in this category represented areas where marginal effects may be present, resulting in

greater uncertainty about the significance of impaired sediment quality.

This category included stations that were usually no t toxic to amphipods but produced

some evidence of toxicity in at least one of the other two bioassays. Areas of no concern

demonstrated no amphipod toxicity and either no toxicity or inconsistent evidence of a moderate

toxic response in the other two tes ts.

The integrated classification strategy was applied to the 241 stations that were assessed

by both the amphipod and HRGS tests. QwikSed test results were not available for 68 of these

stations; a nontoxic QwikSed response was assigned to them for the purpose of applying the

classification strategy.

Areas of potential or high concern were present in each of the strata and comprised 19%

of the area (644 km 2 

### ) of the SCB (Figure VI

- 3). Most of the affected area was classified in the

potential concern category; only 2.7% of the SCB (92 km 2 

### ) was classified in the high concern

category. Among strata, port/industrial and marina areas had the largest total percent of area in

the two categories of concern, 39 and 40%, respectively. Marinas contained the la rgest relative

area and most severe effect, with 27% of the area classified in the high concern category. Areas

of high concern were also present in 10% of port/industrial areas and 9% of river areas.

The spatial extent of toxicity was intermediate amo ng the mid

- depth, POTW, and the

other bays and harbors area strata (22

- 24% of area). Shallow areas were least affected, with only

6% of the area classified in the potential concern category.

### 40

TABLE VI

- 2. Strategy for determining levels of concern from results of three types

of sediment toxicity tests.

Amphipod Toxicity

QwikSed Toxicity HRGS Toxicity 

### High Moderate Nontoxic

High High Concern High Concern High Concern

High Potential High Concern High Concern Potential

Concern

Nontoxic High Concer n High Concern Potential

Concern

High High Concern High Concern Potential

Concern

Moderate Potential High Concern High Concern Potential

Concern

Nontoxic High Concern High Concern No Concern

High High Concern High Concern Potential

Concern

Nontoxic Potential High Concern High Concern No Concern

Nontoxic High Concern Potential

Concern No Concern



# Stratum Shallow Mid-depth River POTW Ports/Industrial Marinas Other Harbor SCBPercent of Area



## 0 20 40 60 80 100

6% 24%

13% 22% 39% 40%

22% 19% High Concern

Potential Concern



### FIGURE VI

- 3. Percent of area of concern based upon joint toxicity classification.

### 41 **VII. DISCUSSION**

Sediment toxicity evaluations were successfully conducted using the amphipod,

QwikSed, and HRGS tests. These tests met the performance criteria specified in the Bight’98

Quality Assurance Plan for 98% of the samples. An interlaboratory comparison demonstrated

that the amphipod toxicity results were c omparable among laboratories.

The successful application of three toxicity indicators during Bight’98 has enabled two of

the objectives of the study to be attained: (1) estimation of the area of toxic sediments among

various strata of the SCB and (2) c omparison of the responses of different toxicity test methods.

The final objective, evaluation of relationships between toxicity and other indicators of sediment

quality, cannot be accomplished at this time because the analysis of chemistry and benthic

bi ology samples is still in progress. The results of this evaluation will be presented in a separate

report that integrates the results of all indicators.

The toxicity results indicate that the majority (81%) of the SCB had good sediment

quality in 1998 . Toxicity was generally most severe (greatest test response) and most prevalent

(largest percent of area affected) in developed areas of bays and harbors (port/industrial and

marina strata), a result that is consistent with previous studies in southern C alifornia (Anderson

et al. 1988, Fairey

et al. 1998) , and throughout the nation (Long 2000, in press ) . Greater

toxicity is to be expected in bays and harbors because these locations receive contaminant inputs

from many s ources (e.g., urban runoff, industrial spills, boating activity), which adsorb onto the

fine

- grained bottom sediments. Most bays and harbors are depositional environments, which

facilitate the retention and accumulation of contaminated sediments over time

.

The presence of toxicity was unambiguous in 3% of the SCB; these were identified as

areas of high concern in the integrated classification strategy because high amphipod mortality

was measured or multiple indicators detected toxicity. These areas were located primarily in

marinas, port/industrial areas, and near the Los Angeles River. Variable or weaker toxicity

results were obtained for a larger number of stations (classified as areas of potential concern),

representing 16% of the SCB and distributed among all of the strata. These areas would be

classified as toxic if the data were interpreted according to the criteria used in NOAA or EMAP

studies. The classification of these stations as areas of potential concern in this report reflects a

lower lev el of confidence in the ecological significance of the data, which is prompted by the

lack of corroborating data describing contaminant concentrations and benthic community

composition at these stations. These data are needed to provide the weight of evid ence necessary

to determine whether the toxicity results are ecologically significant or associated with

anthropogenic activities.

The identification of 379 km 2 

### of mid

- depth areas in the potential concern category,

primarily based upon toxicity to amphip ods, is in contrast to the 1994 SCBPP results that showed

no acute toxicity in the same region (Bay et al. 1998) . A different amphipod test species

( Ampelisca abdita

) was in the 1994 and differential sensitivity between these species may have

be responsible for some of the difference in results between studies. Recent studies in California

indicate that tests with E. estuarius are more likely to detect sediment toxicity than tests with A.

### 42

abdita (B. Anderson, pers. Comm.). Evidence of i nterstitial water toxicity in this area was

observed during the SCBPP, however. Most of these toxic Bight’98 mid

- depth stations were

located in Santa Barbara and Ventura counties, where large point sources of contaminants are

absent. Toxicity at these st ations may be related to the presence of numerous natural oil “seeps,”

or flows, in the area. The highest P450 HRGS response to sediments (903 μ g B[a]PEq/g) was

measured for a station located off of Coal Oil Point, a known seep area. Forthcoming sediment

chemical analysis results may help to reveal the influence of natural hydrocarbons on SCB

sediment toxicity.

The Bight’98 amphipod toxicity results for bays and harbors (13

- 38% of the area,

depending upon the stratum) fell within the range reported in previous studies by NOAA and the

State Water Resources Control Board. Studies conducted between 1992 and 1994 reported

toxicity in 66% of San Diego Bay (Fairey et al. 1998) , 14% of Los Angeles/Long Beach Harbor

(Long 2000

, in press ) , and 58% of other small bays and marinas (Anderson et al. 1997)

. Amphipod toxicity throughout the SCB (14.8% of the area) was higher than the national average

for estuarine areas of 5.9% reported by Long (2000).

A pronoun ced temporal difference in the extent of amphipod toxicity for San Diego Bay

was observed. Nine percent of San Diego Bay was toxic to E. estuarius in 1998, while tests with

a different species, Rhepoxynius abronius , detected toxicity in 66% of the bay (Fairey et al.

1998)

.

The use of different amphipod test species is a potential cause of the temporal differences

in toxicity results for San Diego Bay and the SCB. Most of the earlier tests were conducted with

either Rhepoxynius abroniu s or Ampelisca abdita . The relative sensitivity of E. estuarius and

these species has been studied to a limited extent. Sensitivity to single contaminants varies

between species in an unpredictable manner. For example, E. estuarius is less sensitive tha n R.

abronius to fluoranthene and cadmium (DeWitt et al. 1989) , yet is slightly more sensitive to

DDT. All three species have a similar sensitivity to crude oil (Weston 1996) . The sensitivity of

E. estuarius to contamina ted field sediments has been found to be similar to other amphipods

(DeWitt et al. 1989, Schlekat

et al. 1995) . Analyses of sediment toxicity data from many field

studies indicate that R. abronius and A. abdita respond similarly to sedime nts having comparable

contaminant concentrations (Field et al. 1999) . Thus, differences in contaminant sensitivity

between species may not be a significant factor in field studies, perhaps because the joint toxicity

of multiple contaminan

ts obscures species

- specific differences. Concurrent tests of southern

California sediments using multiple amphipod species are needed to determine whether the

choice of amphipod species has a significant influence on toxicity estimates for the SCB.

The HRGS test has been applied previously to bay and harbor sediments in southern

California and throughout the nation. The Bight’98 results indicate a greater prevalence of

samples with potential or high toxicity (7.2%) compared to studies of southern Calif ornia coastal

bays (0%) or studies throughout the nation (0

- 3.4%) as reported by Anderson et al. (1999) and

Long ( 2000, in press ) . The QwikSed test is a relatively new method that has not been applied in

other regional surveys.

### 43

A broad level of agreement was found among the three test methods (amphipod survival,

QwikSed, and HRGS). All of these tests were in agreement for most of the samples classified as

nontoxic and each test also identified the greatest toxicity in bay and harbor areas. However, the

test methods responded differently to most of the sediments where toxicity was present. This

conclusion is supported by the lack of strong correlations between the test results and the low

percentage of samples that wer e identified as toxic by more than one indicator. Such differences

are often observed in regional sediment toxicity studies, where samples with diverse physical and

chemical characteristics are evaluated (Long et al . 1998).

Differences in sediment tox icity test responses are to be expected, considering that each

of the test methods is likely to vary in sensitivity to specific contaminants. For example, the

HRGS test is sensitive to a select group of biologically active organics (dioxins, furans, PAHs,

and PCBs), but not to metals or pesticides. Sublethal tests using phytoplankton or invertebrate

larvae are often more sensitive to short

- term chemical exposure than are adult crustaceans

(Lapota et al . 1999). Such differences in test responses among ind icators is desirable in

sediment quality surveys as it indicates that each method is contributing new information about

the potential toxicity of the sample. The use of only one toxicity test method cannot provide a

complete measurement of the toxicity of a sample to the diversity of benthic organisms

inhabiting the SCB.

Some of the differences in toxicity test responses may also be due to undesired artifacts

or interferences related to the laboratory test procedure. Sample preparation steps required by

each test method, such as sediment homogenization, elutriate preparation, or solvent extraction,

can alter the partitioning and/or bioavailability of sediment contaminants and can lead to

different responses among tests. The relative role of artifactual versus biological variations in

producing the differences in Bight’98 test responses is difficult to determine at present, as

corresponding sediment chemistry and benthic community data have not yet been analyzed.

The diversity of responses obtained with multiple indicators presents a challenge for

those tasked with making an assessment of sediment quality, who must find a way to reconcile

conflicting responses from multiple indicators. The Bight’98 toxicity study used a relatively

conservative strat

egy that weighted the results based upon assumptions about the ecological

significance of each indicator’s response. The efficacy of this strategy for predicting impaired

sediment quality is not known at present, but will be evaluated through comparisons with benthic

community responses and sediment chemistry in subsequent Bight’98 regional monitoring

program reports. Until these comparisons have been made, our assessments of the categories of

concern should be viewed as preliminary.

### 44 **VIII. CONCLUSIONS**

The Bight’98 sediment toxicity study produced the most comprehensive regional

assessment of sediment quality in the SCB. This study marks the first time that offshore and

bay/harbor sediment quality has been assessed at the same time, using the same me thods. Two

recently developed test methods, QwikSed and P450 HRGS, were used for the first time in many

locations. The first interlaboratory comparison of sediment toxicity test results among southern

California laboratories was also conducted as part of Bight’98. Analysis of the results by the

Toxicology Committee, representing the participating laboratories and other survey partners, has

resulted in the following conclusions:

• **Sediment toxicity was evident in 19% of the sediments of the SCB study area.**

Sediment samples from stations representing 644 km 2 

### produced toxicity in at least one of

the tests. The majority (552 km 2 

### ) of this area was classified in the potential concern category,

representing areas where toxicity was less severe or inconsistentl y detected among the indicators.

Toxicity was more severe in sediments representing 2.7% of the SCB (92 km 2 

### ) that were

classified as areas of high concern; these sediments were almost exclusively located within

harbors and bays.

• **Sediments located with in bays and harbors or near river mouths contained the** 

**greatest severity of toxicity.**

While evidence of acute or sublethal toxicity was detected in every stratum sampled, each

indicator detected the strongest responses or the greatest extent of toxicity within bays and

harbors. The strongest toxic responses in the amphipod test (<50% survival) were produced by

sediments from marina, port/industrial, and river mouth areas. QwikSed toxicity was highest in

POTW outfall, marina, and other bay/harbor areas. Strong HRGS responses (>60 μ g/g

Benzo[a]pyrene equivalents, a level associated with degraded infauna) were most frequently

produced by sediments from port/industrial areas, although similar responses were produced by a

few stations in marina, river mouth, mid

- depth, and shallow areas. Each test method identified

shallow areas as having the lowest percentage of toxic area.

• **Different patterns of response were obtained for each toxicity indicator.**

While each of the tests produced a similar toxicity clas sification for most of the stations,

different response patterns were evident. Correlation analysis indicated only a weak relationship

between the amphipod and HRGS results for the same stations, and no relationship was found

between QwikSed and the other tests. A relatively low rate of agreement (8

- 26%) was observed

between pairs of tests in classifying a station as toxic; and no station was classified as toxic by all

three tests. These results indicate that each test method responded to different chara cteristics of

the sediment and provided new information about the sediment samples.

### 45

• **Amphipod toxicity test data were of high quality and comparable among all of the**

**participating laboratories.**

Each of the participating laboratories produced results tha t met test acceptability criteria.

An interlaboratory comparison demonstrated that the results from all seven participating

laboratories were usually not significantly different from one another. The variability in

reference toxicant results among the la boratories was the same as the within

- laboratory

variability. This result indicates the use of multiple laboratories did not result in increased

measurement error during the study.

### 46 **IX. RECOMMENDATIONS**

• **Determine the relationship between indicator threshold exceedence and benthic** 

**community effects.**

The choice of toxicity classification thresholds is usually based upon statistical

considerations (e.g., significantly different from control) or arbitrary numeric values (e.g., 50%

response). Such thr esholds provide an important element of standardization to toxicity

assessments, but their correspondence to biological impact is poorly understood. Acute toxicity

test responses are generally assumed to have high ecological relevance, but the significanc e of a

20% change in organism growth or metabolism during a toxicity test is more difficult to

determine. Once the Bight’98 benthic ecology data are available, analyses should be conducted

to determine the relationship between toxicity test response and b enthic effects. The results of

these analyses should be used to evaluate (and improve) the response thresholds and weighting

used to produce the toxicity assessments presented in this report. Understanding the predictive

ability of sediment toxicity will be beneficial to the planning of future monitoring activities.

• **Identify the cause of toxicity in bays/harbors and mid**

**- depth areas.** 

The greatest spatial extent and severity of toxicity was present in bays and harbors,

confirming the results of earlier t oxicity surveys. The persistent nature of sediment toxicity in

areas such as Newport Bay and San Pedro Bay indicates that sediment quality is not improving.

Sediment chemistry data should be analyzed and additional toxicity identification studies

conduct ed to determine the cause of toxicity in these areas so that potential sources can be

identified and load reduction/site cleanup activities effectively planned. Follow

- up toxicity

studies should also be conducted in the mid

- depth area of Santa Barbara and Ventura counties.

The presence of acute sediment toxicity in this area was not expected due to the absence of large

point source discharges and may be due to natural factors (e.g., oil seeps). Confirmation of the

toxicity results for this area may help elucidate some of the non

- anthropogenic factors causing

variability in test response, thus facilitating improved application and interpretation of toxicity

tests in other programs.

• **Investigate causes of non**

**- concordance between different sediment toxicity**  **tests.**

The variability in results between the different Bight’98 sediment toxicity test methods

creates uncertainty in the interpretation of the data. The cause of this variability is unknown,

although it is probably the result of several factors, such as species

- specific differences in

contaminant sensitivity and differences in contaminant exposure related to test sample handling.

The efficacy of these tests for predicting impaired sediment quality needs to be evaluated so the

results can be used appro priately in environmental management decisions. An essential first step

is to compare the toxicity results to the sediment contamination and benthic community data

among samples. Following this step, field and laboratory experiments should be conducted t o

evaluate issues such as contaminant sensitivity, test reproducibility, and the influence of non

contaminant sediment factors.

### 47

• **Conduct periodic regional surveys to assess temporal trends.**

The Bight’98 sediment toxicity study identified a larger amou nt of toxicity in offshore

strata and less toxicity in San Diego Bay compared to prior studies. The cause of these trends

has not yet been determined, but changes in sediment quality, natural variability, and differences

in test methods may all play a rol e. A long

- term database of sediment toxicity data using

comparable methodology is needed in order to differentiate between changes in sediment quality

and natural/measurement variability. Distinguishing between natural and anthropogenic effects

is needed to select appropriate management actions in response to the detection of toxicity. The

establishment of a long

- term sediment toxicity database will also permit the evaluation of the

correspondence between sediment toxicity, which measures present conditi ons, and benthic

community effects, which integrate effects over longer timescales. Future regional surveys

should continue to compare the performance of rapid and inexpensive assays, such as HRGS and

QwikSed. These methods provide cost

- effective alterna tives to conventional tests of

contamination and toxicity and could facilitate the collection of more spatially or temporally

extensive data. But additional study of these alternative methods is needed in order to evaluate

their relevance to ecological im pacts.

### 48 **X. LITERATURE CITED**

American Society for Testing and Materials (ASTM). 1998. E 1924

- 97 standard guide for

conducting toxicity tests with bioluminescent dinoflagellates. Biological Effects and

Environmental Fate; Biotechnology; Pesticides. Annual Book of ASTM Standards, Volume

11.06. West Conshohocken, PA.

Anderson, B., J. Hunt, S. Tudor, J. Newman, R. Tjeerdema, R. Fairy, J. Oakden, C. Bretz, C.

Wilson, F. LaCaro, G. Kapahi, M. Stephenson, M. Puckett, J. Anderson, E. Long, T. F leming,

and K. Summers. 1997. Chemistry, toxicity and benthic community conditions in sediments of

selected southern California bays and estuaries. Report to California State Water Resources

Control Board. Sacramento, CA. 141 pp. + Appendices

Anderson, J.W ., S.M. Bay, and B.E. Thompson. 1988. Characteristics and effects of

contaminated sediments from Southern California. Final Report to California State Water

Resources Control Board. Westminster, CA. 120 pp.

Anderson, J.W., J.M. Jones, J. Hameedi, E. Long, and R.H. Tukey. 1999. Comparative analysis

of sediment extracts from NOAA's bioeffects studies by the biomarker, P450 Report Gene

System. Marine Environmental Research 48:407

- 425.

Anderson, J.W., S.S. Rossi, R.H. Tukey, T. Vu, and L.C. Quattrochi. 1995. A biomarker, P450

RGS, for assessing the induction potential of environmental samples. Environmental Toxicology

and Chemistry 14:1159

- 1169.

Bay, S.M., D.J. Greenstein, A.W. Jirik, and J.S. Brown. 1998. Southern California Bight 1994

Pilot Project: VI. Sedi

ment toxicity. Southern California Coastal Water Research Project.

Westminster, CA. 56 pp.

Bight’98 Steering Committee. 1998. Southern California Bight 1998 regional monitoring survey

(Bight’98): Coastal Ecology Workplan. Southern California Coastal Water Research Project.

Westminster, CA. 67 pp.

DeWitt, T.H., R.C. Swartz, and J.O. Lamberson. 1989. Measuring the acute toxicity of estuarine

sediments. Environmental Toxicology and Chemistry 8:1035

- 1048.

Fairey, R., C. Roberts, M. Jacobi, S. Lamerdin, R. Clar k, J. Downing, E. Long, J. Hunt, B.

Anderson, J. Newman, R. Tjeerdema, M. Stephenson, and C. Wilson. 1998. Assessment of

sediment toxicity and chemical concentrations in the San Diego Bay region, California, USA.

Environmental Toxicology and Chemistry 17:1 570

- 1581.

Fairey, R.S., C. Bretz, S. Lamerdin, J.W. Hunt, B.S. Anderson, S. Tudor, C.J. Wilson, F. LaCaro,

M.D. Stephenson, H.M. Puckett, and E.R. Long. 1996. Chemistry, toxicity, and benthic

community conditions of selected sediments of the San Diego Bay region. State Water Resources

Control Board. Sacramento, CA.162 pp.

Field, L.J., D.D. MacDonald, S.B. Norton, C.G. Severn, and C.G. Ingersoll. 1999. Evaluating

sediment chemistry and toxicity data using logistic regression modeling. Environmental

Toxicolog y and Chemistry 18:1311

- 1322.

### 49

Long, E.R. 2000. Spatial extent of sediment toxicity in U.S. estuaries and marine bays.

Environmental Monitoring and Assessment

. in press.

Microbics Corp. 1995. Microtox Acute Toxicity Comparison & Inhibition Test. Microbi cs

Corporation. Carlsbad, CA. 30pp.

Schlekat, C.E., K.J. Scott, R.C. Swartz, B. Albrecht, L. Antrim, K. Doe, S. Douglas, J.A. Ferretti,

D.J. Hansen, D.W. Moore, C. Mueller, and A. Tang. 1995. Interlaboratory comparison of a 10

- day sediment toxicity test method using Ampelisca abdita, Eohaustorius estuarius and

Leptocheirus plumulosus

. Environmental Toxicology and Chemistry 14: 2163

- 2174.

Stevens, D.L., Jr. 1997. Variable density grid

- based sampling designs for continuous spatial

populations. Envirometrics 8: 167

- 195.

Stevens, D.L., Jr., and T.M. Kincaid. 1997. Variance estimation for subpopulation parameters

from samples of spatial environmental populations. Proceedings of the American Statistical

Association Section on Statistics and the Environment. Ame rican Statistical Association.

Alexandria, VA.

Thompson, B.E., J.D. Laughlin, and D.T. Tsukada. 1987. 1985 reference site survey.

Contribution 202. Southern California Coastal Water Research Project. Westminster, CA. 50 pp.

Thompson, B.E., J.D. Laughlin, and D.T. Tsukada. 1993. 1990 reference site survey.

Contribution Number 269. Southern California Coastal Water Research Project. Westminster,

CA. 105 pp.

Thursby, G.B., J. Heltshe, and K.J. Scott. 1997. Revised approach to toxicity test acceptability

crit eria using a statistical performance assessment. Environmental Toxicology and Chemistry

16:1322

- 1329.

U.S. Environmental Protection Agency (U.S. EPA). 1991. Evaluation of dredged material

proposed for ocean disposal (testing manual). Environmental Protecti on Agency. Washington,

DC.

U.S. Environmental Protection Agency (U.S. EPA). 1994. Methods for assessing the toxicity of

sediment

- associated contaminants with estuarine and marine amphipods. EPA/600/R

- 94/025.

Office of Research and Development, U.S. Enviro nmental Protection Agency. Narragansett, RI.

140 pp.

Weston, D.P. 1996. Further development of a chronic Ampelisca abdita bioassay as an indicator

of sediment toxicity. RMP Contribution #17. San Francisco Estuary Institute. Richmond, CA. 26

pp.

Word, J.Q. , A.J. Mearns, and D.A. McDermott. 1979. 60

- Meter control survey off southern

California. Contribution Number 133. Southern California Coastal Water Research Project.

Westminster, CA. 58 pp.

### A

- 1

**APPENDIX A. PARTICI PANTS IN THE SOUTHER**  **N CALIFORNIA BIGHT**

**199 8 REGIONAL MONITORIN**  **G PROGRAM (BIGHT ’98 ).**  **a** 

### Denotes

**participants in the sediment toxicity component.**

AES Corporation

Algalita Marine Research Foundation

Aliso Water Management Authority (AWMA)

Aquatic Bioassay and Consulting Laboratories, Inc.

Califor nia Coastal Conservancy

Central Coast Regional Water Quality Control Board

Channel Islands National Marine Sanctuary (CINMS)

Chevron USA Products Company

Cities and County of Riverside Stormwater Program

City of Long Beach

City of Los Angeles Environmental Monitoring Division (CLAEMD) a



### City of Los Angeles Stormwater Division

City of Oceanside

City of Oxnard

City of San Diego a



### City of Santa Barbara

City of Ventura

Columbia Analytical Services a



### Commission for Environmental Cooperation

Divers Involved Voluntar

ily in Environmental Rehabilitation & Safety (DIVERS)

Encina Wastewater Authority

Goleta Sanitation District

Houston Industries, Inc.

Instituto de Investigaciones Oceanologicas, Universidad Autonoma de Baja California

(UABC)

Los Angeles Department of Water and Power

Los Angeles County Department of Beaches & Harbors

Los Angeles County Department of Health Services

Los Angeles Regional Water Quality Control Board a



### Los Angeles County Sanitation Districts (LACSD)

Marine Environmental Consultants (MEC) a



### Marine Corps Base Camp Pendleton

National Fisheries Institute of Mexico (SEMARNAP)

NOAA

- NOS International Programs Office

NRG Energy, Inc.

Orange County Environmental Health Division

Orange County Public Facilities and Resources Department (OCPFRD)

Orange County Public Health Laboratory

Orange County Sanitation District (OCSD) a



### San Bernadino County Stormwater Program

San Diego County Dept. of Environmental Health

### A

- 2

**APPENDIX A (continued). Participants in the Southern California Bight 1998**

**Regional Monitoring Progra m (Bight ’98).**  **a** 

### Denotes participants in the sediment

**toxicity component.**

San Diego Interagency Water Quality Panel (Bay Panel)

San Diego Regional Water Quality Control Board

San Elijo Joint Powers Authority

Santa Ana Regional Water Quality Control Board

Santa Barbara Public Health Department

Santa Monica Bay Restoration Project

Southeast Regional Reclamation Authority (SERRA)

Southern California Coastal Water Research Project (SCCWRP) a



### Southern California Edison (SCE)

Southern California Marine Institute (SCMI)

State Water Resources Control Board (SWRCB) a



### Surfrider Foundation

USC Wrigley Institute for Environmental Studies (WIES)

University of California, Santa Barbara

University of California, Davis, Marine Pollution Studies Lab a



### US EPA Region IX

US EPA Office of Research and Development

US Geological Survey

US Navy, Space & Naval Warfare Systems Center, San Diego (USN) a



### Ventura County Health Department

### B

- 1

**APPENDIX B. INTERLABORATORY COMP**  **ARISON OF SEDIMENT**

**TOXICITY TESTS WITH THE AMPHIPOD**  **EOHAUSTORIUS EST UARIUS** 

**Abstract**

The sediment toxicity assessment program of the Southern California Bight 1998

Regional Monitoring Survey (Bight ’98) was a coordinated effort with seven laboratories

sharing the responsibility for conducting 10

- d amphipod tests ( Eohausto rius estuarius ).

An interlaboratory comparison exercise was conducted prior to testing Bight ’98 field

samples. In order to assess laboratory performance with samples collected in the field,

sediments from four stations in Los Angeles/Long Beach Harbor w ere tested along with

the animal collection site control. All laboratories successfully performed the sediment

test and associated reference toxicant test. Statistically significant differences in

amphipod mean survival were found among some laboratories for the field

- collected

sediments. However, precision of the results did not appear to be reduced by having

multiple laboratories participate in the testing. The laboratories demonstrated excellent

concordance (Kendall’s W = 0.91) in ranking the field

- c ollected sediments by toxicity.

Agreement on classifying the sediments into the Bight ’98 toxicity categories (not toxic,

moderately toxic, highly toxic) was good for sediments with mean survival values that

differed sufficiently from the threshold.

**Intr oduction** 

Regional surveys such as Bight ’98 require good QA/QC programs to ensure

quality results. The number of potential variables in the results increases with the

number of participating organizations. A good QA/QC program ensures that the sharing

of responsibilities is a positive feature of the program and does not compromise the

quality of the data. As part of the Bight ’98 QA/QC plan, an interlaboratory comparison

exercise was conducted in order to assess competence and comparability among the

l aboratories prior to the start of the Bight ’98 sampling and testing program. Although a

majority of the laboratories had experience with performing 10

- d amphipod tests, most

had not yet worked with Eohaustorius. One laboratory, however, had extensive

ex perience with Eohaustorius and contributed valuable knowledge to the group.

The Bight ’98 planning process necessitated an accelerated preparation schedule

for the laboratories. The committee recommended that each laboratory complete the

interlaborato ry comparison and two additional preliminary tests before beginning the

Bight ’98 testing. Thus, the exercise had to fulfill dual roles: (1) to allow the laboratories

to gain experience working with Eohaustorius and, at the same time, (2) to provide

compa rison data for QA assessment. Since Bight ’98 toxicity samples were not split

among laboratories, the interlaboratory exercise provided the only opportunity to

### B

- 2

compare test results from different laboratories using split samples of field

- collected

sedimen ts.

The nature of toxicity testing presents some challenges to an interlaboratory

comparison. Toxicity is not an indicator that is measured directly; rather it is assessed

based upon a biological response. Interlaboratory agreement for some environmen tal

indicators (i.e., directly measured chemical parameters) can be assessed by distributing a

coded sample of a standard reference material with a known value (Burton et al. 1996).

The results are assessed based upon how close the laboratories are to the known value

(accuracy) as well as by examining interlaboratory variability in the answers (precision).

However, the choice of measures for the assessment of interlaboratory agreement in

toxicity testing is somewhat more limited. While clean reference se diments are

frequently included in bioassays as a negative control (expected to have no toxic

response), reference sediments having “known” toxicity values are not available.

It is important to assess the ability of the participating laboratories to de tect

responses in contaminated sediments (Schlekat et al

. 1995). Thus, in order to make the

most valuable interlaboratory comparison, it is important to include field sediments with

a range of expected toxicity responses. The sediments are compared on a relative basis

instead of an absolute one. The closest approach to a “true” value is a consensus value

based upon the results of the group. Therefore, results from testing field sediments in

multiple toxicology laboratories are judged on the precision of the results rather than on

accuracy.

This paper presents the results of the interlaboratory comparison exercise

conducted prior to the start of Bight ’98 sampling. The first objective of the study was to

assess whether each laboratory was able to per form the 10

- d Eohaustorius test in an

acceptable manner. This criterion was measured using two methods: (1) by evaluating

attainment of test acceptability criteria and (2) by comparing relative reference toxicant

performance. The second objective was to assess the degree of agreement in the test

responses observed by the laboratories. This objective was accomplished by comparing

the toxicity results of the field

- collected sediments.

**Methods**

**Experimental Design**

The design of the exercise was as follow s: four field

- collected sediments were

collected and homogenized by one agency and then divided among seven laboratories for

toxicity testing. Each laboratory also concurrently tested sediment from the animal

collection site (negative control) and a speci fied cadmium reference toxicant series.

Several important steps were taken in order to control experimental variables outside of

the individual laboratories: (1) test sediments and reference toxicant stocks were

simultaneously distributed from one centra l source using identical containers. (2) test

organisms were obtained from a common commercial supplier out of one collection batch

### B

- 3

and distributed to each of the laboratories, and (3) each laboratory began the bioassay on

the same day.

**Sample Collection**

**and Handling**

Sediment sampling was conducted on May 7, 1998. Sediment samples were

collected from four stations in Los Angeles/Long Beach Harbor: LAH1 (33 ° 46.564 ′

, 118 ° 14.608 ′ ), LAH2 (33 ° 45.343 ′ , 118 ° 16.787 ′ ), LAH3 (33 ° 45.529 ′ , 118 ° 11.684 ′ ), and

LAH4 (33 ° 43.890 ′ 118 ° 9.955 ′ ) (Figure B 1). These sites were chosen by screening

existing amphipod toxicity data (SWRCB 1996) for stations with a wide range in toxicity

response. Sediment samples were collected with a 0.1 m 2 

### modified van Veen grab. At a

given station, multiple grab samples were taken to provide 12 L of sediment. A plastic

(high

- density polyethylene [HDPE]) scoop was used to collect sediment from the top 4

cm of the undisturbed surface material in the grab. Contact with sediment within 1 cm of

the side of the grab was avoided in order to minimize contamination. The sediment was

transported back to the laboratory in polycarbonate containers on ice. Once back at the

laboratory, the sediment was homogenized in a polyethylene bucket using a n overhead

mixer. The homogenized sediment was transferred to 950 mL and 250 mL polyethylene

containers.

The laboratories participating in the exercise were: Aquatic Bioassay and

Consulting, Marine Pollution Studies Laboratory, City of Los Angeles Env ironmental

Monitoring Division, City of San Diego Ocean Monitoring Program, MEC Analytical

Systems, County Sanitation Districts of Orange County, and SCCWRP. Each laboratory

received 1.2 L of sediment from each of the four stations. The sediment was ship ped

overnight to each laboratory or picked up directly by the laboratory.

Test organisms, Eohaustorius estuarius , were obtained from Northwest Aquatic

Sciences (collection site: Beaver Creek, Oregon). The animals were collected on May 6,

1998, and del

ivered to each laboratory by Federal Express on May 7, 1998. The

overlying water in the sediment was approximately 20 ppt. These containers were

emptied into larger tubs at each laboratory and 20 ppt water and aeration were added.

The amphipods were hel

d in constant illumination at 15 ° C until the initiation of the test.

### B

- 4

**FIGURE B**  **1. Locations of sites sampled in Los Angeles/Long Beach Harbor for**

**interlaboratory comparison.**

**Test Procedures**

Sediment toxicity was determined using a 10

- d amphipod survival test

(EPA/600/R

- 94/025). Sediment toxicity tests were conducted in 1 L glass test containers.

Sediment was added to the test containers 1 d prior to the start of the test (May 11).

Sediment samples were thoroughly mixed and then added to the te st containers to form a

sediment layer approximately 2 cm deep. Filtered seawater (20 ppt) was slowly added

until a final volume of 800 mL was reached. Pipettes connected to an air source provided

aeration. Test containers were then allowed to equilibra te overnight. Each sample

consisted of five randomly arranged replicates, along with an extra container to provide

samples for water quality. For each batch of samples tested, a negative control

(consisting of test animal collection site sediment) was in cluded.

At the start of the test (May 12, 1998), amphipods were added randomly until a

total of 20 animals per container was present. Tests were conducted at 15 ° C under

constant illumination. Test animals were exposed to the sediment samples for 10 d. Test

containers were checked daily for air and for any dead animals or animals stuck to the

surface of the water. Any floating animals were submerged by gently pushing them

beneath the surface with a probe. At the end of the exposure period (May 22, 1 998) the

sediment was screened through a 0.5 mm screen and the number of surviving amphipods

was recorded. 3

4

2 1

San Pedro Long Beach Dominguez Channel Los

Angeles

River

### B

- 5 Concurrently with the sediment toxicity test, a cadmium reference toxicity test

was conducted. A standard cadmium stock solution (1,000 mg/L) wa s prepared and

distributed to the participating laboratories. The aqueous phase reference toxicant test

consisted of three replicates of 5 dilutions, plus control. The dilutions were 0.32, 1.00,

3.20, 5.60, and 10.00 mg/L. A sample of the 10.00 mg/L con centration was analyzed for

verification purposes. At the beginning of the test (May 12, 1998), 10 amphipods per

replicate were added randomly to each test container and exposed to the reference

toxicant for 4 d. At the end of 4 d (May 16, 1998) the numb er of surviving animals was

recorded and the LC 50 

### (median lethal concentration) was calculated.

Initial water quality (temperature, pH, dissolved oxygen, salinity, and total

ammonia) was recorded from the overlying water from the extra test container.

Interstitial water quality was also obtained from sediment collected from the same

container centrifuged at 3,000 g for 20 minutes. Temperature, pH, salinity, and dissolved

oxygen of the overlying water were also recorded at the end of the exposure period . All

water quality measurements were measured with laboratory

- approved equipment and

procedures. Water quality measurements for the reference toxicant test were similar to

the sediment phase of the test.

**Data Analysis**

Absolute agreement was assessed by comparing mean survival among the

laboratories. The mean percent of survival rate was calculated for each sediment sample

(grouped by laboratory). The data were then normalized by dividing the sample mean by

the appropriate control mean. This value, a percentage of the control response, reduces

the variation in results due to differences in control survival and allows the comparison

between different tests. T

- tests were conducted versus the appropriate control to

determine significance at the 95% lev el.

The degree of agreement among the laboratories for absolute values of survival

was first assessed by analysis of variance (ANOVA) for data meeting assumptions of

normality, otherwise by a Kruskal

- Wallis ANOVA on ranks. In cases where significant

d ifferences were found, Tukey pairwise multiple comparison tests were conducted to

detect specific differences among the laboratories.

The relative agreement of the laboratories was assessed by ranking the mean

survival values within a laboratory. The degree of association of toxicity rankings

between laboratories was assessed by Kendall’s coefficient of concordance (W) (Siegel

and Castellan, 1988). The field sediments were ranked in order of toxicity for each

laboratory, with a value of 1.0 assigned t o the sediment with the highest survival rate and

a value of 4.0 assigned to the sediment with the lowest survival rate. Kendall’s W ranges

from 0.0 (no degree of association) to 1.0 (perfect association).

Sediment toxicity was defined by two criteria: (1) a statistically significant

difference between the sample and control and (2) a minimum percentage difference

### B

- 6

between the sample and control. Samples that were significantly different from the

control and had a 20% response relative to the control (su rvival less than 80% of the

control) were classified as toxic. This measure of toxicity represents a 90% power to

determine statistical significance in survival between control and sample (SAIC 1994).

Toxic samples were further classified as moderately t oxic (50 to 79% survival) or highly

toxic (less than 50% survival). Toxicity classification systems with three or more tiers

have been used for amphipod test results as well as for other bioassay species (Long et al

. 1998).

Mean survival was calculated for each cadmium concentration of the reference

toxicant test. Reference toxicant test LC 50 

### values were calculated with the Spearman

Karber method. A control chart was prepared by plotting the LC50 values for the seven

laboratories. A test result within two standard deviations of the mean was considered

acceptable.

**Results**

**Assessment of Laboratory Performance**

The participating laboratories successfully met all test acceptability criteria for the

amphipod test (Table B 1). Each laboratory had nea rly 100% survival in the animal

collection site control sediment. In addition, each laboratory obtained at least 90%

survival in the reference toxicant seawater control (mean=97.6, Table 1). The

laboratories reported that all experiments were run within the parameters of the test

protocol (including water quality) and that no tests needed to be repeated.

**TABLE B**  **1. Laboratory control performance. Test protocol requirements are from**

**EPA (1994).**

Lab 

## Sediment

Mean Control

Survival (%) Test

Conditi ons

Conformed to

Protocol? 

## Reference

**Toxicant**

Control Survival (%)

1 99 Yes 100

2 100 Yes 100

3 98 Yes 97

4 96 Yes 90

5 97 Yes 100

6 100 Yes 100

7 97 Yes 97

### B

- 7

**Interlaboratory Comparability**

Reference Toxicant Tests

A wide range of sensitivity to cadmium was observed between the highest and the

lowest values. The LC 50 

### values ranged from 1.76 to 9.43 μ g/L, approximately a factor of

five (Table B2). In spite of the overall range, five of the seven laboratory results were

within ±28% of the mean val ue (5.39 μ g/L). The lowest LC 50 

### value stood out since more

than a factor of two separated it from the rest of the group. The LC 50 

### 95% confidence

intervals for the other six laboratories at least partially overlapped one another.

TABLE B2. Cadmium ref erence toxicant results obtained during the

interlaboratory comparison exercise.

Lab LC50 (mg/L) 95% CI

1 1.8 1.6

- 1.9

2 9.4 7.1

- 12.6

3 4.0 3.2

- 5.0

4 5.7 4.6

- 7.0

5 5.9 5.0

- 7.0

6 6.3 4.6

- 8.6

7 4.7 3.9

- 5.7

In order to better dete rmine the significance of the reference toxicant variability,

the LC 50 

### values from this interlaboratory test were compared to the results of numerous

Eohaustorius reference toxicant tests conducted by a single laboratory. The variability

and the range of the interlaboratory tests were similar to the intralaboratory variability

and range observed in the single laboratory data (Figure B2). The LC50 values of the

interlaboratory test fell within two standard deviations of the mean calculated from the

intrala boratory test data.

Sediment Tests

The sediments from the four sampling locations produced a wide range of mean

survival values, which was the intent of the study design. Consensus survival means

ranged from 11 to 80% (Figure B3). Each of the laborat ories was able to statistically

discriminate between the control sediment and the field

- collected sediments. Overall, 27

of the 28 possible t

- tests between control sediments and field sediments were significant

(p<0.05). These data demonstrated that each of the laboratories could discriminate

between the control sediment and moderately contaminated field sediments.

For each sediment type, all laboratories reported data that fell within two standard

deviations of the consensus mean. The standard deviati on of the laboratory means ranged

### B

- 8

from 9.9 to 16.4 for the four sediment types. Coefficients of variation ranged from 12.4

to 124%. The high CV value (for Station LAH1) was mainly due to low mean survival,

which has the effect of inflating the coefficien t of variation.

FIGURE B2. Cadmium reference toxicant median lethal concentrations

(LC 50 

### ) for the interlaboratory exercise (filled circles) and for a single

laboratory (open circles).LC50 (mg/L)



### 0

5 10 15 20

Intralab mean 2 SD



### Intralab Interlab

### B

- 9 LAH 1

Laboratory 1 2 3 4 5 6 7% Survival (% of Control)

0 20 40 60 80 100

Mean



### LAH 2

Laboratory 1 2 3 4 5 6 7% Survival (% of Control)

0 20 40 60 80 100

Mean 79.5 11.0 + 2 s.d.

+ 2 s.d.

- 2 s.d.



### FIGURE B3. Survival results for Eohaustorius estuarius exp osed to field

sediments (LAH1

- LAH4). Bars represent the mean of 5 replicates tested at

each laboratory. Error bars are one standard deviation. Solid reference

line is the consensus mean of the seven laboratories. Dashed reference

lines are ±2 standard deviations from the consensus mean.

### B

- 10 Laboratory 1 2 3 4 5 6 7% Survival (% of Control)

0 10 20 30 40 50 60 70 80 90 100

Mean Laboratory 1 2 3 4 5 6 7% Survival (% of Control)

0 10 20 30 40 50 60 70 80 90 100

Mean 

### LAH3

LAH4 33.3

80.0 + 2 s.d.

-2 s.d.

-2 s.d.



### FIGURE B3. (Continued). l

### B

- 11 Analysis of variance detected statistically significant differences between some of

the laboratories for each of the four sediment types (p<0.05). Overall, 25% (21 out of 84)

of the possible Tukey pairwise comparisons showed significant differences (Table B3).

Two minor trends were observed in the statistical differences. First, Lab 7 had the lowest

or second lowest survival rate for each sediment type. However, the Tukey analysis

g rouped Lab 7 together with at least three of the other laboratories for each sediment

type, indicating that the survival results were not markedly different from other

laboratories. Second, one laboratory (Lab 5) was different from all the other laborator ies

for station LAH4. However, this was the only case where a single laboratory was left

ungrouped and that three laboratories were grouped together statistically in all other

cases. Furthermore, this same laboratory (Lab 5) was the only laboratory that did not

differ significantly from any other laboratories for field sediments LAH1, LAH2, and

LAH3 (indicated in Table B3 by two lines that connect to the other laboratories).

TABLE B3. Laboratories arranged by order of survival results for each field

sediment. Laboratories not significantly different from one another (Tukey

pairwise comparison, p>0.05) are connected by solid lines.

Sediment Laboratory Number

Highest Survival Lowest Survival

LAH1 

## 4 5 2 1 3 6 7

LAH2 

## 2 1 6 3 5 4 7

LAH3 

## 4 2 5 3 6 7 1

LAH4 

## 1 2 3 4 6 7 5

Since the sediments were tested blindly, ranking the four field sediments by

survival for each laboratory provided a way to asse ss the ability of the participating

laboratories to distinguish between sediments. Each of the seven laboratories ranked

LAH1 and LAH3 as the most toxic and next most toxic, respectively (Table B4). Four of

the laboratories ranked the sediments in exactl y the same order and a fifth only differed

in that LAH2 and LAH4 were tied in the rankings. The Kendall coefficient of

concordance was 0.91, indicating a high level of agreement (p<0.01) between

laboratories.

### B

- 12

TABLE B4. Rank of field sediments by am phipod survival. 1 = highest

survival, 4 = lowest survival.

Laboratory

Sediment

Type Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7

Station LAH1 4 4 4 4 4 4 4

Station LAH2 2 1.5 2 2 1 1 2

Station LAH3 3 3 3 3 3 3 3

Station LAH4 1 1.5 1 1 2 2 1

The sediments were classified according to level of toxicity by employing the

proposed ratings for the Bight ’98 study (non

- toxic, moderately toxic, highly toxic). All

seven laboratories classified LAH1 sediment as highly toxic (Table B5). All s even

laboratories classified LAH3 as toxic, with five classifying it as highly toxic and two as

moderately toxic. The classification results were more variable for the two stations with

a survival mean of approximately the same value as the moderate toxic ity threshold

(80%). Three of the seven laboratories classified LAH2 as moderately toxic; four

classified it as not toxic. Four laboratories classified LAH4 as moderately toxic, three as

not toxic.

TABLE B5. Comparison of individual laboratory class ifications of samples

tested. N=nontoxic ( 3 80% survival), MT=moderately toxic (50

- 79% survival,

significantly different from the control (t

- test, p<0.05)), HT=highly toxic

(<50% survival, significantly different from the control (t

- test, p<0.05)).

b) S ample Tested

LAH1 LAH2 LAH3 LAH4

1 HT N HT N

2 HT N MT N

3 HT N HT N

4 HT MT MT MT

5 HT MT HT MT

6 HT N HT MT

7 HT MT HT MT

### B

- 13 **Discussion**

All of the participating laboratories successfully completed the interlaboratory

comparison exercise. Eac h participating laboratory demonstrated that it was capable of

performing the Eohaustorius estuarius 10

- d sediment test and associated 96

- h reference

toxicant test. In addition, each laboratory demonstrated an excellent capability to

discriminate statisti

cally between the control sediment and field

- collected sediments.

These achievements indicated that no major problems occurred with either the general

laboratory conditions or the handling techniques employed by the personnel in each

laboratory.

The g ood performance of the laboratories is especially noteworthy considering the

fact that many did not have experience with Eohaustorius estuarius. This demonstrates

that the species is well suited as a test organism in 10

- d sediment tests. The test can be

learned and applied quickly by laboratories having basic familiarity with marine toxicity

tests.

**Interlaboratory Agreement**

When multiple laboratories are involved in a study, there is the potential for lower

precision in the results than could be obtain ed by a single laboratory. However, the

findings of the current study do not indicate that precision was sacrificed. The

interlaboratory range observed for the reference toxicant LC 50 

### results fell within the

intralaboratory range of one very experienced laboratory. The data points produced by

multiple laboratories were indistinguishable from the points produced by the single

laboratory. This result is noteworthy given that this was the first time some of the

laboratories had conducted an amphipod test.

The influence of laboratory conditions and techniques over variations in mean

survival values would indicate a finding of consistent bias in the results. However, a

strong bias was not observed in the performance of any of the laboratories. Analysis o f

variance did detect some significant differences between the laboratories for the field

collected sediments, but most (75%) of the differences in pairwise comparisons were not

significant. The pairwise comparisons that were significant were distributed among the

possible combinations. These results support a finding that the variability observed in the

comparison exercise was the result of variability inherent in the test itself.

The laboratories demonstrated an excellent ability to rank field sedim ents by

toxicity. Classification of sediments into the Bight ’98 categories also showed

reasonably good agreement considering that the grand mean of each of two stations

(LAH2 and LAH4) was very close to the classification threshold. It is important to n ote

that the laboratories had 96% agreement on whether the response to the field sediment

was statistically different from the control response.

### B

- 14

**Comparison to Previous Interlaboratory Studies**

The results of our interlaboratory study compare well to a previous interlaboratory

study using the same species. Schlekat et al

. (1995) reported similar findings of

agreement for an interlaboratory comparison involving eight laboratories and using

multiple dilutions of a contaminated sediment sample. As in our study, the investigators

found some significant differences between the mean survival values for individual

laboratories. Also, as we did, they found very good agreement in the ability of the

laboratories to rank toxicity levels of the sediments tested ( Kendall’s W = 0.93). The

largest difference observed between an individual mean survival and the consensus mean

survival was 35.9%; in our study, the maximum was 25.4%.

We can compare the relative level of interlaboratory variability observed for the

Bight ’98 interlaboratory study to previous 10

- d amphipod test interlaboratory studies. In

the Bight ’98 study, the coefficient of variation (CV) ranged from 2% (for the control

sediment) to 123% and decreased markedly as mean survival increased. The C.V . for a

given level of survival was very similar to that observed in other interlaboratory

comparisons (Figure B4). This is an indication that the amount of variability among

laboratories observed in the current study was within expected values.

Mean Survival (%) 0 20 40 60 80 100Coefficient of Variation (%)



### 0 20 40 60 80 100 120 140 160



### Bight'98

Other Eohaustorius

Rhepoxynius

Ampelisca

FIGU RE B4. Interlaboratory variability as a function of mean survival for

three commonly applied 10

- d amphipod tests. In addition to the current

study, data are from Mearns et al. (1986) and Schlekat et al. (1995).

### B

- 15

**Implications for Cooperative Regional Mon itoring Programs** 

Our results show that cooperative toxicity testing can provide data of comparable

quality to similar testing performed by a single laboratory. Some improvement may be

reasonably expected as laboratories gain more experience working with the test species.

Thus, we can expect that the laboratories will perform the Eohaustorius tests adequately

and have levels of variability equal to or less than those observed in the current study.

The uncertainty associated with the classification of a ny one station is reduced

when many stations are grouped together to provide an assessment of a geographic area,

as they are in regional monitoring projects such as Bight ’98. Including more stations in

a subpopulation decreases the potential that misclas sifications will have a significant

influence on the assessment of the subpopulation.

**LITERATURE CITED**

Bay, S.M., D.J. Greenstein, A.W. Jirik, and J.S. Brown. 1998. Southern California Bight

1994 Pilot Project: VI. Sediment Toxicity. Southern Califo rnia Coastal Water Research

Project. Westminster, CA.

Burton, G.A. Jr., T.J Norberg

- King., C.G. Ingersoll, D.A. Benoit, G.T. Ankley, P.V.

Winger, J. Kubitz, J. Lazorchak, M. Smith, E. Greer, F.J. Dwyer, D.J. Call, K.E. Day, P.

Kennedy, and M. Stinson. 1 996. Interlaboratory study of precision: Hyalella Azteca and

Chironomus Tentans freshwater sediment toxicity assays. Environmental Toxicology and

Chemistry 15:1335

- 1343.

California State Water Resources Control Board (SWRCB), Division of Water Quality,

Bay Protection and Toxic Cleanup Program. 1996. Sediment Chemistry, Toxicity, and

Benthic Community Conditions in Selected Water Bodies of the Los Angeles Region

Final Report. Sacramento, CA.

Long, E.R., L.J. Field, and D.D. MacDonald. 1998. Predict ing toxicity in marine

sediments with numerical sediment quality guidelines. Environmental Toxicology and

Chemistry 17:714

- 727.

Mearns, A.J. R.C. Swartz, J.M. Cummins, P.A. Dinnel, P. Plesha, and P.M. Chapman.

1986. Inter

- laboratory comparison of a sed iment toxicity test using the marine amphipod

Rhepoxynius abronius

. Marine Environmental Research 19:13

- 37.

SAIC. 1994. Southern California Coastal Water Research Project Environmental

Monitoring and Assessment Program: Sediment Toxicity Test Results. Submitted to

Southern California Coastal Water Research Project. Westiminster, CA.

### B

- 16

SCBPP Steering Committee. 1998. Southern California Bight 1994 Pilot Project: I.

Executive summary. Southern California Coastal Water Research Project. Westminster,

CA.

S chlekat, C.E., K.J. Scott, R.C. Swartz, B. Albrecht, L. Antrim, K. Doe, S. Douglas, J.A.

Ferretti, D.J. Hansen, D.W. Moore, C. Mueller, and A. Tang. 1995. Interlaboratory

comparison of a 10

- day sediment toxicity test method using Ampelisca abdita

, Eohaust orius estuarius , and Leptocheirus plumulosus. Environmental Toxicology and

Chemistry 14:2163

- 2174.

United States Environmental Protection Agency (USEPA), Office of Research and

Development. Methods for Assessing the Toxicity of Sediment

- associated Co ntaminants

with Estuarine and Marine Amphipods. 1994. EPA/600/R

- 94/025. Washington, DC.

**ACKNOWLEDGEMENTS**

The authors wish to thank the laboratory personnel of the following organizations

who conducted the bioassays for this study: Aquatic Bioassay a nd Consulting

Laboratories, Marine Pollution Studies Laboratory, City of Los Angeles Environmental

Monitoring Division, City of San Diego Ocean Monitoring Program, MEC Analytical

Systems, the County Sanitation Districts of Orange County, and SCCWRP. We th ank

Darrin Greenstein for coordinating the logistics of the exercise, Tim Mikel for assisting

with the reference toxicant data analysis, and Molly Leecaster for her input regarding

statistical tests. Dario Diehl (SCCWRP) as well as the skipper and crew of the Ocean

Sentinel (County Sanitation Districts of Los Angeles County) assisted in the collection of

field sediments. We are grateful to Brian Anderson for sharing his insights into working

with Eohaustorius estuarius as a test species.

### C

- 1

-

**APPENDIX C. M ICROTOX STORAGE EXPERIMENT** 

**INTRODUCTION**

The time that a water sample is held prior to toxicity test initiation can have a

significant effect on toxicity test results and the potential for effects increases with time.

Changes to sample constituents are inevitable. For example, sample constituent alteration

may occur due to changes in temperature, pH, adsorption of chemicals to holding vessel

walls, or biological activity (APHA 1998). Sample holding and preservation techniques

can only retard the rate o f these changes, which include changes due to biological

activity, hydrolysis of chemical compounds, and reduced constituent volatility (APHA

1998).

As part of the Bight’98 regional monitoring survey conducted in July

- August,

1998, sediment samples were collected for acid volatile sulfides (AVS) analysis and pore

water toxicity testing. The samples were sent to the U.S. EPA Laboratory in Newport,

Oregon, for processing and AVS analysis. Pore water was removed by centrifugation. A

sub

- sample of the por

e water was removed, placed in borosilicate glass vials with Teflon

lids, and stored at 1.5

–1.8° C prior to being shipped to the Orange County Sanitation

District (OCSD) for toxicity testing. Pore water samples were held until all sediments

were receive d and processed. Some pore water samples were held several weeks prior to

shipping. Upon receipt, the samples were frozen at

- 20° C until thawed for testing using the MicrotoxTM Rapid Toxicity Testing System

(Azur Environmental, Carlsbad, CA). The pore water samples were held for

approximately 6 months prior to testing.

Due to concerns over the possible effects of the extended sample holding time on

Microtox test results, a storage experiment was conducted. The purpose of this

experiment was to deter

mine if sample storage time has a significant effect on pore water

toxicity from marine sediment samples as measured using the MicrotoxTM Rapid Toxicity

Testing System.

**METHODS**

The study was conducted at the OCSD Bioassessment Laboratory (Fountain

Vall ey, CA). Sediment samples for pore water extraction were collected from two

locations on March 23, 1999. One was an offshore site (Station 37) not expected to show

significant toxicity and the other a Newport Bay site (Station 2137), which was expected

to produce a toxic response. The sediment samples were collected using a 0.1 m2

modified Van Veen grab sampler. Samples were collected using a stainless steel scoop

and placed into a HDPE container. Only the top 2 cm of sediment was collected. The

sampl es were kept in wet ice in a cooler on the boat and were subsequently transported to

the OCSD for pore water extraction. The pore water was extracted within 24 h of

sediment collection. The pore water was extracted by centrifugation (10,000 rpm for 10

### C

- 2

-

mi nutes), decanted, and stored in glass jars with Teflon

- lined lids. Following extraction

from the sediments, the pore water samples were kept frozen at

- 20° C until thawed for

testing. Once thawed, the samples were kept refrigerated at 4° C ±1° C for the duration

of the study.

Testing was conducted using the MicrotoxTM Comparison Test Method

(Microbics Corp. 1995). The Microtox system uses a luminescent bacterium, Vibrio

fischeri (strain NRRL B

- 11177), to measure aquatic phase toxicity. The light emitt ed by

the bacteria as a byproduct of metabolic processes is used as a measure of biological

activity. Exposure to toxicants alters the metabolic processes, thus affecting light

production. Toxicity is assessed by exposing the bacteria to a sample and mea suring the

light production relative to a control sample. A reduction in light output is assumed to be

proportional to the toxicity of a sample.

The Comparison Test Method uses multiple replicates of the sample at a single

concentration (in this case, 100% sample) and compares light output against a control

(clean seawater) to measure relative acute toxicity of the sample. Light readings are

taken at time zero, five, and fifteen minutes. The time zero light measurement is taken as

the initial reading to which subsequent light measurements are compared. Different toxic

constituents can affect the bacteria at different rates. Some toxicants (e.g., phenol) cause

an immediate reduction in light production, while others act over a longer time period.

Tak ing light measurements at both times is recommended (Microbics Corp. 1992).

In this study, three replicates of sample were compared against three replicates of

control water. The control water was clean seawater obtained from the Kerckhoff Marine

Labor atory, operated by the California Institute of Technology (Corona Del Mar, CA).

The seawater was filtered through a 2 μm filter and stored at 4° C ±1° C. The test was

conducted using the Microtox® M500 Analyzer, at a temperature of 15° C. Testing was

in itiated one

- week from pore water extraction and continued at one

- week intervals for

four weeks and then monthly for four additional months.

**RESULTS**

In general, the magnitude and pattern of changes in toxicity over time were

similar for both stations and for both 5 and 15

- minute measurements. Neither station

showed toxicity at the initial time period (one

- week); luminescence was greater than or

approximately equal to the control luminescence (Table C1, Figure C1).

A marked decrease was found in li ght production relative to the control from

week one to week two for both samples. Toxicity then varied within a relatively narrow

range through a holding time of one month. At the two

- month testing, light production

increased in the Station 37 sample to 93% of the control. However, the sample from

Station 2137 showed no significant change.

### C

- 3

- After three and four months of storage, the light production of the Station 37

sample was greater than that of the control. However, light production decreased t o

below the toxic threshold of 95% of the control at the final testing period (five months).

The Station 2137 sample had a sharp increase in light production at the three

- month

sampling interval, but was still below the toxic threshold. Light production subsequently

declined for Station 2137 (four months) and then remained approximately unchanged at

five months.

**DISCUSSION**

The results of this study indicate that toxicity tends to increase approximately

20% with a storage time of two weeks or greater. Toxicity then decreases at two to three

months’ storage time, but increases again at four months. The cause of the variability

was not identified.

In this study, significant changes were seen in toxicity after samples were held for

only two

- weeks. S ome of the samples taken in the Bight ’98 project were held in the

processing laboratory up to four

- weeks before delivery to the testing laboratory. All

samples received by the testing laboratory were held frozen for approximately six

- months

prior to toxi city testing. The results of this storage experiment indicate Microtox test

results would have been compromised even if testing was initiated immediately upon

receipt of the samples due to the holding time in the processing laboratory.

The porewater s amples in the Bight ’98 study were held frozen at

– 20°C by the

testing laboratory prior to testing. While, there is no one method of preservation that

satisfactorily prevents the degradation of all constituents in a water sample, the effect of

pore water freezing relative to Microtox test results has not been investigated. This

method of preservation may have also altered the chemical nature of the sample and thus

the toxicity test results.

The results of this storage study on the toxicity of marine pore water toxicity, as

measured by the MicrotoxTM system, indicate that the length of time the Bight ’98

sediment porewater samples were held prior to testing was sufficient to significantly

affect Microtox toxicity test results. Therefore, the results of Microtox testing of

sediment porewater in the Bight’98 project were excluded from regional toxicity

assessments.

### C

- 4

-

Table C1. Results of sample storage time on pore water toxicity measured

by Microtox. Values reported are light production expressed as a

percentage of the seawater control response for either a 5 or 15 minute

exposure.

Station 37 Station 2137

Storage Time Date Tested 5 min. 15 min. 5 min. 15 min.

1 week 3/31/99 105.5 105.9 100.5 99.1

2 weeks 4/07/99 84.9 81.6 79.5 72.3

3 weeks 4/14/99 87.9 87.0 82.0 78.8

4 weeks 4/20/99 83.4 81.7 82.5 77.7

2 months 5/14/99 90.3 93.3 79.6 79.1

3 months 6/15/99 106.5 109.3 94.4 92.7

4 months 7/19/99 100.7 106.0 86.5 79.8

5 months 8/17/99 93.6 92.3 87.7 80.9

### C

- 5

- Time Week1 Week2 Week3 Week4 Month2 Month3 Month4 Month5% Control Response

0 10 20 30 40 50 60 70 80 90 100 110

Station 37

Station 2137



### Figure C1. Change of toxicity over the five

- month study period. Fifteen

minute reading values are shown.

**LITERATURE CITED**

APHA. 1998. Standard Methods for the Examination of Water and Wastewater, 20 th



### Edition. American Public Health Association. Washington, D.C.

Microbics Corp. 1992. Microtox Manual, Volume I. Micribics Corporation.

Carlsbad, CA. 85 pp.

Microbics Corp. 1995. Microtox® Acute Toxicity Comparison and Inhibition Test

for Fresh Water and Marine Samples. Micribics Corporation. Carlsbad, CA. 30pp.

### D

- 1

-

**APPENDIX D. TEST RESULTS BY STATION** 

Test Response b

Station Longitude Latitude Strata a Depth

(m) Location Amphipod

(%) QwikSed

(%) H RGS

(μg/g) Fines

(%) TOC

(%)

2268 119.8000 34.4007 Bath

- 120 31 Goleta 89.0 100.0 22.8 27.0 0.68

2381 118.76 93 34.0025 Bath

- 120 40 Los Angeles Co. 103.2 97.6 4.8 61.9 0.94

2384 118.5039 33.9299 Bath

- 120 47 Los Angeles Co. 100.0 na 10.7 49.8 0.77

2387 118.4522 33.8483 Bath

- 120 75 Los Angeles Co. 101.0 100.0 14.4 45.1 0.88

2394 118.2491 33.6511 Bath

- 120 42 Los Angeles Co. 101.0 88.7 2.8 16.5 0.50

2396 118.1494 33.6481 Bath

- 120 31 Orange Co. 90.6 na 2.1 16.8 0.32

2398 118.1430 33.6193 Bath

- 120 41 Orange Co. 97.9 na 2.8 15.6 0.26

2400 118.0954 33.6033 Bath

- 120 52 Orange Co. 93.8 97.7 2.3 23.3 0.29

2401 117.957 5 33.5917 Bath

- 120 41 Orange Co. 100.0 na 2.1 46.4 0.53

2403 117.8029 33.5187 Bath

- 120 89 San Diego Co. 96.4 84.7 2.2 75.4 0.86

2405 117.5586 33.2946 Bath

- 120 58 San Diego Co. 91.8 100.0 3.1 67.0 0.78

2407 117.5237 33.2603 Bath

- 120 58 San Diego Co. 93.8 na 2.3 58.5 0.69

2408 117.3618 33.1051 Bath

- 120 83 San Diego Co. 92.8 100.0 3.5 59.3 0.66

2410 117.3546 32.7722 Bath

- 120 87 San Diego Co. 84.5 100.0 3.2 46.9 1.26

2411 117.3430 32.7526 Bath

- 120 89 San Diego Co. 78.4 na 4.5 52.9 0.68

2412 117.2963 32.7 214 Bath

- 120 62 San Diego Co. 99.0 100.0 4.1 46.6 0.67

2413 117.2784 32.6905 Bath

- 120 49 San Diego Co. 95.9 100.0 3.8 28.7 0.48

2418 117.3148 32.5953 Bath

- 120 100 San Diego Co. 100.0 na 6.1 24.5 0.48

2419 117.2636 32.5894 Bath

- 120 56 San Diego Co. 101.0 100.0 7.0 10.7 0.39

2356 120.0739 34.4477 Bath

- 120 44 Santa Barbara Co. 97.8 na 35.0 30.5 0.74

2357 119.9408 34.4064 Bath

- 120 57 Santa Barbara Co. 74.7 100.0 77.0 74.3 1.82

2358 119.9730 34.3968 Bath

- 120 75 Santa Barbara Co. 76.8 100.0 23.0 42.3 1.15

2360 119.8752 34.3940 Bath

- 120 46 Santa Barbara Co. 69.7 100.0 902.9 49.4 2.82

2362 119.6681 34.3683 Bath

- 120 55 Santa Barbara Co. 84.0 100.0 12.6 71.6 1.41

2363 119.6281 34.3556 Bath

- 120 45 Santa Barbara Co. 97.0 100.0 2.6 0.6 0.12

2364 119.6568 34.343 4 Bath

- 120 14 Santa Barbara Co. 94.0 na 5.6 21.6 0.33

2365 119.5623 34.3447 Bath

- 120 45 Santa Barbara Co. 73.0 100.0 9.5 55.1 0.75

2366 119.6109 34.3309 Bath

- 120 57 Santa Barbara Co. 75.8 100.0 15.9 46.1 0.72

2367 119.5332 34.3168 Bath

- 120 61 Santa Barb ara Co. 92.0 na 7.2 98.9 1.52

2368 119.4633 34.3014 Bath

- 120 45 Ventura Co. 68.0 na 15.0 98.5 1.39

2369 119.4252 34.3040 Bath

- 120 33 Ventura Co. 72.0 100.0 10.9 96.0 1.21

2371 119.5101 34.2641 Bath

- 120 76 Ventura Co. 80.0 na 18.5 94.8 1.58

2372 119.602 0 34.2399 Bath

- 120 107 Ventura Co. 90.9 100.0 15.3 40.5 0.63

2374 119.4911 34.1880 Bath

- 120 98 Ventura Co. 71.7 na 17.1 75.7 1.42

2132 119.2272 34.1549 Bath

- 30 3 Channel Is. Harbor 101.1 100.0 2.8 2.0 0.06

2377 118.9403 34.0389 Bath

- 30 19 Los Angeles Co

. 95.9 na 2.6 11.3 0.18

2378 118.9225 34.0325 Bath

- 30 27 Los Angeles Co. 94.8 100.0 2.3 15.3 0.23

2379 118.8368 34.0192 Bath

- 30 17 Los Angeles Co. 68.0 na 1.4 5.1 0.18

2380 118.7601 34.0183 Bath

- 30 20 Los Angeles Co. 94.8 na 4.6 29.4 0.64

2382 118.5926 34.0231 Bath

- 30 23 Los Angeles Co. 97.9 na 6.8 43.4 0.71

### D

- 2

- Test Response b

Station Longitude Latitude Strata a Depth

(m) Location Amphipod

(%) QwikSed

(%) H RGS

(μg/g) Fines

(%) TOC

(%)

2383 118.5133 34.0118 Bath

- 30 10 Los Angeles Co

. 97.9 100.0 5.7 20.7 0.31

2385 118.4572 33.9068 Bath

- 30 25 Los Angeles Co. 97.9 100.0 4.2 7.1 0.15

2386 118.4245 33.8781 Bath

- 30 16 Los Angeles Co. 101.1 na 6.5 6.8 0.16

2388 118.1487 33.7483 Bath

- 30 13 Los Angeles Co. 87.8 92.7 4.8 65.5 0.85

2389 118 .3218 33.7095 Bath

- 30 20 Los Angeles Co. 103.1 na 3.1 6.4 1.75

2390 118.2647 33.6935 Bath

- 30 25 Los Angeles Co. 100.0 93.8 12.6 60.3 1.50

2391 118.1378 33.7095 Bath

- 30 19 Los Angeles Co. 97.9 na 8.1 51.3 0.49

2392 118.1554 33.7051 Bath

- 30 21 Los Angeles Co. 102.1 na 2.7 19.8 0.26

2393 118.0883 33.6829 Bath

- 30 17 Orange Co. 96.9 na 1.0 1.7 0.07

2395 118.0541 33.6727 Bath

- 30 13 Orange Co. 102.1 na 1.0 8.3 0.12

2397 118.1260 33.6501 Bath

- 30 30 Orange Co. 101.0 100.0 3.1 24.4 0.56

2399 117.9948 33.6347 B ath

- 30 12 Orange Co. 101.0 na 0.8 13.2 0.14

2402 117.8302 33.5574 Bath

- 30 30 San Diego Co. 95.4 100.0 3.9 50.2 1.19

2404 117.6589 33.4221 Bath

- 30 21 San Diego Co. 96.9 na 1.7 36.5 0.27

2406 117.4719 33.2814 Bath

- 30 11 San Diego Co. 87.6 100.0 1.6 28.5 0 .23

2409 117.2820 33.0041 Bath

- 30 7 San Diego Co. 94.8 na 1.0 0.6 0.07

2414 117.1898 32.6770 Bath

- 30 9 San Diego Co. 99.0 na 1.6 0.3 0.15

2415 117.1823 32.6587 Bath

- 30 14 San Diego Co. 100.0 na 0.9 6.4 0.10

2417 117.2146 32.6250 Bath

- 30 24 San Diego Co

. 98.0 na 1.1 1.5 0.23

2354 120.3128 34.4589 Bath

- 30 17 Santa Barbara Co. 103.3 na 5.2 16.4 0.34

2355 120.3367 34.4519 Bath

- 30 16 Santa Barbara Co. 101.4 84.4 9.7 12.0 0.25

2359 119.8648 34.3987 Bath

- 30 26 Santa Barbara Co. 94.9 na 89.8 20.5 0.66

2361 119.5701 34.3994 Bath

- 30 13 Santa Barbara Co. 98.0 na 5.9 13.7 0.19

2370 119.3618 34.2986 Bath

- 30 11 Santa Barbara Co. 96.8 na 2.8 20.6 0.28

2373 119.3851 34.2208 Bath

- 30 28 Ventura Co. 96.0 100.0 8.1 71.5 0.54

2375 119.3554 34.1880 Bath

- 30 27 Ventura C o. 100.0 100.0 6.7 62.4 0.45

2376 119.3464 34.1788 Bath

- 30 26 Ventura Co. 96.9 100.0 4.2 50.8 0.36

2189 118.5596 33.9533 LPOTW 63 Hyperion WWTP na na 9.0 19.0 0.80

2190 118.5287 33.9479 LPOTW 50 Hyperion WWTP 104.3 100.0 13.9 33.4 0.64

2191 118.5607 33 .9370 LPOTW 98 Hyperion WWTP 90.6 86.0 15.7 17.8 0.77

2192 118.5191 33.9445 LPOTW 48 Hyperion WWTP 103.2 5.0 6.1 35.2 0.58

2194 118.5215 33.9204 LPOTW 57 Hyperion WWTP na na 12.7 42.0 0.76

2195 118.5250 33.9109 LPOTW 59 Hyperion WWTP 103.2 1.9 17.8 43.1 0.78

2196 118.5534 33.9033 LPOTW 72 Hyperion WWTP na na 7.8 19.0 1.79

2197 118.5008 33.9039 LPOTW 53 Hyperion WWTP 104.3 100.0 9.8 55.4 0.85

2198 118.5238 33.8872 LPOTW 57 Hyperion WWTP na na 5.9 22.0 1.35

2199 118.5349 33.8816 LPOTW 52 Hyperion WWTP 96.9 50.6 7.4 15.8 1.53

2200 118.4290 33.7479 LPOTW 51 JWPCP na na 15.9 51.0 3.64

2201 118.3924 33.7207 LPOTW 86 JWPCP 98.9 100.0 49.4 64.7 2.75

2202 118.3725 33.7221 LPOTW 48 JWPCP 89.2 100.0 16.7 72.2 2.06

2204 118.3390 33.6969 LPOTW 76 JWPCP na na 5 8.4 66.0 3.09

2205 118.3058 33.6769 LPOTW 64 Orange Co. WWTP 101.1 100.0 15.2 40.6 1.10

2207 118.0659 33.6126 LPOTW 33 Orange Co. WWTP na na 1.0 6.0 0.13

### D

- 3

- Test Response b

Station Longitude Latitude Strata a Depth

(m) Location Amphipod

(%) QwikSe d

(%) H RGS

(μg/g) Fines

(%) TOC

(%)

2208 118.0565 33.6022 LPOTW 37 Orange Co. WWTP 96.9 100.0 1.7 19.0 0.26

2209 118.0629 33.5920 LPOTW 65 Orange Co. WWTP 103.1 0.2 3.7 25.2 0.43

2210 118.0412 33.5902 LPOTW 45 Orange Co. WWTP na na 2.2 24.0 0.40

2211 118.0100 33.5880 LPOTW 41 Orange Co. WWTP na na 2.1 26.0 0.44

2212 117.9893 33.5901 LPOTW 40 Orange Co. WWTP 101.0 100.0 2.5 37.6 0.39

2213 117.9778 33.5715 LPOTW 60 Orange Co. WWTP na na 1.0 11.0 0.23

2214 117.3011 32.6899 LPOTW 74 Point Lom a WWTP 88.7 100.0 3.3 52.5 0.74

2217 117.3106 32.6627 LPOTW 86 Point Loma WWTP 101.0 nv 3.3 46.8 0.59

2218 117.2798 32.6664 LPOTW 59 Point Loma WWTP 56.7 100.0 3.5 44.6 0.65

2219 117.3314 32.6548 LPOTW 106 Point Loma WWTP na na 6.4 36.0 1.01

2220 117.2 800 32.6597 LPOTW 60 Point Loma WWTP na na 8.6 40.0 0.49

2134 118.0622 33.7154 Marina 3 Anaheim Bay 77.3 79.8 18.4 77.7 2.99

2129 119.2278 34.1732 Marina 5 Channel Is. Harbor 55.7 85.1 18.6 79.1 2.44

2130 119.2233 34.1718 Marina 3 Channel Is. Harbor 39. 8 86.6 23.1 90.7 2.54

2131 119.2247 34.1628 Marina 3 Channel Is. Harbor 105.4 79.1 8.9 90.2 1.28

2149 117.7042 33.4616 Marina 4 Dana Point Harbor 88.7 100.0 5.4 31.7 0.67

2150 117.7043 33.4624 Marina 2 Dana Point Harbor 97.9 100.0 4.2 42.3 0.69

2151 11 7.6990 33.4598 Marina 5 Dana Point Harbor 99.0 100.0 16.9 76.8 1.43

2421 118.2409 33.7663 Marina 14 Los Angeles Harbor 85.7 100.0 60.0 84.6 1.84

2443 118.4562 33.9820 Marina 3 Marina del Rey Harbor 65.9 100.0 36.7 84.7 1.64

2444 118.4486 33.9830 Marina 3 Marina del Rey Harbor 79.1 100.0 10.4 90.3 1.59

2445 118.4554 33.9776 Marina 3 Marina del Rey Harbor 70.3 100.0 9.6 79.8 1.42

2446 118.4422 33.9775 Marina 3 Marina del Rey Harbor 102.2 94.4 10.8 58.5 1.07

2447 118.4478 33.9766 Marina 3 Marina del Rey Harbor 103.3 100.0 14.9 77.3 1.42

2448 118.4468 33.9695 Marina 5 Marina del Rey Harbor 107.7 45.7 40.5 57.3 1.35

2449 118.4550 33.9657 Marina 4 Marina del Rey Harbor 87.9 99.2 95.0 61.0 2.31

2423 117.2491 32.7807 Marina 3 Mission Bay 97.9 59.3 6.2 42.2 1.18

2424 117.2475 32.7665 Marina 6 Mission Bay 100.0 100.0 2.2 7.2 0.22

2425 117.2356 32.7673 Marina 4 Mission Bay 97.9 100.0 24.3 69.5 3.48

2136 117.9272 33.6189 Marina 6 Newport Harbor 32.2 100.0 80.7 87.6 1.96

2137 117.9239 33.6130 Marina 4 Newport Harbor 37.4 39.6 22.1 83.7 2.21

2138 117.9141 33.6141 Marina 5 Newport Harbor 9.0 82.1 6.7 93.0 1.46

2141 117.9022 33.6114 Marina 3 Newport Harbor 59.4 100.0 14.3 88.6 1.49

2142 117.9100 33.6077 Marina 3 Newport Harbor 21.9 25.3 10.1 89.6 1.18

2143 11 7.9063 33.6070 Marina 3 Newport Harbor 36.1 81.1 8.5 91.6 1.27

2144 117.9006 33.6075 Marina 3 Newport Harbor 70.8 79.1 11.3 81.3 1.03

2145 117.8887 33.6038 Marina 3 Newport Harbor 65.6 72.4 7.9 88.3 1.33

2146 117.8874 33.6019 Marina 4 Newport Harbor 85. 4 100.0 9.5 64.1 0.95

2147 117.8927 33.6012 Marina 3 Newport Harbor 52.1 92.3 12.4 86.1 1.14

2148 117.8797 33.5944 Marina 7 Newport Harbor 89.6 79.4 4.8 21.3 0.54

2221 117.2051 32.7279 Marina 4 San Diego Harbor 82.3 100.0 20.3 68.7 0.86

2222 117.2259 3 2.7188 Marina 5 San Diego Harbor 82.3 100.0 9.4 74.2 0.99

2223 117.2305 32.7154 Marina 4 San Diego Harbor 88.5 80.5 16.2 76.2 1.11

2224 117.2341 32.7131 Marina 5 San Diego Harbor 96.9 100.0 11.3 40.7 0.62

### D

- 4

- Test Response b

Station Longitude Lati tude Strata a Depth

(m) Location Amphipod

(%) QwikSed

(%) H RGS

(μg/g) Fines

(%) TOC

(%)

2225 117.2302 32.7134 Marina 4 San Diego Harbor 88.5 72.2 14.6 58.1 1.03

2226 117.2317 32.7111 Marina 5 San Diego Harbor 86.5 98.3 19.7 91.4 1.73

2227 117 .2080 32.7237 Marina 9 San Diego Harbor 97.9 76.0 12.0 51.1 0.93

2228 117.1782 32.7241 Marina 5 San Diego Harbor 101.0 100.0 47.1 45.9 0.73

2438 117.1017 32.6223 Marina 3 San Diego Harbor 79.6 84.5 4.4 67.7 0.92

2128 119.2594 34.2461 Marina 3 Ventura Ha rbor 80.0 87.8 17.7 95.4 1.74

2229 117.1760 32.7090 Other 12 San Diego Harbor 97.9 1.3 15.6 43.1 0.93

2230 117.1787 32.7025 Other 4 San Diego Harbor 65.7 nv 5.8 11.8 0.20

2231 117.1566 32.6947 Other 13 San Diego Harbor 93.8 82.1 9.1 31.8 0.64

2233 117. 1518 32.6858 Other 9 San Diego Harbor 99.0 94.0 19.4 37.9 0.45

2235 117.1369 32.6408 Other 4 San Diego Harbor 71.4 100.0 5.0 43.3 0.64

2238 117.1287 32.6254 Other 3 San Diego Harbor 86.7 52.5 3.4 57.6 0.97

2239 117.1451 32.6824 Other 11 San Diego Harbor 100.0 66.9 15.9 34.6 0.72

2240 117.1541 32.6675 Other 3 San Diego Harbor 88.8 86.2 4.7 44.3 0.55

2241 117.1365 32.6703 Other 4 San Diego Harbor 97.9 69.1 7.7 19.4 0.52

2242 117.1498 32.6650 Other 4 San Diego Harbor 91.8 92.2 5.5 32.6 0.74

2243 117.142 7 32.6645 Other 4 San Diego Harbor 95.9 96.0 5.2 35.1 0.49

2244 117.1318 32.6597 Other 3 San Diego Harbor 100.0 51.9 3.5 21.2 0.29

2245 117.1427 32.6508 Other 4 San Diego Harbor 65.7 100.0 5.4 59.6 0.78

2247 117.1247 32.6423 Other 3 San Diego Harbor 89. 8 32.8 5.0 43.3 0.58

2249 117.1281 32.6213 Other 3 San Diego Harbor 75.5 100.0 10.4 73.9 1.35

2433 117.2092 32.7224 Other 9 San Diego Harbor 96.9 61.3 18.5 72.3 1.17

2434 117.1836 32.7249 Other 3 San Diego Harbor 101.0 92.3 13.4 44.2 0.71

2435 117.2229 32.7115 Other 12 San Diego Harbor 102.1 68.4 6.1 49.5 0.55

2436 117.1831 32.7150 Other 11 San Diego Harbor 100.0 27.8 25.2 55.9 1.36

2164 118.0826 33.7303 Other 3 Anaheim Bay 93.8 100.0 9.9 22.1 0.42

2152 118.1627 33.7593 Other 6 Los Angeles Harbor 1 01.0 92.9 7.2 68.2 0.81

2153 118.1578 33.7535 Other 5 Los Angeles Harbor 96.9 100.0 8.9 37.0 0.64

2154 118.1559 33.7490 Other 11 Los Angeles Harbor 96.9 100.0 6.5 34.4 0.50

2155 118.1675 33.7433 Other 12 Los Angeles Harbor 88.7 100.0 19.6 87.3 1.82

2156 118.1713 33.7400 Other 13 Los Angeles Harbor 84.5 100.0 19.6 88.9 1.73

2157 118.1532 33.7423 Other 10 Los Angeles Harbor 77.3 100.0 9.2 47.9 0.65

2158 118.2086 33.7283 Other 21 Los Angeles Harbor 90.7 68.2 16.2 82.1 1.63

2159 118.2104 33.7225 Ot her 15 Los Angeles Harbor 80.4 90.3 19.8 71.3 1.98

2160 118.2047 33.7236 Other 14 Los Angeles Harbor 84.5 42.2 14.8 73.9 2.27

2161 118.2017 33.7235 Other 14 Los Angeles Harbor 90.7 72.8 12.8 77.7 2.26

2162 118.2417 33.7135 Other 14 Los Angeles Harbo r 90.3 100.0 5.6 90.0 1.15

2163 118.1668 33.7279 Other 15 Los Angeles Harbor 89.7 97.3 11.1 75.5 1.36

2167 118.1577 33.7356 Other 13 Los Angeles Harbor 88.7 97.6 16.8 86.6 1.68

2168 118.2506 33.7119 Other 27 Los Angeles Harbor 80.2 100.0 5.3 80.7 1.8 5

2426 118.2314 33.7342 Other 11 Los Angeles Harbor 99.0 100.0 4.7 38.2 0.46

2427 118.2355 33.7309 Other 9 Los Angeles Harbor 52.0 100.0 6.2 89.4 1.47

2428 118.2585 33.7188 Other 23 Los Angeles Harbor 67.7 100.0 3.5 99.9 1.09

2188 118.0888 33.7338 P ort 11 Anaheim Bay 85.6 87.2 31.2 85.7 1.84

### D

- 5

- Test Response b

Station Longitude Latitude Strata a Depth

(m) Location Amphipod

(%) QwikSed

(%) H RGS

(μg/g) Fines

(%) TOC

(%)

2169 118.2784 33.7682 Port 11 Los Angeles Harbor 79.4 91.1 181. 5 83.2 2.54

2170 118.2560 33.7643 Port 13 Los Angeles Harbor 16.5 99.6 128.4 77.7 2.96

2172 118.2427 33.7492 Port 11 Los Angeles Harbor 78.4 89.8 45.7 55.0 0.93

2173 118.2377 33.7473 Port 12 Los Angeles Harbor 89.7 100.0 14.8 70.4 0.96

2174 118.2666 33 .7343 Port 3 Los Angeles Harbor 97.8 100.0 24.6 42.6 1.09

2175 118.2267 33.7409 Port 11 Los Angeles Harbor 85.6 83.6 12.7 71.5 0.87

2176 118.2611 33.7310 Port 18 Los Angeles Harbor 60.2 100.0 3.2 95.8 1.09

2177 118.2426 33.7348 Port 16 Los Angeles Harbo r 77.3 100.0 3.3 84.0 1.15

2178 118.2710 33.7280 Port 14 Los Angeles Harbor 91.8 100.0 23.3 87.1 2.53

2179 118.2103 33.7390 Port 26 Los Angeles Harbor 78.4 100.0 13.7 88.4 1.39

2182 118.2623 33.7238 Port 19 Los Angeles Harbor 87.6 100.0 2.7 75.4 0.68

2 184 118.2691 33.7206 Port 15 Los Angeles Harbor 91.8 98.3 20.0 88.4 2.83

2185 118.1998 33.7332 Port 23 Los Angeles Harbor 89.7 100.0 21.6 82.9 1.50

2186 118.1930 33.7314 Port 15 Los Angeles Harbor 88.7 96.1 9.7 52.5 0.81

2187 118.1840 33.7312 Port 15 Lo s Angeles Harbor 82.5 96.9 7.5 50.1 0.90

2430 118.2245 33.7691 Port 18 Los Angeles Harbor 66.3 77.5 19.9 83.7 1.56

2431 118.2241 33.7534 Port 12 Los Angeles Harbor 94.8 100.0 30.5 77.6 1.05

2432 118.2304 33.7508 Port 16 Los Angeles Harbor 87.6 77.3 22.2 93.1 1.45

2251 117.1621 32.7023 Port 9 San Diego Harbor 76.0 100.0 84.9 73.0 1.99

2252 117.1529 32.6919 Port 11 San Diego Harbor 104.2 100.0 18.2 16.7 0.59

2253 117.1381 32.6881 Port 8 San Diego Harbor 88.9 96.1 67.9 66.5 1.57

2254 117.1632 32.6773 Po rt 5 San Diego Harbor 98.0 100.0 97.1 35.6 0.66

2255 117.1294 32.6780 Port 11 San Diego Harbor 96.9 100.0 36.6 59.8 1.18

2256 117.1359 32.6769 Port 8 San Diego Harbor 100.0 100.0 16.6 65.9 1.26

2257 117.1341 32.6768 Port 9 San Diego Harbor 90.8 100.0 34 .8 77.8 1.63

2258 117.1321 32.6759 Port 11 San Diego Harbor 91.8 100.0 59.5 71.9 1.44

2259 117.1247 32.6702 Port 11 San Diego Harbor 96.9 97.6 31.3 69.0 1.24

2260 117.1300 32.6672 Port 4 San Diego Harbor 73.5 83.1 5.5 27.7 0.51

2262 117.1229 32.6515 Po rt 10 San Diego Harbor 78.5 86.2 27.9 74.8 1.64

2263 117.1760 32.7161 Port 13 San Diego Harbor 88.2 100.0 115.1 73.6 1.28

2264 117.1328 32.6854 Port 10 San Diego Harbor 89.8

- 99.0 123.0 73.4 1.99

2265 117.1403 32.6839 Port 11 San Diego Harbor 84.9 100.0 14.0 13.8 0.35

2439 117.1895 32.7261 Port 3 San Diego Harbor 84.4 78.6 26.4 53.2 1.03

2440 117.1748 32.7185 Port 10 San Diego Harbor 103.1 na 19.1 37.4 0.50

2441 117.2380 32.6912 Port 16 San Diego Harbor 87.9 84.6 32.3 79.9 1.97

2442 117.2371 32.6892 Port 13 San Diego Harbor 80.8 79.3 72.4 77.6 1.99

2302 117.7764 33.5100 River 50 Ballona Creek 88.2 na 4.2 78.4 1.02

2303 117.7819 33.5187 River 48 Ballona Creek 90.3 na 3.6 71.5 0.83

2304 117.7696 33.5213 River 14 Ballona Creek 93.5 na 1.3 1.1 0.08

23 05 118.4711 33.9757 River 8 Ballona Creek 104.3 na 3.8 9.9 0.18

2306 118.4536 33.9359 River 14 Ballona Creek 100.0 na 1.8 1.7 0.17

2307 118.4767 33.9623 River 15 Ballona Creek 101.1 na 3.9 15.2 0.21

2311 118.1850 33.7555 River 14 Los Angeles River 61.9 na 55.1 71.1 2.20

2312 118.6649 34.0202 River 30 Malibu Creek 101.0 na 12.8 61.0 1.26

### D

- 6

- Test Response b

Station Longitude Latitude Strata a Depth

(m) Location Amphipod

(%) QwikSed

(%) H RGS

(μg/g) Fines

(%) TOC

(%)

2314 118.6825 34.027 1 River 11 Malibu Creek 102.1 na 6.2 14.6 0.24

2308 119.0902 34.0831 River 74 Mugu Lagoon 63.9 na 3.4 59.6 1.22

2310 119.0891 34.0916 River 18 Mugu Lagoon 92.8 na 5.1 36.3 0.25

2315 117.2697 32.7695 River 19 San Diego River 100.0 na 1.1 0.0 0.07

2317 1 17.2757 32.7671 River 23 San Diego River 100.0 na 1.2 1.0 0.08

2318 118.1269 33.7237 River 12 San Gabriel River 94.8 na 4.3 11.7 0.22

2319 118.1435 33.7371 River 12 San Gabriel River 94.8 na 12.5 66.6 0.99

2320 118.1215 33.7330 River 7 San Gabriel River 96.9 na 10.8 6.4 0.15

2321 118.1335 33.7293 River 12 San Gabriel River 88.7 na 8.8 26.8 0.42

2325 117.9872 33.6277 River 13 San Gabriel River 97.9 na 1.2 16.0 0.14

2326 117.9562 33.6227 River 8 San Gabriel River 92.8 na 2.1 16.8 0.08

2328 119.2877 34. 2191 River 15 Santa Clara River 82.8 na 3.7 40.2 0.37

2329 119.2764 34.2501 River 11 Santa Clara River 100.0 100.0 3.9 0.5 0.18

2330 119.2808 34.2399 River 12 Santa Clara River 93.8 na 4.0 34.8 0.38

2331 119.2887 34.2456 River 16 Santa Clara River 92.5 na 4.1 91.6 0.92

2339 119.2848 34.2523 River 15 Santa Clara River 92.8 na 4.1 72.8 0.73

2335 117.1548 32.5447 River 18 Tijuana River 101.0 na 1.6 17.2 0.16

2338 119.3413 34.2699 River 19 Ventura River 100.0 na 3.4 24.0 0.31

2340 119.3416 34.2651 River 20 Ventura River 104.3 na 3.4 15.1 0.20

2450 118.1994 33.7603 River

- grad 5 Los Angeles River 7.2 na 88.5 70.6 3.44

2451 118.1742 33.7519 River

- grad 12 Los Angeles River 64.9 na 33.8 64.7 0.97

2453 117.9749 33.6279 River

- grad 11 San Gabriel River 95.4 na 1.2 30.3 0.12

2454 117.7553 33.5090 River

- grad 10 San Juan Creek 86.6 na 2.3 18.9 0.09

2289 117.3585 33.1025 SPOTW 78 Encina 96.8 100.0 3.9 62.5 0.66

2290 117.3588 33.1162 SPOTW 187 Encina 87.2 100.0 8.3 81.0 1.94

2266 119.8217 34.3982 SPOTW 41 Goleta na na 51.4 52.0 1.43

2267 119.8254 34.4060 SPOTW 14 Goleta 92.0 100.0 14.5 16.1 0.34

2301 119.8330 34.4011 SPOTW 21 Goleta na na 48.5 25.0 0.57

2286 117.3885 33.1667 SPOTW 22 Oceanside 95.0 98.7 1.8 22.5 0.25

2288 117.3844 33.1586 SPOTW 33 Oceanside 9 5.0 98.4 3.4 70.4 0.42

2273 119.1984 34.1305 SPOTW 14 Oxnard 56.7 nv 5.5 27.7 0.23

2274 119.1975 34.1238 SPOTW 16 Oxnard na na 2.4 6.0 0.14

2275 119.1955 34.1307 SPOTW 13 Oxnard 99.0 40.6 4.7 8.1 0.15

2276 119.1828 34.1237 SPOTW 14 Oxnard na na 5.8 7.0 0.15

2291 117.3526 33.0999 SPOTW 69 San Elijo 97.9 91.2 3.6 62.0 0.70

2293 117.2923 32.9995 SPOTW 23 San Elijo 92.5 86.2 1.0 0.8 1.82

2269 119.6674 34.3971 SPOTW 21 Santa Barbara na na 10.8 41.0 0.90

2272 119.6595 34.3982 SPOTW 23 Santa Barbara na na 11.7 23.0 0.44

2277 117.7649 33.5073 SPOTW 35 Terminal Island 98.0 100.0 6.8 56.7 0.73

2295 117.2987 32.9904 SPOTW 41 Terminal Island 97.8 100.0 3.8 36.7 0.41

2297 118.2353 33.7229 SPOTW 12 Terminal Island 88.3 93.2 10.4 78.4 1.40

2298 118.2340 33.728 9 SPOTW 11 Terminal Island 87.2 100.0 8.7 69.8 0.93

2299 118.2340 33.7206 SPOTW 13 Terminal Island 91.4 87.4 12.3 65.2 1.36

2300 118.2391 33.7180 SPOTW 12 Terminal Island 95.8 100.0 3.6 86.1 1.34

### D

- 7

-

a 

### Strata D efinitions:

Bath

- 30 = O ffshore areas of sha llow depth (5

- 30 m)

Bath

- 120 = O ffshore areas of mid depth (30

- 120 m)

River = S ites near river mouths

River

- gradient = S ites along a gradient of river influence

LPOTW = A reas near the outfall of a large publicly owned treatment work s (POTW)

SPOTW = A reas near the outfall of a small publicly owned treatment work s

Port = P ort and industrial areas

Marina = M arina areas

Other = H arbor areas of mixed use

b 

### Indicator R esponses :

Amphipod = Percent of control survival

QwikSed = Percent of control l uminescence

H RGS = μg/g benzo [a] pyrene equivalents

na = N o sample was collected for the given analysis.

nv = N o valid data were obtained from testing

### E

- 8



## APPENDIX E. TOXICITY TEST RESULTS MAPPED BY

**INDICATOR**

### E

- 9

### E

- 10

### E

- 11

### E

- 12

### E

- 13

### E

- 14

### E

- 15

### E

- 16

### F

- 1

**A PPENDIX F. RELATIONSHIP BETWEEN SEDIMENT CHARACTERISTICS** 

**AND INDICATOR RESPONSES**

Percent Fines

0 10 20 30 40 50 60 70 80 90 100Amphipod Survival (% of Control)

0 20 40 60 80 100 120

r = -0.555



### Total Organic Carbon (%) 0 1 2 3 4Amphipod Survival (% of Control)

0 20 40 60 80 100 120

r = -0.488

### F

- 2 Percent Fines 0 20 40 60 80 100QwikSed Response (% of Control)

0 20 40 60 80 100 120

r = -0.037

Total Organic Carbon (%) 0 1 2 3 4QwikSed Response (% of Control)

0 20 40 60 80 100 120

r = -0.127

### F

- 3 Percent Fines (%) 0 20 40 60 80 100P450 Response (μg B[a]P eq/g)

0 20 40 60 80 100 120

r = 0.534



### Total Organic Carbon (%) 0 1 2 3 4P450 Response (μg B[a]P eq/g)

0 20 40 60 80 100 120

r = 0.702